WO2008127655A1 - Anti-alpha 6 beta 4 integrin antibodies and uses therof - Google Patents

Anti-alpha 6 beta 4 integrin antibodies and uses therof Download PDF

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Publication number
WO2008127655A1
WO2008127655A1 PCT/US2008/004714 US2008004714W WO2008127655A1 WO 2008127655 A1 WO2008127655 A1 WO 2008127655A1 US 2008004714 W US2008004714 W US 2008004714W WO 2008127655 A1 WO2008127655 A1 WO 2008127655A1
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WIPO (PCT)
Prior art keywords
seq
antibody
fragment
polynucleotide
integrin
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PCT/US2008/004714
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French (fr)
Inventor
Karen Mclachlan
Christilyn Graff
Heather Huet
Ling Ling Chen
Xianjun Cao
Beth Browning
Anne Cheung
Rachel Rennard
Steven Miklasz
Veronica Gabarra
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Biogen Idec Ma Inc.
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Publication of WO2008127655A1 publication Critical patent/WO2008127655A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM

Definitions

  • ⁇ 6 ⁇ 4 integrin is a laminin receptor expressed on basal epithelial cells.
  • the primary function of ⁇ 6 ⁇ 4 integrin is to maintain the integrity of epithelia, especially the epidermis.
  • studies have shown that expression of ⁇ 6 ⁇ 4 also persists in aggressive carcinomas and that its expression is linked to the behavior of these tumors. It is believed to play a pivotal role in functions associated with carcinoma progression and has also been implicated in the formation of carcinomas. Dajee et al., Nature 421:639-643 (2003).
  • ⁇ 6 ⁇ 4 integrins belong to the integrin family of cell surface receptors.
  • the integrin family serve cellular adhesion functions.
  • the receptors form a link between the extracellular matrix and the cytoskeleton through their binding to various extracellular components.
  • Each integrin receptor is a heterodimer composed of an ⁇ and a ⁇ subunit. At least 18 ⁇ chains and eight ⁇ chains have been characterized.
  • the molecular architecture of the ⁇ 6 ⁇ 4 integrin comprises an ⁇ 6 subunit which is composed of a heavy and light chain linked by a disulfide bond and a ⁇ 4 subunit.
  • the distinguishing structural feature of the ⁇ 6 ⁇ 4 integrin is the atypical cytoplasmic domain of the ⁇ 4 subunit.
  • Two pairs of fibronectin type III repeats separated by a connecting segment characterize the ⁇ 4 domain and it is distinct both in size (approximately 1000 amino acids) and structure from any other integrin subunits.
  • ⁇ 6 ⁇ 4 integrin functions primarily as an adhesion receptor in normal epithelia. It mediates the formation on the basal epithelial cell surface of stable adhesive structures known as hemidesmosomes (HDs) that serve an essential mechanical function.
  • the hemidesmosomes link the intermediate filament cytoskeleton with laminins in the basement membrane (BM). Borradori, L. and Sonnenberg, A., J Invest. Dematol. //2:411-418 (1999).
  • BM basement membrane
  • DiPersio et al J. Cell ScL //5:3051-3062 (2000).
  • mutations in the ⁇ 4 subunit also cause epidermal blistering. Vidal et al., Nature Genetics /0:229-234 (1995).
  • ⁇ 6 ⁇ 4 integrins have also been shown to play a role in the formation, migration, invasion and survival of carcinomas.
  • ⁇ 6 ⁇ 4 integrins have been shown to exist in two "functional states," one as an adhesion receptor localized in the HDs and two, as a signaling competent receptor localized with F-actin in lamellipodia and filopodia during carcinoma migration and invasion. It has been shown to cooperate with specific growth factors, such as EGF, to facilitate signaling and promote the migration and survival of carcinoma cells. Without being bound by theory, it is believed that EGF can increase PKC- ⁇ -mediated phosphorylation of the ⁇ 4 cytoplasmic domain.
  • This phosphorylation leads to disruption of the HDs and mobilization of ⁇ 6 ⁇ 4 to lamellipodia and its association with F-actin.
  • the release of ⁇ 6 ⁇ 4 from the HDs activates its signaling capacity, which may occur by its association with growth factor receptors such as erbB2, Met, and Ron or lipid rafts.
  • the key signaling event mediated by ⁇ 6 ⁇ 4 is activation of PI3-K.
  • the ⁇ 6 ⁇ 4-mediated activation of PB-K and its downstream effectors Akt, mTOR and Rac have been shown to have profound consequences on the biology of carcinoma cells.
  • the regulation of mTOR by ⁇ 6 ⁇ 4 in particular, has been shown to influence the translation of growth factors such as VEGF that can function to further amplify PB-K activation, thus sustaining and enhancing ⁇ 6 ⁇ 4-mediated functions.
  • the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59- B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the reference monoclonal Fab antibody fragment is M59-B05.
  • the reference monoclonal Fab antibody fragment is M61-C03.
  • the ⁇ 6 ⁇ 4 integrin epitope is contained within amino acids 446-686 of SEQ ID NO:101.
  • the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the antibody or fragment competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the antibody or fragment thereof comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the heavy chain variable region (VH) of the antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
  • VH heavy chain variable region
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the light chain variable region (VL) of the antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92.
  • VL light chain variable region
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ BD NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ DD NO: 47, SEQ ID NO: 57, SEQ DD NO: 67, SEQ DD NO: 77, and SEQ DD NO: 87.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ DD NO: 22, SEQ DD NO: 32, SEQ DD NO: 42, SEQ DD NO: 52, SEQ DD NO: 62, SEQ DD NO: 72, SEQ DD NO: 82 and SEQ DD NO:92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ DD NO: 92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences identical, except for 20 or fewer conservative amino acid substitutions each, to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ ID NO: 92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ ID NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ DD NO: 92.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region- 1 (VH-CDRl) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88.
  • the VH-CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-2 (VH-CDR2) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79 and SEQ ID NO: 89.
  • the VH-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-3 (VH-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ED NO: 60, SEQ ID NO: 70, SEQ BD NO: 80, and SEQ ID NO: 90.
  • the VH-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region- 1 (VL-CDRl) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93.
  • the VL-CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region-2 (VL-CDR2) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 24, SEQ BD NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84, and SEQ ID NO: 94.
  • the VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region-3 (VL-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, and SEQ ID NO: 95.
  • the VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO:
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90, except for one, two, three, or four amino acid substitutions in at least one of said VH-CDRs.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VH of the antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ED NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ BD NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95, except for one, two, three, or four amino acid substitutions in at least one of said VL-CDRs.
  • the invention provides an isolated antibody or fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the VL of the antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95.
  • VH framework regions and/or VL framework regions are human, except for five or fewer amino acid substitutions.
  • the above-described antibodies or fragments thereof bind to a linear epitope or a non-linear conformation epitope
  • the above-described antibodies or fragments thereof are multivalent, and comprise at least two heavy chains and at least two light chains.
  • the above-described antibodies or fragments thereof are multispecific. In further embodiments, the above-described antibodies or fragments thereof are bispecific.
  • the heavy and light chain variable domains are fully human, hi further embodiments, the heavy and light chain variable domains are from a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65- Al 1, M66-H09 and M67-F05.
  • the heavy and light chain variable domains are murine.
  • the heavy and light chain variable domains are from a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the above-described antibodies or fragments thereof are humanized.
  • the above-described antibodies or fragments thereof are chimeric.
  • the above-described antibodies or fragments thereof are primatized.
  • Fab fragments Fab fragments, Fab' fragments, F(ab) 2 fragments, or Fv fragments.
  • the above-described antibodies are single chain antibodies.
  • the above-described antibodies or fragments thereof comprise light chain constant regions selected from the group consisting of a human kappa constant region and a human lambda constant region.
  • the above-described antibodies or fragments thereof comprise a heavy chain constant region or fragment thereof.
  • the heavy chain constant region or fragment thereof is human IgG4.
  • the above-described antibodies or fragments thereof specifically bind to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) which is less than the K D for said reference monoclonal antibody, hi further embodiments, the dissociation constant (K D ) is no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 ⁇ 3 M, 10 "3 M, 5 x Kr 4 M, 10 "4 M, 5 x 10 "5 M, 10 '5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x Kr 9 M, 10 "9 M, 5 x 10 "10 M, 10 “10 M, 5 x 10 "H M, 10 11 M, 5 x 10 "12 M, 10 "12 M, 5 x 10 "
  • the above-described antibodies or fragments thereof preferentially bind to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof .
  • the above described antibodies or fragments thereof bind to ⁇ 6 ⁇ 4 integrin expressed on the surface of a cell.
  • the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
  • the above described antibodies or fragments thereof block laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin association with growth factor receptors.
  • the growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin-mediated PB-K activation.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin-mediated activation of the Ras/MAPK signaling pathway.
  • the above described antibodies or fragments thereof inhibit ⁇ 6 ⁇ 4 integrin-mediated cell proliferation or tumor cell growth.
  • the above described antibodies or fragments thereof induce apoptosis.
  • the above described antibodies or fragments thereof further comprise a heterologous polypeptide fused thereto.
  • the above described antibodies or fragments thereof are conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents
  • the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents
  • the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemi
  • Certain embodiments of the invention include an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the amino acid sequence of the VH polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and where an antibody or antigen binding fragment thereof comprising the VH polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid sequence of the VH polypeptide is selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
  • the nucleotide sequence encoding the VH polypeptide is optimized for increased expression without changing the amino acid sequence of the VH polypeptide.
  • the optimization comprises identification and removal of splice donor and splice acceptor sites and/or optimization of codon usage for the cells expressing the polynucleotide.
  • the nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 66, SEQ ID NO: 76, and SEQ ID NO: 86.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, where the amino acid sequence of the VL polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ED NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82, and SEQ ID NO: 92; and where an antibody or antigen binding fragment thereof comprising the VL polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid sequence of the VL polypeptide is selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82, and SEQ ID NO: 92.
  • the nucleotide sequence encoding the VL polypeptide is optimized for increased expression without changing the amino acid sequence of said VL polypeptide.
  • the optimization comprises identification and removal of splice donor and splice acceptor sites and/or optimization of codon usage for the cells expressing the polynucleotide
  • the nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ DD NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81, and SEQ ID NO: 91.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the amino acid sequence of the VH polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ E) NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and where an antibody or antigen binding fragment thereof comprising said VH polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, where the amino acid sequence of the VL polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO: 92; and wherein an antibody or antigen binding fragment thereof comprising said VL polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDRl amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88; and where an antibody or antigen binding fragment thereof comprising the VH-CDRl specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VH- CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ BD NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR2 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, and SEQ ID NO: 89; and where an antibody or antigen binding fragment thereof comprising the VH-CDR2 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • VH- CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, and SEQ ID NO: 89.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • VH- CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDRl amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93; and where an antibody or antigen binding fragment thereof comprising the VL-CDRl specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VL-CDRl amino acid sequence is selected from the group consisting of: SEQ ED NO: 23, SEQ ID NO: 33, SEQ ED NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR2 amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ED NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ED NO: 74, SEQ ED NO: 84 and SEQ ED NO: 94; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR2 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ED NO: 24, SEQ ED NO: 34, SEQ ID NO: 44, SEQ ED NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84 and SEQ ID NO: 94.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ED NO: 25, SEQ ED NO: 35, SEQ ED NO: 45, SEQ ED NO: 55, SEQ ED NO: 65, SEQ ED NO: 75, SEQ ED NO: 85, and SEQ ED NO: 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ED NO: 25, SEQ ED NO: 35, SEQ ED NO: 45, SEQ ED NO: 55, SEQ ED NO: 65, SEQ ED NO: 75, SEQ ED NO: 85, and SEQ ED NO: 95.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the VH polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ED NOs: 18, 19, and 20; SEQ ED NOs: 28, 29; and 30; SEQ ED NOs: 38, 39, and 40; SEQ ED NOs: 48, 49, and 50; SEQ ED NOs: 58, 59, and 60; SEQ ED NOs: 68, 69, and 70; SEQ ED NOs: 78, 79, and 80; and SEQ ID NOs: 88, 89 and 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ED NOs: 23, 24, and 25; SEQ ED NOs: 33, 34, and 35; SEQ ED NOs: 43, 44, and 45; SEQ ED NOs: 53, 54, and 55; SEQ ED NOs: 63, 64, and 65; SEQ ED NOs: 73, 74, and 75; SEQ ED NOs: 83, 84, and 85; and SEQ ID NO: 93, 94, and 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • the above-described polynucleotides further comprise a nucleic acid encoding a signal peptide fused to the antibody VH polypeptide or the antibody VL polypeptide.
  • the above-described polynucleotides further comprise a nucleic acid encoding a heavy chain constant region CHl domain fused to the VH polypeptide, encoding a heavy chain constant region CH2 domain fused to the VH polypeptide, encoding a heavy chain constant region CH3 domain fused to the VH polypeptide, or encoding a heavy chain hinge region fused to said VH polypeptide.
  • the heavy chain constant region is human IgG4.
  • the above-described polynucleotides comprise a nucleic acid encoding a light chain constant region domain fused to said VL polypeptide.
  • the light chain constant region is human kappa.
  • the antibody or antigen-binding fragment thereof comprising a polypeptide encoded by the nucleic acid specifically binds the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the antibody or antigen-binding fragment thereof comprising a polypeptide encoded by the nucleic acid competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the framework regions of the VH polypeptide or VL polypeptide are human, except for five or fewer amino acid substitutions.
  • the invention provides an antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid, that binds to a linear epitope or a non-linear conformational epitope.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is multivalent, and comprises at least two heavy chains and at least two light chains.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is multispecific. In further embodiments, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is bispecific.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid comprises heavy and light chain variable domains which are fully human.
  • the heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid comprises heavy and light chain variable domains which are murine.
  • the heavy and light chain variable domains are identical to those of a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is humanized.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is primatized.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is chimeric.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is fully human.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is an Fab fragment, an Fab' fragment, an F(ab) 2 fragment, or an Fv fragment, hi certain embodiments of the above-described polynucleotides, the antibody or antigen- binding fragment thereof comprising the polypeptide encoded by the nucleic acid is a single chain antibody.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid specifically binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 '2 M, 10 ⁇ 2 M, 5 x 10 "3 M, 10 '3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 "9 M, 1(T 9 M, 5 x 10- 10 M, 10- 10 M, 5 x 1(T” M, 1(T 1 M, 5 x 10 "12 M, 1(T 12 M, 5 x 10
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid preferentially binds to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid binds to ⁇ 6 ⁇ 4 integrin expressed on the surface of a cell.
  • the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by said nucleic acid blocks laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin -mediated activation of PI3-K.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits activation of the Ras/MAPK signaling pathway. [0091] hi some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin association with growth factor receptors.
  • the growth factor receptor is selected from the group consisting of erbB2,
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin-mediated cell proliferation or tumor cell growth.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid induces apoptosis.
  • the above-described polynucleotides further comprise a nucleic acid encoding a heterologous polypeptide.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents.
  • the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
  • the invention provides compositions comprising the above-described polynucleotides.
  • the invention provides vectors comprising the above-described polynucleotides, hi further embodiments, the polynucleotides are operably associated with a promoter. In additional embodiments, the invention provides host cells comprising such vectors. In further embodiments, the invention provides vectors where the polynucleotide is operably associated with a promoter.
  • the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising culturing a host cell containing a vector comprising the above-described polynucleotides, and recovering said antibody, or fragment thereof.
  • the invention provides an isolated polypeptide produced by the above-described method.
  • the invention provides isolated polypeptides encoded by the above-described polynucleotides.
  • the antibody or fragment thereof comprising the polypeptide specifically binds to ⁇ 6 ⁇ 4 integrin.
  • Other embodiments include the isolated antibody or fragment thereof comprising the above- described polypeptides.
  • the invention provides a composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, where the VH encoding polynucleotide and the VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ED NO: 37 and SEQ ID NO: 42; SEQ ED NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ED NO: 62; SEQ ID NO: 67 and 72; SEQ ID NO: 77 and 82; and SEQ ED NO: 87 and 92; and where an antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds ⁇ 6 ⁇ 4 integrin
  • the VH encoding polynucleotide and said VL encoding polynucleotide comprise nucleic acids encoding amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and 72; SEQ DD NO: 77 and 82; and SEQ DD NO: 87 and 92.
  • the invention provides a composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, where the VH encoding polynucleotide and the VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences identical, except for less than 20 conservative amino acid substitutions, to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and 72; SEQ DD NO: 77 and 82; and SEQ DD NO: 87 and 92; and where an antibody or fragment thereof encoded by the VH and VL encoding
  • the VH encoding polynucleotide encodes a VH polypeptide comprising VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ DD NOs: 18, 19, and 20; SEQ DD NOs: 28, 29, and 30; SEQ ED NOs: 38, 39, and 40; SEQ DD NOs: 48, 49, and 50; SEQ BD NOs: 58, 59, and 60; SEQ ED NOs: 68, 69, and 70; SEQ DD NOs: 78, 79 and 80; and SEQ DD NOs: 88, 89, and 90; where the VL encoding polynucleotide encodes a VL polypeptide comprising VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ DD NOs: 23, 24, and 25; SEQ DD NOs:
  • the VL encoding polynucleotide further comprises a nucleic acid encoding a signal peptide fused to the antibody VL polypeptide.
  • the VH encoding polynucleotide further comprises a nucleic acid encoding a heavy chain constant region CHl domain fused to the VH polypeptide, further comprises a nucleic acid encoding a heavy chain constant region CH2 domain fused to the VH polypeptide, further comprises a nucleic acid encoding a heavy chain constant region CH3 domain fused to the VH polypeptide, or further comprises a nucleic acid encoding a heavy chain hinge region fused to the VH polypeptide, hi further embodiments, the heavy chain constant region is human IgG4.
  • the VL encoding polynucleotide further comprises a nucleic acid encoding a light chain constant region domain fused to the VL polypeptide.
  • the light chain constant region is human kappa.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • the framework regions of the VH and VL polypeptides are human, except for five or fewer amino acid substitutions.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides binds to a linear epitope or a non-linear conformational epitope.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is multivalent, and comprises at least two heavy chains and at least two light chains.
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is multispecific. hi further embodiments, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is bispecific. [0114] hi some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are fully human. In further embodiments, the heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03,
  • the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are murine, hi further embodiments, the heavy and light chain variable domains are identical to those of a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is humanized. [0117] hi various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is primatized. [0118] hi various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is chimeric. [0119] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is fully human.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is an Fab fragment, an Fab 1 fragment, an F(ab) 2 fragment, or an Fv fragment.
  • the antibody or antigen- binding fragment thereof comprising the polypeptide encoded by the nucleic acid is a single chain antibody.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid specifically binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 “4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 “6 M, 5 x 10 "7 M, 10 “7 M, 5 x 10 "8 M, 10 “8 M, 5 x 1(T 9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 "12 M, 10 "12 M, 5 x 10 "13 M, 10 "13 M, 5
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid preferentially binds to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid binds to ⁇ 6 ⁇ 4 integrin expressed on the surface of a cell.
  • the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by said nucleic acid blocks laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin-mediated activation of PI3-K.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits activation of the Ras/MAPK signaling pathway.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin association with growth factor receptors.
  • the growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits ⁇ 6 ⁇ 4 integrin-mediated cell proliferation or tumor cell growth.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid induces apoptosis.
  • the above-described compositions, the VH encoding polynucleotide, the VL encoding polynucleotide, or both the VH and the VL encoding polynucleotides further comprise a nucleic acid encoding a heterologous polypeptide.
  • the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
  • the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents.
  • the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
  • the VH encoding polynucleotide is contained on a first vector and the VL encoding polynucleotide is contained on a second vector, hi further embodiments, the VH encoding polynucleotide is operably associated with a first promoter and the VL encoding polynucleotide is operably associated with a second promoter, hi certain other embodiments, the first and second promoters are copies of the same promoter. In further embodiments, the first and second promoters are non-identical.
  • the first vector and the second vector are contained in a single host cell.
  • the first vector and the second vector are contained in separate host cells.
  • the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising culturing the above-described host cells, and recovering the antibody, or fragment thereof.
  • the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising co-culturing separate host cells, and recovering the antibody, or fragment thereof.
  • the invention provides combining the VH and VL encoding polypeptides, and recovering the antibody, or fragment thereof.
  • the invention provides an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, produced by the above-described methods.
  • the invention provides compositions, where the VH encoding polynucleotide and the VL encoding polynucleotide are on the same vector, as well as the vectors therein.
  • the VH encoding polynucleotide and the VL encoding polynucleotide are each operably associated with a promoter.
  • the VH encoding polynucleotide and the VL encoding polynucleotide are fused in frame, are co- transcribed from a single promoter operably associated therewith, and are cotranslated into a single chain antibody or antigen-binding fragment thereof.
  • the VH encoding polynucleotide and said VL encoding polynucleotide are co-transcribed from a single promoter operably associated therewith, but are separately translated.
  • the vectors further comprise an IRES sequence disposed between the VH encoding polynucleotide and the VL encoding polynucleotide.
  • the polynucleotide encoding a VH and the polynucleotide encoding a VL are separately transcribed, each being operably associated with a separate promoter.
  • the separate promoters are copies of the same promoter or the separate promoters are non-identical.
  • the invention provides host cells comprising the above- described vectors. [0143] In other embodiments, the invention provides a method of producing an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, comprising culturing the above-described host cells, and recovering the antibody, or fragment thereof.
  • the invention provides an antibody or fragment thereof which specifically binds ⁇ 6 ⁇ 4 integrin, produced by the above-described methods.
  • the invention provides a method for treating a hyperproliferative disorder in an animal, comprising administering to an animal in need of treatment a composition comprising: a) an isolated antibody or fragment as described above; and b) a pharmaceutically acceptable carrier, hi further embodiments, the hyperproliferative disease or disorder is selected from the group consisting of cancer, a neoplasm, a tumor, a malignancy, or a metastasis thereof.
  • the antibody or fragment thereof specifically binds to ⁇ 6 ⁇ 4 integrin expressed on the surface of a malignant cell, hi further embodiments, the binding of the antibody or fragment thereof to the malignant cell results in growth inhibition of the malignant cell.
  • the antibody or fragment thereof inhibits ⁇ 6 ⁇ 4 integrin phosphorylation or inhibits tumor cell proliferation
  • the tumor cell proliferation is inhibited through the prevention or retardation of metastatic growth.
  • the antibody or fragment thereof inhibits tumor cell migration, hi further embodiments, the tumor cell proliferation is inhibited through the prevention or retardation of tumor spread to adjacent tissues.
  • the hyperproliferative disease or disorder is a neoplasm located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, adrenal gland, parathyroid gland, pituitary gland, testicles, ovary, thymus, thyroid, eye, head, neck, central nervous system, peripheral nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, or urogenital tract.
  • the hyperproliferative disease is cancer, said cancer selected from the group consisting of: epithelial squamous cell cancer, melanoma, leukemia, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, renal cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer.
  • the cancer is selected from the group consisting of stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • the animal is a mammal.
  • the mammal is a human.
  • Figure 1 shows the purification of soluble ⁇ 6Fc ⁇ 4 protein.
  • Figure 2 shows the titration of murine monoclonal antibodies against CHO- human- ⁇ 6 ⁇ 4.
  • Figure 3 shows inhibition of murine antibody binding with ⁇ 6Fc ⁇ 4 protein.
  • Figure 4 shows titration of murine antibodies against colorectal and breast tumor cell lines.
  • Figure 5 shows the titration of IgG4P human antibodies against CHO-human- ⁇ 6 ⁇ 4.
  • Figure 6 shows the titration of IgG4P human antibodies against CHO-murine- ⁇ 6 ⁇ 4.
  • Figure 7 shows specificity of human antibody 59B05.
  • Figure 8 shows the inhibition of human antibody binding with ⁇ 6Fc ⁇ 4.
  • Figure 9 shows titration of human antibody 59B05 binding to breast and colorectal tumor cell lines.
  • Figure 10 shows the inhibition of anchorage independent growth in SW620 colorectal cancer cells.
  • Figure 11 shows the inhibition of anchorage independent growth in MDA-MB-
  • Figure 12 shows the inhibition of adhesion in colorectal cancer cells to SCC25
  • Figure 13 shows induction of apoptosis in human tumor cell lines.
  • Figure 14 shows inhibition of Phospho-AKT in breast tumor cells with human ⁇ 6 ⁇ 4 antibodies.
  • Figure 15 shows inhibition of SW480 colorectal tumor cell line adhesion on purified rat laminin-5 using full-length IgG4 versus Fab versions of human antibodies
  • Figure 16 shows inhibition of anchorage independent growth in BT474 breat cancer cells using full-length IgG4 versus Fab versions of human antibodies 59B05 and 61C03.
  • Figure 17 shows the induction of apoptosis in MCF7 breast cancer cells by human antibody 59B05.
  • Figure 18 shows a western blot of CNBr cleavage products from ⁇ 4 digests.
  • a or “an” entity refers to one or more of that entity; for example, “an ⁇ 6 ⁇ 4 integrin antibody,” is understood to represent one or more ⁇ 6 ⁇ 4 integrin antibodies.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • polypeptide is intended to encompass a singular
  • polypeptide as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • peptides, dipeptides, tripeptides, oligopeptides, "protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post- expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
  • a polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids.
  • Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
  • glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e.g., a serine residue or an asparagine residue.
  • an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • an isolated polypeptide can be removed from its native or natural environment.
  • Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof.
  • fragments include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native antibody or polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein.
  • Variants of ⁇ 6 ⁇ 4 integrin antibodies and antibody polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring Non- naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of ⁇ 6 ⁇ 4 integrin antibodies and antibody polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins.
  • variant polypeptides may also be referred to herein as "polypeptide analogs.”
  • a “derivative" of an ⁇ 6 ⁇ 4 integrin antibody or antibody polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
  • derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • polynucleotide is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA).
  • mRNA messenger RNA
  • pDNA plasmid DNA
  • a polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).
  • PNA peptide nucleic acids
  • nucleic acid refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.
  • isolated nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment.
  • a recombinant polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody contained in a vector is considered isolated for the purposes of the present invention.
  • Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention.
  • Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.
  • polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
  • a "coding region” is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors.
  • any vector may contain a single coding region, or may comprise two or more coding regions, e.g., a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region.
  • a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a nucleic acid encoding an ⁇ 6 ⁇ 4 integrin antibody or fragment, variant, or derivative thereof.
  • Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.
  • the polynucleotide or nucleic acid is DNA.
  • a polynucleotide comprising a nucleic acid which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions.
  • An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s).
  • Two DNA fragments are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed.
  • a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid.
  • the promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells.
  • transcription control elements besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription.
  • Suitable promoters and other transcription control regions are disclosed herein.
  • transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus).
  • Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit ⁇ -globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
  • translation control elements include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
  • a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).
  • mRNA messenger RNA
  • Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.
  • proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature” form of the polypeptide.
  • the native signal peptide e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it.
  • a heterologous mammalian signal peptide, or a functional derivative thereof may be used.
  • the wild- type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse ⁇ -glucuronidase.
  • the present invention is directed to certain ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof.
  • ⁇ 6 ⁇ 4 integrin antibodies encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
  • an antigen binding molecule refers in its broadest sense to a molecule that specifically binds an antigenic determinant. It is understood by those of skill in the art that fragments of mature antibodies can bind specifically to an antigen. Accordingly, an antigen binding molecule, as the term is used herein, includes, but is not limited to, fragments of mature antibodies that bind specifically to a target antigen.
  • An ABM of the present invention need not contain a constant region. If one or more constant region(s) is present, in particular embodiments, the constant region is substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical.
  • the ABMs of the present invention may be glycoengineered to enhance antibody dependent cellular cytotoxicity.
  • antibody and “immunoglobulin” are used interchangeably herein.
  • An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain.
  • Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
  • immunoglobulin comprises various broad classes of polypeptides that can be distinguished biochemically.
  • heavy chains are classified as gamma, mu, alpha, delta, or epsilon, ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ l- ⁇ 4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively.
  • immunoglobulin subclasses e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention. All immunoglobulin classes are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules.
  • IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000- 70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region.
  • Light chains are classified as either kappa or lambda (K, ⁇ ). Each heavy chain class may be bound with either a kappa or lambda light chain, hi general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells.
  • the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
  • variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity.
  • the constant domains of the light chain (CL) and the heavy chain (CHl, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like.
  • the N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy- terminus of the heavy and light chain, respectively.
  • variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen binding site.
  • This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains, hi some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et ai, Nature 563:446-448 (1993).
  • each antigen binding domain is short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment.
  • the remainder of the amino acids in the antigen binding domains referred to as "framework” regions, show less inter-molecular variability.
  • the framework regions largely adopt a ⁇ - sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure.
  • framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
  • the antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope.
  • the amino acids comprising the CDRs and the framework regions, respectively can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, "Sequences of Proteins of Immunological Interest,” Kabat, E., et al, U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol, /95:901-917 (1987), which are incorporated herein by reference in their entireties).
  • CDR complementarity determining region
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • Kabat numbering refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest” (1983).
  • references to the numbering of specific amino acid residue positions in an ⁇ 6 ⁇ 4 integrin antibody or antigen-binding fragment, variant, or derivative thereof of the present invention are according to the Kabat numbering system.
  • VHH the heavy chain variable region
  • VHH variable regions include (a) more hydrophobic amino acids in the light chain contact surface of VH as compared to the corresponding region in VHH, (b) a longer CDR3 in VHH, and (c) the frequent occurrence of a disulfide bond between CDRl and CDR3 in VHH.
  • an antigen binding molecule of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an antigen binding molecule of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.
  • Antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to ⁇ 6 ⁇ 4 integrin antibodies disclosed herein).
  • anti-Id antigen-binding fragments, variants, or derivatives thereof of the invention
  • Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Antibody fragments may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHl, CH2, and CH3 domains. Also included in the invention are antigen- binding fragments also comprising any combination of variable region(s) with a hinge region, CHl, CH2, and CH3 domains.
  • Antibodies or immunospecific fragments thereof of the present invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies, hi another embodiment, the variable region may be condricthoid in origin (e.g., from sharks).
  • human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al. A human antibody is still “human” even if amino acid substitutions are made in the antibody.
  • heavy chain portion includes amino acid sequences derived from an immunoglobulin heavy chain.
  • a polypeptide comprising a heavy chain portion comprises at least one of: a CHl domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.
  • a binding polypeptide for use in the invention may comprise a polypeptide chain comprising a CHl domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHl domain and a CH3 domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain, hi another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain.
  • a binding polypeptide for use in the invention may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain).
  • a CH2 domain e.g., all or part of a CH2 domain.
  • the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer.
  • heavy chain portion-containing monomers of the invention are not identical.
  • each monomer may comprise a different target binding site, forming, for example, a bispecific antibody.
  • the heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules.
  • a heavy chain portion of a polypeptide may comprise a CHl domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule
  • a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule
  • a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
  • the term "light chain portion” includes amino acid sequences derived from an immunoglobulin light chain.
  • the light chain portion comprises at least one of a VL or CL domain.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein may be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide ( ⁇ 6 ⁇ 4 integrin) that they recognize or specifically bind.
  • a target polypeptide ⁇ 6 ⁇ 4 integrin
  • the portion of a target polypeptide which specifically interacts with the antigen binding domain of an antibody is an "epitope," or an "antigenic determinant.”
  • a target polypeptide may comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen.
  • the epitope may include a portion of ⁇ 6 and a portion of ⁇ 4 together, i.e., the epitope can comprise portions of both components in the heterodimer.
  • an "epitope" on a target polypeptide may be or include non-polypeptide elements, e.g., an "epitope may include a carbohydrate side chain.
  • the minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids.
  • Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain.
  • peptide or polypeptide epitope recognized by ⁇ 6 ⁇ 4 integrin antibodies of the present invention contains a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of ⁇ 6 ⁇ 4 integrin.
  • an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind” to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope.
  • the term “specificity” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope.
  • antibody “A” may be deemed to have a higher specificity for a given epitope than antibody "B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
  • preferentially binds it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope.
  • an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (K D ) that is less than the antibody's K D for the second epitope.
  • K D dissociation constant
  • an antibody may be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's K D for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's K D for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope.
  • an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.
  • An antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X 10 "2 sec '1 , 10 ⁇ 2 sec "1 , 5 X 10 '3 sec '1 or 10 ⁇ 3 sec "1 .
  • an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec '1 , or 10 '5 sec '1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec “1 or 10 "7 sec “1 .
  • off rate k(off)
  • An antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) of greater than or equal to 10 3 M "1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
  • an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) greater than or equal to 10 5 M “1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec “1 , or 5 X 10 6 M “1 sec “1 or 10 7 M “1 sec “1 .
  • An antibody is said to competitively inhibit binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope.
  • Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays.
  • An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • the term "affinity” refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28.
  • the term “avidity” refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g. , Harlow at pages 29-34.
  • Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.
  • ⁇ 6 ⁇ 4 integrin antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their cross- reactivity.
  • cross-reactivity refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances.
  • an antibody is cross reactive if it binds to an epitope other than the one that induced its formation.
  • the cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.
  • certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
  • epitopes e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
  • An antibody may be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope.
  • An antibody may be deemed "highly specific" for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.
  • binding affinities include those with a dissociation constant or Kd less than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 “3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 "8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 “10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 "12 M, 10 “12 M, 5 x 10 "13 M, 10 "13 M, 5 x 10 "14 M, 10 “14 M, 5 x 10 "15 M, or 10 "15 M
  • ⁇ 6 ⁇ 4 integrin antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may be "multispecif ⁇ c," e.g., bispecific, trispecific or of greater multispecificity, meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins) at the same time.
  • an ⁇ 6 ⁇ 4 integrin antibody is "monospecific” or “multispecif ⁇ c,” e.g., "bispecific,” refers to the number of different epitopes with which a binding polypeptide reacts.
  • Multispecific antibodies may be specific for different epitopes of a target polypeptide described herein or may be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
  • valency refers to the number of potential binding domains, e.g., antigen binding domains, present in an ⁇ 6 ⁇ 4 integrin antibody, binding polypeptide or antibody. Each binding domain specifically binds one epitope.
  • each binding domain may specifically bind the same epitope, for an antibody with two binding domains, termed “bivalent monospecific,” or to different epitopes, for an antibody with two binding domains, termed “bivalent bispecific.”
  • An antibody may also be bispecific and bivalent for each specificity (termed “bispecific tetravalent antibodies").
  • tetravalent minibodies or domain deleted antibodies can be made.
  • Bispecific bivalent antibodies, and methods of making them are described, for instance in U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated by reference herein.
  • Bispecific tetravalent antibodies, and methods of making them are described, for instance, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated by reference herein. See generally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J.
  • VH domain includes the amino terminal variable domain of an immunoglobulin heavy chain
  • CHl domain includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain.
  • the CHl domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
  • CH2 domain includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat EA et al. op. cit.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
  • Hinge region includes the portion of a heavy chain molecule that joins the CHl domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al, J. Immunol. 757:4083 (1998)).
  • disulfide bond includes the covalent bond formed between two sulfur atoms.
  • the amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
  • the CHl and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
  • chimeric antibody will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant invention) is obtained from a second species.
  • the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.
  • the term “engineered antibody” refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, by partial framework region replacement and sequence changing.
  • the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species.
  • An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody.” It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations set forth in, e.g., U. S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.
  • polypeptides [0222] As used herein the term "properly folded polypeptide” includes polypeptides
  • a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.
  • engineered includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
  • an "in-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs.
  • ORFs polynucleotide open reading frames
  • a recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence.
  • polynucleotides encoding the CDRs of an immunoglobulin variable region may be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.
  • a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • the term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide.
  • the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • shRNA small hairpin RNA
  • siRNA small interfering RNA
  • a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
  • additive effect is one in which the effect of two molecules acting simultaneously is the simple sum of the effects that they would have if acting alone.
  • the term “synergistic effect” is one in which the effect of two molecules is greater than the effect of each molecule individually, or the sum of the individual effects.
  • the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
  • phrases such as "a subject that would benefit from administration of a binding molecule” and "an animal in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of a binding molecule used, e.g., for detection of an antigen recognized by a binding molecule (e.g., for a diagnostic procedure) and/or from treatment, i.e., palliation or prevention of a disease such as cancer, with a binding molecule which specifically binds a given target protein.
  • the binding molecule can be used in unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an isotope.
  • hyperproliferative disease or disorder all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues.
  • Hyperproliferative diseases or disorders include, but are not limited to, precancerous lesions, abnormal cell growths, benign tumors, malignant tumors, and "cancer.”
  • the hyperproliferative disease or disorder e.g., the precancerous lesion, abnormal cell growth, benign tumor, malignant tumor, or "cancer” comprises cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • hyperproliferative diseases, disorders, and/or conditions include, but are not limited to neoplasms, whether benign or malignant, located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital tract.
  • neoplasms in certain embodiments, express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • the diseases involve cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • tumor or tumor tissue refer to an abnormal mass of tissue that results from excessive cell division, in certain cases tissue comprising cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • a tumor or tumor tissue comprises "tumor cells” which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumors, tumor tissue and tumor cells may be benign or malignant.
  • a tumor or tumor tissue may also comprise "tumor-associated non- tumor cells", e.g., vascular cells which form blood vessels to supply the tumor or tumor tissue. Non-tumor cells may be induced to replicate and develop by tumor cells, for example, the induction of angiogenesis in a tumor or tumor tissue.
  • malignancy refers to a non-benign tumor or a cancer.
  • cancer connotes a type of hyperproliferative disease which includes a malignancy characterized by deregulated or uncontrolled cell growth.
  • examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers are noted below and include: squamous cell cancer (e.g.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer
  • cancer includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).
  • primary malignant cells or tumors e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor
  • secondary malignant cells or tumors e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor.
  • Cancers conducive to treatment methods of the present invention involves cells which express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin.
  • cancers or malignancies include, but are not limited to: Acute
  • the method of the present invention may be used to treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above.
  • Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where nonneoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976).
  • Such conditions in which cells begin to express, over-express, or abnormally express ⁇ 6 ⁇ 4 integrin are particularly treatable by the methods of the present invention.
  • Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • Hyperplastic disorders which can be treated by the method of the invention include, but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocyte hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperp
  • Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell.
  • Metaplastic disorders which can be treated by the method of the invention include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation.
  • Dysplastic disorders which can be treated by the method of the invention include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysiali
  • Additional pre-neoplastic disorders which can be treated by the method of the invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
  • benign dysproliferative disorders e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia
  • leukoplakia keratoses
  • Bowen's disease keratoses
  • Farmer's Skin Farmer's Skin
  • solar cheilitis solar keratosis
  • the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
  • Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythro leukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, lipos
  • Naturally occurring alpha6/beta4 integrin ( ⁇ 6 ⁇ 4 integrin) is a heterodimer single- pass type I membrane protein composed of one alpha and one beta subunit that are non- covalently linked together.
  • Full-length ⁇ 6 consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain.
  • An integrin ⁇ 6 heavy chain and an integrin ⁇ 6 light chain are also present in the protein.
  • ⁇ 6 is also known in the art by the names ITGA6, VLA-6 and CD49f antigen. Six isoforms of human ⁇ 6 have been reported, however, two of the isoforms are dominant.
  • VYVYMNQQGR 361 WNNVKPiRLN GTKDSMFGIA VKNIGDINQD GYPDIAVGAP YDDLGKVF ⁇ Y
  • VYVYMNQQGR 361 WNNVKPiRLN GTKDSMFGIA VKNIGDINQD GYPDIAVGAP YDDLGKVF ⁇ Y
  • HGSANGINTK 421 PTQVLKGISP YFGYSIAGNM DLDRNSYPDV AVGSLSDSVT IFRSRPVINI
  • FCDGRLRGHEKFGSCQQGv AATFTKDFHY ⁇ VFGAPGTYNWKGLLFLTSVSYTDPDQFV YKTRPPREQPDTFPDVMMNSYLGFSLDSGKG ⁇ VSKDEITFVSGAPRANHSGAWLLKRD MKSAHLLPEH ⁇ FDGEGLASSFGYDVAWDLNKDGWQD ⁇ VIGAPQYFDRDGEVGGAVY VYMNQQGRWNNVKPIRLNGTKDSMFGIA VKNIGDINQDGYPDIAVGAP YDDLGKVFIY HGSANGiNTKPTQVLKGisPYFGYSiAGNMDLDRNSYPDv AVGSLSDSVTIFRSRPV ⁇ NIQ KTiTVTPNRiDLRQKTACGAPSGiCLQ VKSCFEYT ANP AGYNPSIS ⁇ VGTLEAEKERRKSG
  • mouse ⁇ 6 polypeptide has been reported with the following sequences in
  • FASTA Q61739
  • ⁇ 6 polypeptide domain designations used herein are defined as follows:
  • Full-length ⁇ 4 consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain.
  • a von Willebrand factor type A (VWFA) domain exists in the extracellular domain and four fibronectin type-Ill domains and one Calx-beta domain exist in the cytoplasmic domain.
  • ⁇ 4 is also known in the art by the names ITGB4, GP150 and CD104 antigen. Five isoforms of human ⁇ 4 exist, however 3 isoforms are dominant.
  • isoform 4 A or isoform 3 sequence and has the accession number AAC51632 in Genbank.
  • APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
  • SDHLDTPMLR 781 SGNLKGRDVV RWKVTNNMQR PGFATHAASI NPTELVPYGL SLRLARLCTE
  • PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
  • APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
  • SDHLDTPMLR 781 SGNLKGRDW RWKVTNNMQR PGF ATHAASI NPTELVPYGL SLRLARLCTE
  • PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
  • HLVNGRMDFA 1381 FPGSTNSLHR MTTTSAAAYG THLSPHVPHR VLSTSSTLTR DYNSLTRSEH
  • RAGLFQHPLQ 1741 SEYSSISTTH TSATEPFLVD GPTLGAQHLE AGGSLTRHVT QEFVSRTLTT
  • APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
  • SDHLDTPMLR 781 SGNLKGRDVV RWKVTNNMQR PGFATHAASI NPTELVPYGL SLRLARLCTE
  • PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
  • FASTA sp_vs
  • mouse ⁇ 4 polypeptide (isoform 1) has been reported with the following and has the accession number NP_001005608 in Genbank.
  • Full-Length mouse ⁇ 4 (isoform 1) (SEQ ID NO: 15):
  • VHLRAVEDID 421 GTHVCQLAKE DQGGNIHLKP SFSDGLRMDA SVICDVCPCE LQKEVRSARC
  • ICNGRGYCEC 601 GRCHCNQQSL YTDTTCEINY SAIRLGLCED LRSCVQCQAW GTGEKKGRAC
  • VNGRMDFAYP 1381 GSANSLHRMT AANVAYGTHL SPHLSHRVLS TSSTLTRDYH SLTRTEHSHS
  • ⁇ 4 polypeptide domain designations used herein are defined as follows:
  • the present invention is also directed to ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof which bind specifically, preferentially, or competitively to non-human ⁇ 6 ⁇ 4 integrin proteins, e.g., ⁇ 6 ⁇ 4 integrin from rodents or non-human primates.
  • ⁇ 6 ⁇ 4 integrin is expressed in a large number of tumor cells, including, but not limited to certain of the following: bladder tumors (Oncol. Rep. 7:13-16 (2000)); breast tumors (Breast Cancer Res. 9:203 (2007) and Exp. Cell Res. 312:3822-3834 (2006)); colon tumors, (Exp. Cell Res. 266:1-10 (2001)); gastric tumors (Am. J. Pathol. 149:781- 793 (1996)); lung tumors (J. Cell Biochem. 55:409-418 (1994)); ovarian tumors (Hum. Pathol. 34:803-808 (2003)); pancreatic tumors,(J. Cell Sci.
  • the present invention is directed to ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof.
  • the present invention includes at least the antigen-binding domains of certain monoclonal antibodies, and fragments, variants, and derivatives thereof shown in Tables 4 and 5 .
  • Table 4 lists human anti-human ⁇ 6 ⁇ 4 integrin Fab regions identified from a phage display library.
  • Table 5 lists murine anti-human ⁇ 6 ⁇ 4 integrin monoclonal antibodies identified by hybridoma technology.
  • the term "antigen binding domain” includes a site that specifically binds an epitope on an antigen (e.g., an epitope of ⁇ 6 ⁇ 4 integrin).
  • the antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
  • the present invention is more specifically directed to an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the ⁇ 6 ⁇ 4 integrin antibody specifically binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61- C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61- C03, M65-A11, M66-H09 and M67-F05
  • a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B
  • the invention is further drawn to an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the ⁇ 6 ⁇ 4 integrin antibody competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05
  • a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to
  • the invention is also drawn to an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the ⁇ 6 ⁇ 4 integrin antibody comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin.
  • a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05
  • a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to ⁇ 6 ⁇ 4 integrin
  • determining which amino acids, or epitope, of ⁇ 6 ⁇ 4 integrin to which the antibody or antigen binding fragment binds can be determined by epitope mapping protocols as described herein as well as methods known in the art (e.g. double antibody-sandwich ELISA as described in "Chapter 11 - Immunology,” Current Protocols in Molecular Biology, Ed. Ausubel et ai, v.2, John Wiley & Sons, Inc. (1996)). Additional epitope mapping protocols may be found in Morris, G.
  • Epitope Mapping Protocols New Jersey: Humana Press (1996), which are both incorporated herein by reference in their entireties. Epitope mapping can also be performed by commercially available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wisconsin)).
  • antibodies produced which bind to any portion of ⁇ 6 ⁇ 4 integrin can then be screened for their ability to act as an antagonist of ⁇ 6 ⁇ 4 integrin for example, to inhibit binding of laminin to ⁇ 6 ⁇ 4 integrin, to inhibit activation of the PI3-K/Akt pathway including but not limited to Rac, mTOR and Akt, to inhibit activation of the Ras/MAPK signaling pathway, to inhibit the interaction of ⁇ 6 ⁇ 4 with growth factor receptors including but not limited to erbB2, Met, and Ron, to inhibit integrin clustering in lipid rafts, to inhibit the interaction with cytoskeletal components, to induce apoptosis, to inhibit receptor tyrosine kinase (RTK) activity, to promote caspase cleavage, to induce integrin internalization or to inhibit tumor cell growth, proliferation, motility or metastasis.
  • Antibodies can be screened for these and other properties according to methods described in detail in the Examples. Other functions of antibodies of the present invention can be
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids of SEQ ID NOs: 1-15, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ID NO:1-15.
  • amino acids of a given epitope of SEQ ID NOs:l-15 as described may be, but need not be contiguous or linear, hi certain embodiments, at least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of ⁇ 6 or ⁇ 4 integrin as expressed on the surface of a cell or as a soluble fragment, e.g., fused to an IgG Fc region.
  • At least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ID NOs: 1-15, where non-contiguous amino acids form an epitope through protein folding.
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of a portion of ⁇ 6 and a portion of ⁇ 4.
  • At least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of an ⁇ 6 sequence selected from the group consisting of SEQ ID NOs: 1-8 and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of a ⁇ 4 sequence selected from the group consisting of SEQ ID NOs: 1
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or noncontiguous amino acids of SEQ ID NOs:l-15 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety may be included such that the ⁇ 6 ⁇ 4 integrin antibody binds with higher affinity to modified target protein than it does to an unmodified version of the protein. Alternatively, the oc6 ⁇ 4 integrin antibody does not bind the unmodified version of the target protein at all.
  • the present invention is directed to an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically binds to a ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or an ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) which is less than the K D for a given reference monoclonal antibody.
  • K D dissociation constant
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds specifically to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above; or binds to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above with an affinity characterized by a dissociation constant K D of less than about 5 x 10 "2 M, about 10
  • the antibody or fragment thereof preferentially binds to a human ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, relative to a murine ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, hi another particular aspect, the antibody or fragment thereof preterentially binds to one or more ⁇ 6 ⁇ 4 integrin polypeptides or fragments thereof, e.g., one or more mammalian ⁇ 6 ⁇ 4 integrin polypeptides.
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "2 sec “1 , 10 "2 sec “1 , 5 X 10 "3 sec “1 or 10 '3 sec “1 .
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 "5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X 10 "7 sec “1 or 10 "7 sec “1 .
  • an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 10 3 M "1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
  • an antibody, or antigen- binding fragment, variant, or derivative thereof of the invention binds ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 10 5 M "1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec “1 , or 5 X 106 M “1 sec “1 or 10 7 M “1 sec “1 .
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof as described herein is an antagonist of ⁇ 6 ⁇ 4 integrin activity.
  • binding of an antagonist ⁇ 6 ⁇ 4 integrin antibody to ⁇ 6 ⁇ 4 integrin as expressed on a tumor cell inhibits binding of laminin to ⁇ 6 ⁇ 4 integrin, inhibits activation of the PB-K/ Akt pathway, or inhibits tumor cell proliferation, motility or metastasis.
  • an ⁇ 6 ⁇ 4 integrin antibody e.g., an antibody of the invention is a bispecific ⁇ 6 ⁇ 4 integrin antibody, e.g., a bispecific antibody, minibody, domain deleted antiDody, or fusion protein having binding specificity for more than one epitope, e.g., more than one antigen or more than one epitope on the same antigen.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody has at least one binding domain specific for at least one epitope on a target polypeptide disclosed herein, e.g., ⁇ 6 ⁇ 4 integrin.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody has at least one binding domain specific for an epitope on a target polypeptide and at least one target binding domain specific for a drug or toxin.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody has at least one binding domain specific for an epitope on a target polypeptide disclosed herein, and at least one binding domain specific for a prodrug.
  • a bispecific ⁇ 6 ⁇ 4 integrin antibody may be a tetravalent antibody that has two target binding domains specific for an epitope of a target polypeptide disclosed herein and two target binding domains specific for a second target.
  • a tetravalent bispecific ⁇ 6 ⁇ 4 integrin antibody may be bivalent for each specificity.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can comprise a constant region which mediates one or more effector functions.
  • binding of the Cl component of complement to an antibody constant region may activate the complement system.
  • Activation of complement is important in the opsonisation and lysis of cell pathogens.
  • the activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity.
  • antibodies bind to receptors on various cells via the Fc region, with a Fc receptor binding site on the antibody Fc region binding to a Fc receptor (FcR) on a cell.
  • FcR Fc receptor
  • Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • certain embodiments of the invention include an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
  • certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain .domains.
  • one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted.
  • certain antibodies for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG4 heavy chain constant region, which is altered to eliminate glycosylation, referred to elsewhere herein as "agly" antibodies. While not being bound by theory, it is believed that "agly" antibodies may have an improved safety and stability profile in vivo. Methods of producing aglycosylated antibodies, having desired effector function are found for example in WO 2005018572, which is incorporated by reference in its entirety.
  • the Fc portion may be mutated to decrease effector function using techniques known in the art.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization.
  • constant region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin.
  • modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility.
  • the resulting physiological profile, bioavailability and other biochemical effects of the modifications such as tumor localization, biodistribution and serum half- life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
  • Modified forms of ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein.
  • both the variable and constant regions of ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof are fully human.
  • Fully human antibodies can be made using techniques that are known in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in US patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human antibodies can likewise be produced by various display technologies, e.g., phage display or other viral display systems, as described in more detail elsewhere herein.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made or manufactured using techniques that are known in the art.
  • antibody molecules or fragments thereof are "recombinantly produced," i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention will not elicit a deleterious immune response in the animal to be treated, e.g., in a human.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made.
  • Non-human antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen- binding properties of the parent antibody, but which is less immunogenic in humans.
  • This may be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues.
  • CDRs complementarity determining regions
  • De-immunization can also be used to decrease the immunogenicity of an antibody.
  • the term "de-immunization” includes alteration of an antibody to modify T cell epitopes ⁇ see, e.g., WO9852976A1, WO0034317A2).
  • VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence.
  • CDRs complementarity-determining regions
  • VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function.
  • binding polypeptides e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function.
  • Typically, between 12 and 24 variant antibodies are generated and tested.
  • Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art.
  • an ⁇ 6 ⁇ 4 integrin antibody e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, chickens, hamsters, goats, donkeys, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al, in: Monoclonal Antibodies and T-CeIl Hybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporated by reference in their entireties).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
  • Monoclonal antibodies can be prepared using ⁇ 6 ⁇ 4 integrin knockout mice to increase the regions of epitope recognition.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology as described elsewhere herein.
  • antibodies are raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen ⁇ e.g., purified ⁇ 6 ⁇ 4 integrin or cells or cellular extracts comprising ⁇ 6 ⁇ 4 integrin) and an adjuvant.
  • This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes.
  • the resulting antibodies may be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs).
  • the lymphocytes are obtained from the spleen.
  • lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g., a myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • an immortal tumor cell line e.g., a myeloma cell line
  • hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell.
  • the resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies, which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal.”
  • Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established.
  • culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen.
  • the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab') 2 fragments may be produced recombinantly or by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments). F(ab') 2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain. [0303] Those skilled in the art will also appreciate that DNA encoding antibodies or antibody fragments (e.g., antigen binding sites) may also be derived from antibody libraries, such as phage display libraries.
  • such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M 13 binding domains expressed from phage with Fab, Fv OE DAB (individual Fv region from light or heavy chains) or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al, Nat. Biotechnol. 75:1287 (2000); Wilson et al, Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al, J. Immunol. Methods 248:31 (2001)).
  • cell surface libraries can be screened for antibodies (Boder et al, Proc. Natl. Acad. Sci. USA 97: 10701 (2000); Daugherty et al, J. Immunol Methods 243:211 (2000)).
  • Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
  • phage display methods functional antibody domains are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL regions are amplified or otherwise isolated from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries.
  • the DNA encoding the VH and VL regions are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).
  • the vector is electroporated in E. coli and the E.
  • Phage used in these methods are typically filamentous phage including fd and M 13 and the VH or VL regions are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to an antigen of interest i.e., an ⁇ 6 ⁇ 4 integrin polypeptide or a fragment thereof
  • an antigen of interest i.e., an ⁇ 6 ⁇ 4 integrin polypeptide or a fragment thereof
  • can be selected or identified with antigen e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • phage display methods that can be used to make the antibodies include those disclosed in Brinkman et al, J. Immunol. Methods 752:41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol 24:952-958 (1994); Persic et al, Gene 187:9-18 (1997); Burton et al, Advances in Immunology 57:191-280 (1994); PCT Application No.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, J. Immunol. Methods /25:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR- grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5) :489-498 (1991); Studnicka et al, Protein Engineering 7 ⁇ :805-814 (1994); Roguska. et al, PNAS 97:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
  • Human antibodies are particularly desirable for therapeutic treatment of human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. Ln particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a desired target polypeptide.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.” hi this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al, Bio/Technology 72:899-903 (1988). See also, U.S. Patent No. 5,565,332.) [0312] Further, antibodies to target polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" target polypeptides using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate antiidiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
  • antiidiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a desired target polypeptide and/or to bind its ligands/receptors, and thereby block its biological activity.
  • DNA encoding desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the isolated and subcloned hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as, but not limited to, E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins.
  • the isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al, U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least one heavy or light chain CDR of an antibody molecule.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least two CDRs from one or more antibody molecules.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least three CDRs from one or more antibody molecules.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least four CDRs from one or more antibody molecules.
  • an ⁇ 6 ⁇ 4 integrin antibody of the invention comprises at least five CDRs from one or more antibody molecules.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. MoI. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, e.g., ⁇ 6 ⁇ 4 integrin.
  • a desired polypeptide e.g., ⁇ 6 ⁇ 4 integrin.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain antibody. Techniques for the assembly of functional Fv fragments in E coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).
  • Yet other embodiments of the present invention comprise the generation of human or substantially human antibodies in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference).
  • transgenic animals e.g., mice
  • the homozygous deletion of the antibody heavy- chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge.
  • Another preferred means of generating human antibodies using SCDD mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies may also be isolated and manipulated as described herein.
  • lymphocytes can be selected by micromanipulation and the variable genes isolated.
  • peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated.
  • Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay.
  • Ig-producing B cells can be micromanipulated into a tube and the VH and VL genes can be amplified using, e.g. , RT-PCR.
  • the VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g. , eukaryotic or prokaryotic cells) for expression.
  • antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
  • Antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques as described herein.
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises a synthetic constant region wherein one or more domains are partially or entirely deleted ("domain-deleted antibodies").
  • compatible modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed ( ⁇ CH2 constructs).
  • ⁇ CH2 constructs domain deleted constructs or variants wherein the entire CH2 domain has been removed
  • a short connecting peptide may be substituted for the deleted domain to provide flexibility and freedom of movement for the variable region.
  • Domain deleted constructs can be derived using a vector encoding an IgGi human constant domain (see, e.g., WO 02/060955 A2 and WO02/096948A2). This vector is engineered to delete the CH2 domain and provide a synthetic vector expressing a domain deleted IgGj constant region.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are minibodies.
  • Minibodies can be made using methods described in the art (see, e.g., US patent 5,837,821 or WO 94/09817A1).
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits association between the monomelic subunits.
  • the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization.
  • Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • the constant regions of the disclosed antibodies may be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody.
  • Yet other embodiments comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it may be desirable to insert or replicate specific sequences derived from selected constant region domains.
  • the present invention also provides antibodies that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e.g., the VH regions and/or VL regions) described herein, which antibodies or fragments thereof immunospecifically bind to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment or variant thereof.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an ⁇ 6 ⁇ 4 integrin antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions.
  • the variants encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH region, VH-CDRl, VH-CDR2, VH-CDR3, VL region, VL-CDRl, VL-CDR2, or VL-CDR3.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge.
  • Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains ( e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind an ⁇ 6 ⁇ 4 integrin polypeptide).
  • mutations can be introduced only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations may be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen, indeed some such mutations do not alter the amino acid sequence whatsoever. These types of mutations may be useful to optimize codon usage, or improve a hybridoma's antibody production.
  • Codon-optimized coding regions encoding ⁇ 6 ⁇ 4 integrin antibodies of the present invention are disclosed elsewhere herein.
  • non-neutral missense mutations may alter an antibody's ability to bind antigen.
  • the location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement.
  • One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e.g., improvements in antigen binding activity or change in antibody specificity).
  • the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecif ⁇ cally bind at least one epitope of an ⁇ 6 ⁇ 4 integrin polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.
  • the present invention also provides for nucleic acid molecules encoding ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2, or VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VH immunoglobulin heavy chain variable region
  • VH-CDRl, VH-CDR2, and VH- CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a heavy chain variable region of the invention has VH-CDRl, VH-CDR2, or VH-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 6.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2, or VL-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VL immunoglobulin light chain variable region
  • VL-CDRl, VL-CDR2, and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a light chain variable region of the invention has VL-CDRl, VL-CDR2, or VL-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 6.
  • N nucleotide sequence
  • P polypeptide sequence
  • sequence identity between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide.
  • sequence identity is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide.
  • whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
  • BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
  • an antibody or antigen-binding fragment comprising the
  • VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the nucleotide sequence encoding the VH polypeptide is altered without altering the amino acid sequence encoded thereby.
  • the sequence may be altered for improved codon usage in a given species, to remove splice sites, or the remove restriction enzyme sites. Sequence optimizations such as these are described in the examples and are well known and routinely carried out by those of ordinary skill in the art.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2, and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2, and VH-CDR3 groups shown in Table 6.
  • VH immunoglobulin heavy chain variable region
  • an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the VH polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ED NOs: 78, 79, and 80; and SEQ ID NOs: 88, 89 and 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 “4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 '11 M, 5 x 10- 12 M, 10- 12 M,
  • an antibody or antigen-binding fragment comprising the
  • VL encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2, and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL- CDR2, and VL-CDR3 groups shown in Table 6.
  • VL immunoglobulin light chain variable region
  • an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2, and VL-CDR3 regions are encoded by nucleotide sequences which are identical to the nucleotide sequences which encode the VL-CDRl, VL-CDR2, and VL-CDR3 groups shown in Table 6.
  • an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs:83, 84, and 85; and SEQ ID NOs:93, 94 and 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 3 M, 5 x 10 "4 M, lO "4 M, 5 x 10 "5 M, W 5 M, 5 x 10 "6 M, 10 "6 M, 5 x 10 ⁇ 7 M, 10 "7 M, 5 x Kr 8 M, 1(T 8 M, 5 x ICT 9 M, 10 '9 M, 5 x 10 "10 M, lO '10 M, 5 x lO '11 M, 1(T 11 M, 5
  • K D dissociation constant
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VH at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a VH-encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76 and 86.
  • an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH of the invention, where the amino acid sequence of the VH is selected from the group consisting of SEQ ED NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • the present invention further includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH of the invention, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76 and 86.
  • an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 1(T 3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 "6 M, 10 “6 M, 5 x 1(T 7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 “9 M, 1(T 9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 1(T 11 M, 5 x 10 "12 M
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a VL- encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21, 31, 41, 51, 61, 71, 81 and 91.
  • an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • the present invention further includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL of the invention, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 21, 31, 41, 51, 61, 71, 81 and 91.
  • an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybrid
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 "3 M, 10 '3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x l( ⁇ 6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 “8 M, 5 x 10 "9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 " 12 M,
  • any of the polynucleotides described above may further include additional nucleic acids, encoding, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
  • the present invention includes compositions comprising the polynucleotides comprising one or more of the polynucleotides described above.
  • the invention includes compositions comprising a first polynucleotide and second polynucleotide wherein said first polynucleotide encodes a VH polypeptide as described herein and wherein said second polynucleotide encodes a VL polypeptide as described herein.
  • composition which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide, wherein the VH polynucleotide and the VL polynucleotide encode polypeptides, respectively at least 80%, 85%, 90% 95% or 100% identical to reference VH and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, and 87 and 92.
  • compositions which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide at least 80%, 85%, 90% 95% or 100% identical, respectively, to reference VL and VL nucleic acid sequences selected from the group consisting of SEQ ID NOs: 16 and 21, 26 and 31, 36 and 41, 46 and 51, 56 and 61, 66 and 71, 76 and 81, and 86 and 91.
  • an antibody or antigen-binding fragment comprising the VH and VL encoded by the polynucleotides in such compositions specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention also includes fragments of the polynucleotides of the invention, as described elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated by the invention.
  • the polynucleotides may be produced or manufactured by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 77:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen- binding fragment, variant, or derivative thereof may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody or other ⁇ 6 ⁇ 4 integrin antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g.
  • a cDNA clone from a cDNA library that encodes the antibody or other ⁇ 6 ⁇ 4 integrin antibody may then be cloned into replicable cloning vectors using any method well known in the art.
  • nucleotide sequence and corresponding amino acid sequence of the ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • a polynucleotide encoding ⁇ 6 ⁇ 4 integrin antibody, or antigen- binding fragment, variant, or derivative thereof can be composed of single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide encoding an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • “Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin ⁇ e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
  • the present invention is further directed to isolated polypeptides which make up ⁇ 6 ⁇ 4 integrin antibodies, and polynucleotides encoding such polypeptides.
  • ⁇ 6 ⁇ 4 integrin antibodies of the present invention comprise polypeptides, e.g., amino acid sequences encoding ⁇ 6 ⁇ 4 integrin-specific antigen binding regions derived from immunoglobulin molecules.
  • a polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide having a certain amino acid sequence.
  • the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), where at least one of VH-CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2 or VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VH immunoglobulin heavy chain variable region
  • VH-CDRl, VH-CDR2 and VH-CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2 and VH-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a heavy chain variable region of the invention has VH-CDRl, VH-CDR2 and VH-CDR3 polypeptide sequences related to the groups shown in Table 6, supra. While Table 6 shows VH-CDRs defined by the Kabat system, other CDR definitions, e.g., VH-CDRs defined by the Chothia system, are also included in the present invention.
  • an antibody or antigen- binding fragment comprising the VH specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and VH-CDR3 groups shown in Table 6.
  • VH immunoglobulin heavy chain variable region
  • an antibody or antigen-binding fragment comprising the VH specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and VH-CDR3 groups shown in Table 6, except for one, two, three, four, five, or six amino acid substitutions in any one VH-CDR.
  • VH immunoglobulin heavy chain variable region
  • CDRs e.g., VH-CDR-3
  • additional substitutions may be made in the CDR, as long as the a VH comprising the VH-CDR specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid substitutions are conservative.
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90, except for one, two, three, four, five or six amino acid substitutions in at least one of said VH-CDRs.
  • VH immunoglobulin heavy chain variable region
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90.
  • VH immunoglobulin heavy chain variable region
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57 67, 77 and 87.
  • an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VH polypeptides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of one or more of the VH polypeptides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 '2 M, 10 "2 M, 5 x 10 "3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 '5 M, 10 "5 M, 5 x 10 '6 M, 10 "6 M, 5 x 10 "7 M, 10 "7 M, 5 x 10 "8 M, 10 " 8 M, 5 x 10 '9 M, 10 "9 M, 5 x 10 "10 M, 10 "10 M, 5 x 10 "11 M, 10 "11 M, 5 x 10 "12 M, 10 "12 M, 5
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2 or VL-CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • VL immunoglobulin light chain variable region
  • VL-CDRl, VL-CDR2 and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2 and VL- CDR3 amino acid sequences from monoclonal ⁇ 6 ⁇ 4 integrin antibodies disclosed herein.
  • a light chain variable region of the invention has VL- CDRl, VL-CDR2 and VL-CDR3 polypeptide sequences related to the polypeptides shown in Table 6, supra.
  • VL-CDRs defined by the Kabat system
  • other CDR definitions e.g., VL-CDRs defined by the Chothia system
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL-CDR2 and VL-CDR3 groups shown in Table 6.
  • VL immunoglobulin light chain variable region
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL-CDR2 and VL-CDR3 groups shown in Table 6, except for one, two, three, four, five, or six amino acid substitutions in any one VL-CDR.
  • additional substitutions may be made in the VL-CDR, as long as the a VL comprising the VL-CDR specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the amino acid substitutions are conservative, hi certain embodiments, an antibody or antigen-binding fragment comprising the VL specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ED NOs: 83, 84 and 85; and SEQ ID NOs: 93, 94 and 95, except for one, two, three, four, five or six amino acid substitutions in at least one of said VL-CDRs.
  • VL immunoglobulin heavy chain variable region
  • the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ED NOs: 33, 34, and 35; SEQ ED NOs: 43, 44, and 45; SEQ ED NOs: 53, 54, and 55; SEQ ED NOs: 63, 64, and 65; SEQ ED NOs: 73, 74, and 75; SEQ ED NOs: 83, 84 and 85; and SEQ ED NOs: 93, 94 and 95.
  • VL immunoglobulin heavy chain variable region
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence selected from the group consisting of SEQ ED NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide selected from the group consisting of SEQ ED NOs: 22, 32, 42, 52, 62, 72, 82 and 92.
  • an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, one or more of the VL polypeptides described above specifically or preferentially binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61- C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to ⁇ 6 ⁇ 4 integrin.
  • a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61- C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P
  • an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VL polypeptides described above specifically or preferentially binds to an ⁇ 6 ⁇ 4 integrin polypeptide or fragment thereof, or a ⁇ 6 ⁇ 4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (K D ) no greater than 5 x 10 "2 M, 10 "2 M, 5 x 10 ⁇ 3 M, 10 "3 M, 5 x 10 "4 M, 10 "4 M, 5 x 10 "5 M, 10 "5 M, 5 x 10 6 M, 10 "6 M, 5 x 10 "7 M, 10 '7 M, 5 x 10 '8 M, 10 "8 M, 5 x 10 "9 M, 10 '9 M, 5 x 10 '10 M, 10 "10 M, 5 x lO "11 M, 10 " ⁇ M, 5 x 10 " 12 M, 10 "12
  • an antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a VH polypeptide, and a VL polypeptide, where the VH polypeptide and the VL polypeptide, respectively are at least 80%, 85%, 90% 95% or 100% identical to reference VL and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, and 87 and 92.
  • an antibody or antigen-binding fragment comprising these VH and VL polypeptides specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin.
  • polypeptides described above may further include additional polypeptides, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
  • polypeptides of the invention include polypeptide fragments as described elsewhere.
  • polypeptides of the invention include fusion polypeptide, Fab fragments, and other derivatives, as described herein.
  • compositions comprising the polypeptides described above.
  • ⁇ 6 ⁇ 4 integrin antibody polypeptides as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived.
  • a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence.
  • nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at "non-essential" amino acid regions may be made.
  • a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions
  • a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions.
  • a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, fifteen or fewer, or twenty or fewer individual amino acid substitutions, insertions, or deletions.
  • a polypeptide or amino acid sequence derived from a designated protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.
  • Certain ⁇ 6 ⁇ 4 integrin antibody polypeptides of the present invention comprise, consist essentially of, or consist of an amino acid sequence derived from a human amino acid sequence.
  • certain ⁇ 6 ⁇ 4 integrin antibody polypeptides comprise one or more contiguous amino acids derived from another mammalian species.
  • an ⁇ 6 ⁇ 4 integrin antibody of the present invention may include a primate heavy chain portion, hinge portion, or antigen binding region, hi another example, one or more murine-derived amino acids may be present in a non-murine antibody polypeptide, e.g.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies, or antigen-binding fragments, variants, or analogs thereof are designed so as to not be immunogenic in the animal to which the antibody is administered.
  • an ⁇ 6 ⁇ 4 integrin antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below.
  • a single-chain fv antibody fragment of the invention may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).
  • An ⁇ 6 ⁇ 4 integrin antibody polypeptide of the invention may comprise, consist essentially of, or consist of a fusion protein.
  • Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature.
  • the amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
  • heterologous as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the rest of the entity to which it is being compared.
  • a “heterologous polypeptide” to be fused to an ⁇ 6 ⁇ 4 integrin antibody, or an antigen- binding fragment, variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
  • mutations may be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into ⁇ 6 ⁇ 4 integrin antibodies for use in the diagnostic and treatment methods disclosed herein and screened for their ability to bind to the desired antigen, e.g., ⁇ 6 ⁇ 4 integrin.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
  • ⁇ 6 ⁇ 4 integrin-specif ⁇ c ⁇ 6 ⁇ 4 integrin antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody binding ⁇ 6 ⁇ 4 integrin.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • ⁇ 6 ⁇ 4 integrin-specfic antibodies may be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • Modifications can occur anywhere in the ⁇ 6 ⁇ 4 integrin-specific antibody, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given ⁇ 6 ⁇ 4 integrin-specific antibody. Also, a given ⁇ 6 ⁇ 4 integrin-specific antibody may contain many types of modifications. ⁇ 6 ⁇ 4 integrin-specific antibodies may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching.
  • Cyclic, branched, and branched cyclic ⁇ 6 ⁇ 4 integrin-specific antibodies may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selen
  • the present invention also provides for fusion proteins comprising an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide.
  • a fusion protein of the invention comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the VH regions of an antibody of the invention or the amino acid sequence of any one or more of the VL regions of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence.
  • a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three of the VH-CDRs of an ⁇ 6 ⁇ 4 integrin-specif ⁇ c antibody, or fragments, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL-CDRs of an ⁇ 6 ⁇ 4 integrin-specific antibody, or fragments, variants, or derivatives thereof, and a heterologous polypeptide sequence.
  • the fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of an ⁇ 6 ⁇ 4 integrin-specific antibody of the present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to at least one epitope of ⁇ 6 ⁇ 4 integrin.
  • a fusion protein comprises a polypeptide having the amino acid sequence of at least one VH region of an ⁇ 6 ⁇ 4 integrin-specific antibody of the invention and the amino acid sequence of at least one VL region of an ⁇ 6 ⁇ 4 integrin-specific antibody of the invention or fragments, derivatives or variants thereof, and a heterologous polypeptide sequence.
  • a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of an ⁇ 6 ⁇ 4 integrin-specific antibody and the amino acid sequence of any one, two, three or more of the VL CDRs of an ⁇ 6 ⁇ 4 integrin-specific antibody, or fragments or variants thereof, and a heterologous polypeptide sequence.
  • VH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv or Fab fragment) of the invention.
  • Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.
  • Exemplary fusion proteins reported in the literature include fusions of the T cell receptor (Gascoigne et al, Proc. Natl. Acad. Sci. USA 54:2936-2940 (1987)); CD4 (Capon et al., Nature 537:525-531 (1989); Traunecker et al, Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be fused to heterologous polypeptides to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art.
  • PEG can be conjugated to the ⁇ 6 ⁇ 4 integrin antibodies of the invention to increase their half- life in vivo. Leong, S.R., et al, Cytokine /(5:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weir et al, Biochem. Soc. Transactions 30:512 (2002).
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • Other peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, Cell 37:161 (1984)) and the "flag" tag.
  • Fusion proteins can be prepared using methods that are well known in the art (see for example US Patent Nos. 5,116,964 and 5,225,538). The precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
  • ⁇ 6 ⁇ 4 integrin antibodies of the present invention may be used in non-conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the patient.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be labeled or conjugated either before or after purification, when purification is performed.
  • ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
  • conjugates may also be assembled using a variety of techniques depending on the selected agent to be conjugated.
  • conjugates with biotin are prepared e.g. by reacting a binding polypeptide with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester.
  • conjugates with a fluorescent marker may be prepared in the presence of a coupling agent, e.g. those listed herein, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate.
  • Conjugates of the ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention are prepared in an analogous manner.
  • the present invention further encompasses ⁇ 6 ⁇ 4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent.
  • the ⁇ 6 ⁇ 4 integrin antibodies can be used diagnostically to, for example, monitor the development or progression of a neurological disease as part of a clinical testing procedure to, e.g. , determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, e.g., U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 1, 131 1, 111 In or 99 Tc.
  • An ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged ⁇ 6 ⁇ 4 integrin antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • an ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol. 37:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the ⁇ 6 ⁇ 4 integrin antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay ⁇ see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated by reference herein).
  • the radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
  • An ⁇ 6 ⁇ 4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin- specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG.
  • cytotoxins such as radioisotopes, cytotoxic drugs, or toxins
  • therapeutic agents such as radioisotopes, cytotoxic drugs, or toxins
  • cytostatic agents such as radioisotopes, cytotoxic drugs, or toxins
  • biological toxins such as radioisotopes, cytotoxic drugs
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that decreases vascularization of tumors.
  • the disclosed compositions may comprise binding molecules, e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof coupled to drugs or prodrugs.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin.
  • adjunct treatment e.g., chemotherapy or external radiation
  • radioisotopes include: 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 1 86 Re and 188 Re.
  • the radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death.
  • the isotopes used to produce therapeutic conjugates typically produce high energy ⁇ - or ⁇ -particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof may be directly labeled (such as through iodination) or may be labeled indirectly through the use of a chelating agent.
  • the phrases "indirect labeling” and “indirect labeling approach” both mean that a chelating agent is covalently attached to a binding molecule and at least one radionuclide is associated with the chelating agent.
  • Such chelating agents are typically referred to as bifunctional chelating agents as they bind both the polypeptide and the radioisotope.
  • Particularly preferred chelating agents comprise l-isothiocycmatobenzyl-3- methyldiothelene triaminepentaacetic acid ("MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid (“CHX-DTPA”) derivatives.
  • Other chelating agents comprise P-DOTA and EDTA derivatives.
  • Particularly preferred radionuclides for indirect labeling include 111 In and 90 Y.
  • direct labeling and “direct labeling approach” both mean that a radionuclide is covalently attached directly to a polypeptide (typically via an amino acid residue). More specifically, these linking technologies include random labeling and site-directed labeling. In the latter case, the labeling is directed at specific sites on the polypeptide, such as the N-linked sugar residues present only on the Fc portion of the conjugates. Further, various direct labeling techniques and protocols are compatible with the instant invention.
  • Technetium-99 labeled polypeptides may be prepared by ligand exchange processes, by reducing pertechnate (TcO 4 " ) with stannous ion solution, chelating the reduced technetium onto a Sephadex column and applying the binding polypeptides to this column, or by batch labeling techniques, e.g. by incubating pertechnate, a reducing agent such as SnCl 2 , a buffer solution such as a sodium-potassium phthalate-solution, and the antibodies.
  • a reducing agent such as SnCl 2
  • a buffer solution such as a sodium-potassium phthalate-solution
  • preferred radionuclides for directly labeling antibodies are well known in the art and a particularly preferred radionuclide for direct labeling is 131 I covalently attached via tyrosine residues.
  • Binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecif ⁇ c fragments thereof for use in the diagnostic and treatment methods disclosed herein may be derived, for example, with radioactive sodium or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidizing agent, such as lactoperoxidase, glucose oxidase and glucose.
  • a chemical oxidizing agent such as sodium hypochlorite, chloramine T or the like
  • an enzymatic oxidizing agent such as lactoperoxidase, glucose oxidase and glucose.
  • Patents relating to chelators and chelator conjugates are known in the art.
  • U.S. Patent No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation.
  • U.S. Patent Nos. 5,099,069, 5,246,692, 5,286,850, 5,434,287 and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein by reference in their entireties.
  • compatible metal chelators are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), 1,4,8,11-tetraazatetradecane, 1,4,8,11- tetraazatetradecane- 1 ,4,8, 11 -tetraacetic acid, 1 -oxa-4,7, 12, 15-tetraazaheptadecane- 4,7,12,15-tetraacetic acid, or the like. Cyclohexyl-DTPA or CHX-DTPA is particularly preferred and is exemplified extensively below. Still other compatible chelators, including those yet to be discovered, may easily be discerned by a skilled artisan and are clearly within the scope of the present invention.
  • Compatible chelators including the specific bifunctional chelator used to facilitate chelation U.S. Patent Nos. 6,682,134, 6,399,061, and 5,843,439, incorporated herein by reference in their entireties, are preferably selected to provide high affinity for trivalent metals, exhibit increased tumor-to-non-tumor ratios and decreased bone uptake as well as greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites.
  • target sites i.e., B-cell lymphoma tumor sites.
  • other bifunctional chelators that may or may not possess all of these characteristics are known in the art and may also be beneficial in tumor therapy.
  • binding molecules may be conjugated to different radiolabels for diagnostic and therapeutic purposes.
  • U.S. Patent Nos. 6,682,134, 6,399,061, and 5,843,439 disclose radiolabeled therapeutic conjugates for diagnostic "imaging" of tumors before administration of therapeutic antibody.
  • “In2B8" conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to 1 11 In via a bifunctional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1 :1 mixture of l-isothiocyanatobenzyl-3-methyl-DTPA and 1-methyl- 3-isothiocyanatobenzyl-DTPA.
  • MX-DTPA diethylenetriaminepentaacetic acid
  • 111 In is particularly preferred as a diagnostic radionuclide because between about 1 to about 10 mCi can be safely administered without detectable toxicity; and the imaging data is generally predictive of subsequent 90 Y-labeled antibody distribution.
  • 131 I is a well known radionuclide used for targeted immunotherapy.
  • the clinical usefulness of 131 I can be limited by several factors including: eight-day physical half-life; dehalogenation of iodinated antibody both in the blood and at tumor sites; and emission characteristics (e.g., large gamma component) which can be suboptimal for localized dose deposition in tumor.
  • emission characteristics e.g., large gamma component
  • 90 Y provides several benefits for utilization in radioimmunotherapeutic on applications: the 64 hour half- life of Y is long enough to allow antibody accumulation by tumor and, unlike e.g., 131 I, 90 Y is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range in tissue of 100 to 1,000 cell diameters. Furthermore, the minimal amount of penetrating radiation allows for outpatient administration of 90 Y-labeled antibodies. Additionally, internalization of labeled antibody is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target molecule.
  • cytotoxic drugs particularly those which are used for cancer therapy.
  • a cytotoxin or cytotoxic agent means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy.
  • cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present invention.
  • Exemplary cytotoxins include, in general, cytostatic agents, alkylating agents, anti-metabolites, antiproliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like.
  • Exemplary cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine.
  • alkylating substances such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone
  • nitrosourea compounds such as carmustine, lomustine, or semustine.
  • Other preferred classes of cytotoxic agents include, for example, the maytansinoid family of
  • cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins.
  • Particularly useful members of those classes include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like.
  • cytotoxins that are compatible with the teachings herein include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Hormones and hormone antagonists such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g.
  • tamoxifen, androgens e.g. testosterone
  • aromatase inhibitors e.g. aminogluthetimide
  • One skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
  • cytotoxins comprise members or derivatives of the enediyne family of anti-tumor antibiotics, including calicheamicin, esperamicins or dynemicins. These toxins are extremely potent and act by cleaving nuclear DNA, leading to cell death. Unlike protein toxins which can be cleaved in vivo to give many inactive but immunogenic polypeptide fragments, toxins such as calicheamicin, esperamicins and other enediynes are small molecules which are essentially non-immunogenic. These non-peptide toxins are chemically-linked to the dimers or tetramers by techniques which have been previously used to label monoclonal antibodies and other molecules. These linking technologies include site-specific linkage via the N-linked sugar residues present only on the Fc portion of the constructs. Such site-directed linking methods have the advantage of reducing the possible effects of linkage on the binding properties of the constructs.
  • compatible cytotoxins for preparation of conjugates may comprise a prodrug.
  • prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form.
  • Prodrugs compatible with the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, ⁇ -lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to the more active cytotoxic free drug.
  • Further examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention comprise those chemotherapeutic agents described above.
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein can also be associated with or conjugated to a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme.
  • a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulin
  • binding molecules e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin- specific antibodies or immunospecific fragments thereof disclosed herein comprise cytokines such as lymphokines and interferons.
  • cytokines such as lymphokines and interferons.
  • Another class of compatible cytotoxins that may be used in association with or conjugated to the disclosed binding molecules, e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof, are radiosensitizing drugs that may be effectively directed to tumor or immunoreactive cells. Such drugs enhance the sensitivity to ionizing radiation, thereby increasing the efficacy of radiotherapy.
  • An antibody conjugate internalized by the tumor cell would deliver the radiosensitizer nearer the nucleus where radiosensitization would be maximal.
  • the unbound radiosensitizer linked binding molecules of the invention would be cleared quickly from the blood, localizing the remaining radiosensitization agent in the target tumor and providing minimal uptake in normal tissues.
  • adjunct radiotherapy would be administered in one of three ways: 1.) external beam radiation directed specifically to the tumor, 2.) radioactivity directly implanted in the tumor or 3.) systemic radioimmunotherapy with the same targeting antibody.
  • a potentially attractive variation of this approach would be the attachment of a therapeutic radioisotope to the radiosensitized immunoconjugate, thereby providing the convenience of administering to the patient a single drug.
  • a moiety that enhances the stability or efficacy of a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin -specific antibody or immunospecific fragment thereof can be conjugated.
  • a binding polypeptide e.g., an ⁇ 6 ⁇ 4 integrin -specific antibody or immunospecific fragment thereof
  • PEG can be conjugated to the binding molecules of the invention to increase their half- life in vivo. Leong, S.R., et al., Cytokine 75:106 (2001); Adv. in DrugDeliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).
  • the present invention further encompasses the use of binding molecules, e.g., binding polypeptides, e.g., ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments conjugated to a diagnostic or therapeutic agent.
  • the binding molecules can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the binding molecule, e.g., binding polypeptide, e.g., ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 1, 131 1, 111 In or 99 Tc.
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged binding molecule is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et ah, J. Clin. Pathol. 57:507-520 (1978); Butler, J. E., Meth. Enrymol. 75:482-523 (1981); Maggio, E.
  • EIA enzyme immunoassay
  • the enzyme which is bound to the binding molecule will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- 6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • the binding molecule e.g., binding polypeptide, e.g., ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin- specific antibody or immunospecific fragment thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • a binding molecule e.g., a binding polypeptide, e.g., an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp.
  • RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
  • cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods.
  • PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences.
  • PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
  • DNA typically plasmid DNA
  • DNA may be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques.
  • the DNA may be synthetic according to the present invention at any point during the isolation process or subsequent analysis.
  • the polynucleotides encoding the ⁇ 6 ⁇ 4 integrin antibodies are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of ⁇ 6 ⁇ 4 integrin antibody.
  • an antibody, or fragment, derivative or analog thereof e.g., a heavy or light chain of an antibody which binds to a target molecule described herein, e.g., ⁇ 6 ⁇ 4 integrin
  • an expression vector containing a polynucleotide that encodes the antibody Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides.
  • the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. ScL USA 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a host cell.
  • vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses.
  • vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
  • vector systems may be employed.
  • one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus.
  • Others involve the use of polycistronic systems with internal ribosome binding sites.
  • cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper.
  • the selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. [0436] In particularly preferred embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) synthetic as discussed above, hi one embodiment, this is effected using a proprietary expression vector of Biogen EDEC, Inc., referred to as NEOSPLA (disclosed in U.S. patent 6,159,730).
  • This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence.
  • This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification.
  • any expression vector which is capable of eliciting expression in eukaryotic cells may be used in the present invention.
  • Suitable vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, PSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI).
  • screening large numbers of transformed cells for those which express suitably high levels if immunoglobulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems. Vector systems are also taught in U.S. Pat. Nos.
  • the ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be expressed using polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety.
  • polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety.
  • multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct.
  • IRES internal ribosome entry site
  • the expression vector may be introduced into an appropriate host cell.
  • Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors" Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pp. 470-472 (1988).
  • plasmid introduction into the host is via electroporation.
  • the host cells harboring the expression construct are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis.
  • Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host cells refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene.
  • the terms “cell” and “cell culture” are used interchangeably to denote the source of antibody unless it is clearly specified otherwise.
  • recovery of polypeptide from the “cells” may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
  • host-expression vector systems may be utilized to express antibody molecules for use in the methods described herein.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichi ⁇ ) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
  • the host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein.
  • Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXBI l (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with S V40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells), RAJI (human
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which stably express the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22:817 1980) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sd. USA 77:357 (1980); O'Hare et al, Proc. Natl. Acad. Sd. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, MoI. Cell. Biol. 3:257 (1983)).
  • the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
  • customary chromatography methods for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
  • Genes encoding ⁇ 6 ⁇ 4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can also be expressed non-mammalian cells such as bacteria or insect or yeast or plant cells.
  • Bacteria which readily take up nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self- assemble into tetravalent antibodies (WO02/096948A2).
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione- agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available, e.g. , Pichia pastoris.
  • the plasmid YRp7 for example, (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al, Gene 7:141 (1979); Tschemper et al., Gene 10:157 (1980)) is commonly used.
  • This plasmid already contains the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
  • the presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into nonessential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography ⁇ e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • differential solubility e.g., differential solubility
  • One embodiment of the present invention provides methods for treating a hyperproliferative disease or disorder, e.g. , cancer, a malignancy, a tumor, or a metastasis thereof, in an animal suffering from such disease or predisposed to contract such disease, the method comprising, consisting essentially of, or consisting of administering to the animal an effective amount of an antibody or immunospecific fragment thereof, that binds to ⁇ 6 ⁇ 4 integrin or a variant of ⁇ 6 ⁇ 4 integrin.
  • Suitable antibodies include all antibodies and antigen-specific fragments thereof described herein.
  • Examples include, but are not limited to, an isolated antibody or antigen-binding fragment thereof which specifically binds to the same ⁇ 6 ⁇ 4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, an isolated antibody or antigen- binding fragment thereof which specifically binds to ⁇ 6 ⁇ 4 integrin, where the antibody or fragment thereof competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof inhibits laminin from binding to ⁇ 6 ⁇ 4 integrin.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell inhibits activation of downstream signal transduction molecules involved in cell proliferation, motility and/or metastasis.
  • Such molecules include, but are not limited to PI3 -K, Akt, mTOR and Rac.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits activation of the Ras/MAPK signaling pathway.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits the interaction of ⁇ 6 ⁇ 4 with growth factor receptors such as erbB2, Met, and Ron.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell inhibits integrin clustering in lipid rafts.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits the interaction with cytoskeletal components, induces apoptosis, and induces integrin internalization.
  • an antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits cell proliferation, motility, and/or metastasis.
  • An antibody of the present invention which specifically binds to ⁇ 6 ⁇ 4 integrin or a variant thereof, to be used in treatment methods disclosed herein can be prepared and used as a therapeutic agent that stops, reduces, prevents, or inhibits cellular activities involved in cellular hyperproliferation, e.g., cellular activities that induce the altered or abnormal pattern of vascularization that is often associated with hyperproliferative diseases or disorders.
  • Antibodies or immunospecific fragments thereof of the present invention include, but are not limited to monoclonal, chimeric or humanized antibodies, and fragments of antibodies that bind specifically to tumor-associated proteins such as ⁇ 6 ⁇ 4 integrin.
  • the antibodies may be monovalent, bivalent, polyvalent, or bifunctional antibodies, and the antibody fragments include Fab F(ab') 2 , and Fv.
  • Therapeutic antibodies according to the invention can be used in unlabeled or unconjugated form, or can be coupled or linked to cytotoxic moieties such as radiolabels and biochemical cytotoxins to produce agents that exert therapeutic effects.
  • an antibody, or immunospecific fragment thereof of the invention includes an antigen binding domain.
  • An antigen binding domain is formed by antibody variable regions that vary from one antibody to another. Naturally occurring antibodies comprise at least two antigen binding domains, i.e., they are at least bivalent.
  • the term "antigen binding domain” includes a site that specifically binds an epitope on an antigen (e.g., a cell surface or soluble antigen).
  • the antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
  • the present invention provides methods for treating various hyperproliferative disorders, e.g., by inhibiting tumor growth, in a mammal, comprising, consisting essentially of, or consisting of administering to the mammal an effective amount of a antibody or antigen-binding fragment thereof which specifically or preferentially binds to ⁇ 6 ⁇ 4 integrin, e.g., human ⁇ 6 ⁇ 4 integrin.
  • the present invention is more specifically directed to a method of treating a hyperproliferative disease, e.g., inhibiting or preventing tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation, in an animal, e.g., a mammal, e.g., a human, comprising, consisting essentially of, or consisting of administering to an animal in need thereof an effective amount of a an antibody or immunospecific fragment thereof, which specifically or preferentially binds to one or more epitopes of ⁇ 6 ⁇ 4 integrin.
  • a hyperproliferative disease e.g., inhibiting or preventing tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation
  • an animal e.g., a mammal, e.g., a human
  • an effective amount of a an antibody or immunospecific fragment thereof which specifically or preferentially binds to one or more epitopes of ⁇ 6 ⁇ 4 integrin.
  • the present invention includes a method for treating a hyperproliferative disease, e.g., inhibiting tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation in an animal, e.g., a human patient, where the method comprises administering to an animal in need of such treatment an effective amount of a composition comprising, consisting essentially of, or consisting of, in addition to a pharmaceutically acceptable carrier, an antibody, or immunospecific fragment thereof, which specifically binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids amino acids of SEQ ED NOs: 1-15, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ED NOs:l-15.
  • the amino acids of a given epitope of SEQ ED NOs:l-15 as described may be, but need not be contiguous.
  • the at least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of ⁇ 6 ⁇ 4. integrin as expressed on the surface of a cell.
  • the at least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ED NOs: 1-15, where non-contiguous amino acids form an epitope through protein folding.
  • the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of a portion of ⁇ 6 and a portion of ⁇ 4.
  • At least one epitope of ⁇ 6 ⁇ 4 integrin comprises, consists essentially of, or consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or noncontiguous amino acids of an ⁇ 6 sequence selected from the group consisting of SEQ ID NOs: 1-8 and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of a ⁇ 4 sequence selected from the group consisting of SEQ ID NOs: 1-8
  • the present invention includes a method for treating a hyperproliferative disease, e.g., inhibiting tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation in an animal, e.g., a human patient, where the method comprises administering to an animal in need of such treatment an effective amount of a composition comprising, consisting essentially of, or consisting of, in addition to a pharmaceutically acceptable carrier, an antibody, or immunospecific fragment thereof, which specifically binds to at least one epitope of ⁇ 6 ⁇ 4 integrin, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or non-contiguous amino acids of SEQ ID NOs:l-15 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety may be included such that the binding molecule binds with higher affinity to modified target protein than it does to an unmodified version
  • the present invention provides a method of treating cancer in a human, comprising administering to a human in need of treatment a composition comprising an effective amount of an ⁇ 6 ⁇ 4 integrin-specific antibody or immunospecific fragment thereof, and a pharmaceutically acceptable carrier.
  • Types of cancer to be treated include, but are not limited to, stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • an antibody or fragment thereof binds specifically to at least one epitope of ⁇ 6 ⁇ 4 integral or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above; or binds to at least one epitope of ⁇ 6 ⁇ 4 integrin or fragment or variant described above with an affinity characterized by a dissociation constant K D of less than about 5 x 10 "2 M, about 10 "2 M, about 5 x 10 "3 M, about 10 "3
  • antibodies and fragments thereof of the present invention cross-react with ⁇ 6 ⁇ 4 integrin proteins of other species from which they were raised, e.g., an antibody or fragment thereof which specifically binds to human ⁇ 6 ⁇ 4 integrin also binds to murine ⁇ 6 ⁇ 4 integrin.
  • suitable antibodies or fragments thereof of the present invention include those that are highly species specific.
  • antibodies or immunospecific fragments thereof disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "2 sec “1 , 10 "2 sec “1 , 5 X 10 "3 sec “1 or 10 "3 sec “1 .
  • antibodies or immunospecific fragments thereof disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10 "4 sec “1 , 10 "4 sec “1 , 5 X 10 5 sec “1 , or 10 "5 sec “1 5 X 10 "6 sec “1 , 10 “6 sec “1 , 5 X lO- 7 SeC- 1 Or IO- 7 SeC “1 .
  • off rate k(off)
  • antibodies or immunospecific fragments thereof disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 10 3 M '1 sec “1 , 5 X 10 3 M “1 sec “1 , 10 4 M “1 sec “1 or 5 X 10 4 M “1 sec “1 .
  • antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein bind ⁇ 6 ⁇ 4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 10 5 M "1 sec “1 , 5 X 10 5 M “1 sec “1 , 10 6 M “1 sec '1 , or 5 X 10 6 M “1 sec “1 or 10 7 M “1 sec “1 .
  • one or more binding molecules as described above is an antagonist of ⁇ 6 ⁇ 4 integrin activity, for example, binding of an antagonist ⁇ 6 ⁇ 4 integrin antibody to ⁇ 6 ⁇ 4 integrin as expressed on a tumor cell inhibits binding of laminin, inhibits activation of molecules downstream in the signal transduction pathway, e.g., PI3- K, Akt, mTOR and RAC , or inhibits tumor cell proliferation, motility or metastasis.
  • ⁇ 6 ⁇ 4 integrin activity for example, binding of an antagonist ⁇ 6 ⁇ 4 integrin antibody to ⁇ 6 ⁇ 4 integrin as expressed on a tumor cell inhibits binding of laminin, inhibits activation of molecules downstream in the signal transduction pathway, e.g., PI3- K, Akt, mTOR and RAC , or inhibits tumor cell proliferation, motility or metastasis.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies, or fragments, derivatives, or analogs thereof can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of ⁇ 6 ⁇ 4 integrin.
  • ⁇ 6 ⁇ 4 integrin expression is increased in tumor tissue and other neoplastic conditions.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies or fragments thereof are useful for diagnosis, treatment, prevention and/or prognosis of hyperproliferative disorders in mammals, preferably humans.
  • disorders include, but are not limited to, cancer, neoplasms, tumors and/or as described under elsewhere herein, especially ⁇ 6 ⁇ 4 integrin-associated cancers such as stomach cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • ⁇ 6 ⁇ 4 integrin expression is associated with at least stomach, brain, bladder, colon, lung, breast, pancreatic, ovarian, and prostate tumor tissues. Accordingly, antibodies (and antibody fragments) directed against ⁇ 6 ⁇ 4 integrin may be used to detect particular tissues expressing increased levels of ⁇ 6 ⁇ 4 integrin. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
  • the invention provides a diagnostic method useful during diagnosis of a cancers and other hyperproliferative disorders, which involves measuring the expression level of ⁇ 6 ⁇ 4 integrin protein or transcript in tissue or other cells or body fluid from an individual and comparing the measured expression level with a standard ⁇ 6 ⁇ 4 integrin expression levels in normal tissue or body fluid, whereby an increase in the expression level compared to the standard is indicative of a disorder.
  • One embodiment provides a method of detecting the presence of abnormal hyperproliferative cells, e.g., precancerous or cancerous cells, in a fluid or tissue sample, comprising assaying for the expression of ⁇ 6 ⁇ 4 integrin in tissue or body fluid samples of an individual and comparing the presence or level of ⁇ 6 ⁇ 4 integrin expression in the sample with the presence or level of ⁇ 6 ⁇ 4 integrin expression in a panel of standard tissue or body fluid samples, where detection of ⁇ 6 ⁇ 4 integrin expression or an increase in ⁇ 6 ⁇ 4 integrin expression over the standards is indicative of aberrant hyperproliferative cell growth.
  • abnormal hyperproliferative cells e.g., precancerous or cancerous cells
  • the present invention provides a method of detecting the presence of abnormal hyperproliferative cells in a body fluid or tissue sample, comprising (a) assaying for the expression of ⁇ 6 ⁇ 4 integrin in tissue or body fluid samples of an individual using ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof of the present invention, and (b) comparing the presence or level of ⁇ 6 ⁇ 4 integrin expression in the sample with a the presence or level of ⁇ 6 ⁇ 4 integrin expression in a panel of standard tissue or body fluid samples, whereby detection of ⁇ 6 ⁇ 4 integrin expression or an increase in ⁇ 6 ⁇ 4 integrin expression over the standards is indicative of aberrant hyperproliferative cell growth.
  • the presence of a relatively high amount of ⁇ 6 ⁇ 4 integrin protein in biopsied tissue from an individual may indicate the presence of a tumor or other malignant growth, may indicate a predisposition for the development of such malignancies or tumors, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies of the present invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al, J. Cell Biol. 707:976-985 (1985); Jalkanen, et al, J. Cell Biol. 705:3087-3096 (1987)).
  • Other antibody-based methods useful for detecting protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Suitable assays are described in more detail elsewhere herein.
  • One aspect of the invention is a method for the in vivo detection or diagnosis of a hyperproliferative disease or disorder associated with aberrant expression of ⁇ 6 ⁇ 4 integrin in an animal, preferably a mammal and most preferably a human.
  • diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody or fragment thereof of the present invention, which specifically binds to ⁇ 6 ⁇ 4 integrin; b) waiting for a time interval following the administering for permitting the labeled binding molecule to preferentially concentrate at sites in the subject where ⁇ 6 ⁇ 4 integrin is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of ⁇ 6 ⁇ 4 integrin.
  • Background level can be determined by various methods including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
  • the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of, e.g., 99 Tc.
  • the labeled binding molecule e.g., antibody or antibody fragment, will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W.
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours, hi another embodiment the time interval following administration is 5 to 20 days or 7 to 10 days.
  • Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • the binding molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050).
  • the binding molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument
  • the binding molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography
  • the binding molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • Antibody labels or markers for in vivo imaging of ⁇ 6 ⁇ 4 integrin expression include those detectable by X-radiography, nuclear magnetic resonance imaging (NMR), MRI, CAT-scans or electron spin resonance imaging (ESR).
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR. include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • monitoring of an already diagnosed disease or disorder is carried out by repeating any one of the methods for diagnosing the disease or disorder, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
  • detection methods as disclosed herein are useful as a prognostic indicator, whereby patients continuing to exhibiting enhanced ⁇ 6 ⁇ 4 integrin expression will experience a worse clinical outcome relative to patients whose expression level decreases nearer the standard level.
  • ⁇ 6 ⁇ 4 integrin polypeptide expression level in the first biological sample is measured or estimated and compared to a standard ⁇ 6 ⁇ 4 integrin polypeptide level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder.
  • a standard ⁇ 6 ⁇ 4 integrin polypeptide level is known, it can be used repeatedly as a standard for comparison.
  • biological sample any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing ⁇ 6 ⁇ 4 integrin.
  • biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid), and other tissue sources which contain cells potentially expressing ⁇ 6 ⁇ 4 integrin. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
  • antibodies, or immunospecific fragments of antibodies directed to a conformational epitope of ⁇ 6 ⁇ 4 integrin may be used to quantitatively or qualitatively detect the presence of ⁇ 6 ⁇ 4 integrin gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluoresence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.
  • Cancers that may be diagnosed, and/or prognosed using the methods described above include but are not limited to, stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein may be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and noncompetitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors ⁇ e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4.degree.
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol
  • protein phosphatase and/or protease inhibitors ⁇ e.g.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32p or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • ELISAs e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994) at 11.2.1.
  • the binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction can be determined by competitive binding assays.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen ⁇ e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays.
  • the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
  • ⁇ 6 ⁇ 4 integrin-specific antibodies may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of cancer antigen gene products or conserved variants or peptide fragments thereof.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled ⁇ 6 ⁇ 4 integrin-specific antibody or fragment thereof, preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • a labeled ⁇ 6 ⁇ 4 integrin-specific antibody or fragment thereof preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • Immunoassays and non-immunoassays for ⁇ 6 ⁇ 4 integrin gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding to ⁇ 6 ⁇ 4 integrin or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
  • the biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled ⁇ 6 ⁇ 4 integrin-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the antibody is subsequently labeled.
  • the amount of bound label on solid support may then be detected by conventional means.
  • solid phase support or carrier any support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • the binding activity of a given lot of ⁇ 6 ⁇ 4 integrin-specific antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • SPR Surface plasmon reasonance as performed on BIAcore offers a number of advantages over conventional methods of measuring the affinity of antibody-antigen interactions: (i) no requirement to label either antibody or antigen; (ii) antibodies do not need to be purified in advance, cell culture supernatant can be used directly; (iii) real-time measurements, allowing rapid semi-quantitative comparison of different monoclonal antibody interactions, are enabled and are sufficient for many evaluation purposes; (iv) biospecific surface can be regenerated so that a series of different monoclonal antibodies can easily be compared under identical conditions; (v) analytical procedures are fully automated, and extensive series of measurements can be performed without user intervention.
  • BIAapplications Handbook version AB (reprinted 1998), BIACORE code No. BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE code No. BR-1001-84.
  • SPR based binding studies require that one member of a binding pair be immobilized on a sensor surface.
  • the binding partner immobilized is referred to as the ligand.
  • the binding partner in solution is referred to as the analyte.
  • the ligand is attached indirectly to the surface through binding to another immobilized molecule, which is referred as the capturing molecule.
  • SPR response reflects a change in mass concentration at the detector surface as analytes bind or dissociate.
  • the resulting sensorgram can be divided into three essential phases: (i) Association of analyte with ligand during sample injection; (ii) Equilibrium or steady state during sample injection, where the rate of analyte binding is balanced by dissociation from the complex; (iii) Dissociation of analyte from the surface during buffer flow.
  • the equilibrium phase provides information on the affinity of the analyte- ligand interaction (K D ).
  • BIAevaluation software provides comprehensive facilities for curve fitting using both numerical integration and global fitting algorithms. With suitable analysis of the data, separate rate and affinity constants for interaction can be obtained from simple BIAcore investigations. The range of affinities measurable by this technique is very broad ranging from mM to pM.
  • Epitope specificity is an important characteristic of a monoclonal antibody.

Abstract

The invention relates to antibodies which bind to alpha6beta4 integrin(α6β4 integrin) and uses thereof, in particular in the diagnosis and treatment of cancer. Specific human and murine antibodies which inhibit α6β4 integrin-mediated pro-survival and tumor proliferation pathways, and variants, fragments, and derivatives thereof are provided. Also provided are specific human and murine antibodies which block the ability of the ligand, laminin to bind to α6β4 integrin, as well as fragments, variants and derivatives of such antibodies. The invention also includes polynucleotides encoding the above antibodies or fragments, variants or derivatives thereof, as well as vectors and host cells comprising such polynucleotides. The invention further includes methods of diagnosing and treating cancer using antibodies of the invention.

Description

ANTI-ALPHA 6 BETA 4 INTEGRIN ANTIBODIES AND USES THEREOF
BACKGROUND OF THE INVENTION
[0001] α6β4 integrin is a laminin receptor expressed on basal epithelial cells. The primary function of α6β4 integrin is to maintain the integrity of epithelia, especially the epidermis. Dowlings et al., J. Cell Biol. 134:559-572 (1996). However, studies have shown that expression of α6β4 also persists in aggressive carcinomas and that its expression is linked to the behavior of these tumors. It is believed to play a pivotal role in functions associated with carcinoma progression and has also been implicated in the formation of carcinomas. Dajee et al., Nature 421:639-643 (2003).
[0002] α6β4 integrins belong to the integrin family of cell surface receptors. The integrin family serve cellular adhesion functions. The receptors form a link between the extracellular matrix and the cytoskeleton through their binding to various extracellular components. Each integrin receptor is a heterodimer composed of an α and a β subunit. At least 18 α chains and eight β chains have been characterized.
[0003] The molecular architecture of the α6β4 integrin comprises an α6 subunit which is composed of a heavy and light chain linked by a disulfide bond and a β4 subunit. The distinguishing structural feature of the α6β4 integrin is the atypical cytoplasmic domain of the β4 subunit. Two pairs of fibronectin type III repeats separated by a connecting segment characterize the β4 domain and it is distinct both in size (approximately 1000 amino acids) and structure from any other integrin subunits.
[0004] α6β4 integrin functions primarily as an adhesion receptor in normal epithelia. It mediates the formation on the basal epithelial cell surface of stable adhesive structures known as hemidesmosomes (HDs) that serve an essential mechanical function. The hemidesmosomes link the intermediate filament cytoskeleton with laminins in the basement membrane (BM). Borradori, L. and Sonnenberg, A., J Invest. Dematol. //2:411-418 (1999). In the absence of α6β4 expression in knockout and transgenic mice, skin morphogenesis appears normal but the epidermis detaches in response to mechanical stress, a condition that results in death shortly after birth. DiPersio et al, J. Cell ScL //5:3051-3062 (2000). In humans, mutations in the β4 subunit also cause epidermal blistering. Vidal et al., Nature Genetics /0:229-234 (1995).
[0005] However, α6β4 integrins have also been shown to play a role in the formation, migration, invasion and survival of carcinomas. α6β4 integrins have been shown to exist in two "functional states," one as an adhesion receptor localized in the HDs and two, as a signaling competent receptor localized with F-actin in lamellipodia and filopodia during carcinoma migration and invasion. It has been shown to cooperate with specific growth factors, such as EGF, to facilitate signaling and promote the migration and survival of carcinoma cells. Without being bound by theory, it is believed that EGF can increase PKC-α-mediated phosphorylation of the β4 cytoplasmic domain. This phosphorylation leads to disruption of the HDs and mobilization of α6β4 to lamellipodia and its association with F-actin. The release of α6β4 from the HDs activates its signaling capacity, which may occur by its association with growth factor receptors such as erbB2, Met, and Ron or lipid rafts. The key signaling event mediated by α6β4 is activation of PI3-K. The α6β4-mediated activation of PB-K and its downstream effectors Akt, mTOR and Rac have been shown to have profound consequences on the biology of carcinoma cells. The regulation of mTOR by α6β4, in particular, has been shown to influence the translation of growth factors such as VEGF that can function to further amplify PB-K activation, thus sustaining and enhancing α6β4-mediated functions.
[0006] Thus, there is a need for molecules, including anti-α6β4 antibodies, that could inhibit the α6β4 signaling pathways. The molecules would be useful for treating and/or preventing various neoplastic diseases including cancer and metastases thereof.
BRIEF SUMMARY OF THE INVENTION
[0007] In some embodiments, the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59- B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2. In one embodiment, the reference monoclonal Fab antibody fragment is M59-B05. In another embodiment, the reference monoclonal Fab antibody fragment is M61-C03. In a further embodiment, the α6β4 integrin epitope is contained within amino acids 446-686 of SEQ ID NO:101.
[0008] In some embodiments, the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to α6β4 integrin, where the antibody or fragment competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to α6β4 integrin.
[0009] In some embodiments, the invention provides an isolated antibody or antigen- binding fragment thereof which specifically binds to α6β4 integrin, where the antibody or fragment thereof comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
[0010] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the heavy chain variable region (VH) of the antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
[0011] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the light chain variable region (VL) of the antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92.
[0012] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH of the antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
[0013] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VL of the antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92. [0014] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH of the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ BD NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ DD NO: 47, SEQ ID NO: 57, SEQ DD NO: 67, SEQ DD NO: 77, and SEQ DD NO: 87.
[0015] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VL of the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ DD NO: 22, SEQ DD NO: 32, SEQ DD NO: 42, SEQ DD NO: 52, SEQ DD NO: 62, SEQ DD NO: 72, SEQ DD NO: 82 and SEQ DD NO:92.
[0016] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ DD NO: 92.
[0017] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences identical, except for 20 or fewer conservative amino acid substitutions each, to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ ID NO: 92.
[0018] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH and VL of the antibody or fragment thereof comprise, respectively, amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ ID NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and SEQ DD NO: 72; SEQ DD NO: 77 and SEQ DD NO: 82; and SEQ DD NO: 87 and SEQ DD NO: 92. [0019] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region- 1 (VH-CDRl) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88. In further embodiments, the VH-CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO:
58, SEQ ID NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88.
[0020] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-2 (VH-CDR2) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79 and SEQ ID NO: 89. hi further embodiments, the VH-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO:
59, SEQ ID NO: 69, SEQ ID NO: 79 and SEQ ID NO: 89.
[0021] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH of the antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-3 (VH-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ED NO: 60, SEQ ID NO: 70, SEQ BD NO: 80, and SEQ ID NO: 90. In further embodiments, the VH-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO:
60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90.
[0022] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region- 1 (VL-CDRl) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93. In further embodiments, the VL-CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO:
63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93.
[0023] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region-2 (VL-CDR2) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 24, SEQ BD NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84, and SEQ ID NO: 94. In further embodiments, the VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO:
64, SEQ ID NO: 74, SEQ ID NO: 84, and SEQ ID NO: 94.
[0024] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VL of the antibody or fragment thereof comprises a Kabat light chain complementarity determining region-3 (VL-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, and SEQ ID NO: 95. In further embodiments, the VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO:
65, SEQ ID NO: 75, SEQ ID NO: 85, and SEQ ID NO: 95.
[0025] hi some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH of the antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90, except for one, two, three, or four amino acid substitutions in at least one of said VH-CDRs. [0026] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VH of the antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ED NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ BD NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90.
[0027] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VL of the antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95, except for one, two, three, or four amino acid substitutions in at least one of said VL-CDRs.
[0028] In some embodiments, the invention provides an isolated antibody or fragment thereof which specifically binds to α6β4 integrin, where the VL of the antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95.
[0029] In various embodiments of the above-described antibodies or fragments thereof, the VH framework regions and/or VL framework regions are human, except for five or fewer amino acid substitutions.
[0030] In some embodiments, the above-described antibodies or fragments thereof bind to a linear epitope or a non-linear conformation epitope
[0031] In some embodiments, the above-described antibodies or fragments thereof are multivalent, and comprise at least two heavy chains and at least two light chains.
[0032] hi some embodiments, the above-described antibodies or fragments thereof are multispecific. In further embodiments, the above-described antibodies or fragments thereof are bispecific.
[0033] In various embodiments of the above-described antibodies or fragments thereof, the heavy and light chain variable domains are fully human, hi further embodiments, the heavy and light chain variable domains are from a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65- Al 1, M66-H09 and M67-F05.
[0034] In various embodiments of the above-described antibodies or fragments thereof, the heavy and light chain variable domains are murine. In further embodiments, the heavy and light chain variable domains are from a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
[0035] In various embodiments, the above-described antibodies or fragments thereof are humanized.
[0036] In various embodiments, the above-described antibodies or fragments thereof are chimeric.
[0037] In various embodiments, the above-described antibodies or fragments thereof are primatized.
[0038] hi various embodiments, the above-described antibodies or fragments thereof are fully human.
[0039] In certain embodiments, the above-described antibodies or fragments thereof are
Fab fragments, Fab' fragments, F(ab)2 fragments, or Fv fragments.
[0040] hi certain embodiments, the above-described antibodies are single chain antibodies.
[0041] In certain embodiments, the above-described antibodies or fragments thereof comprise light chain constant regions selected from the group consisting of a human kappa constant region and a human lambda constant region.
[0042] hi certain embodiments, the above-described antibodies or fragments thereof comprise a heavy chain constant region or fragment thereof. In further embodiments, the heavy chain constant region or fragment thereof is human IgG4.
[0043] In some embodiments, the above-described antibodies or fragments thereof specifically bind to an α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) which is less than the KD for said reference monoclonal antibody, hi further embodiments, the dissociation constant (KD) is no greater than 5 x 10"2 M, 10"2 M, 5 x 10~3 M, 10"3 M, 5 x Kr4 M, 10"4 M, 5 x 10"5 M, 10'5 M, 5 x 10"6 M, 10"6 M, 5 x 10"7 M, 10"7 M, 5 x 10"8 M, 10"8 M, 5 x Kr9 M, 10"9 M, 5 x 10"10 M, 10"10 M, 5 x 10"H M, 10 11 M, 5 x 10"12 M, 10"12 M, 5 x 10"13 M, 10"13 M, 5 x 10"14 M, 1(T14 M, 5 x 10"15 M, or 10"15 M. [0044] In some embodiments, the above-described antibodies or fragments thereof preferentially bind to a human α6β4 integrin polypeptide or fragment thereof, relative to a murine α6β4 integrin polypeptide or fragment thereof .
[0045] In some embodiments, the above described antibodies or fragments thereof bind to α6β4 integrin expressed on the surface of a cell. In further embodiments, the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
[0046] In some embodiments, the above described antibodies or fragments thereof block laminin from binding to α6β4 integrin.
[0047] In some embodiments, the above described antibodies or fragments thereof inhibit α6β4 integrin association with growth factor receptors. In certain embodiments, the growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
[0048] In some embodiments, the above described antibodies or fragments thereof inhibit α6β4 integrin-mediated PB-K activation.
[0049] In some embodiments, the above described antibodies or fragments thereof inhibit α6β4 integrin-mediated activation of the Ras/MAPK signaling pathway.
[0050] hi some embodiments, the above described antibodies or fragments thereof inhibit α6β4 integrin-mediated cell proliferation or tumor cell growth.
[0051] hi some embodiments, the above described antibodies or fragments thereof induce apoptosis.
[0052] hi further embodiments, the above described antibodies or fragments thereof further comprise a heterologous polypeptide fused thereto.
[0053] hi some embodiments, the above described antibodies or fragments thereof are conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents, hi further embodiments, the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents, hi further embodiments, the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemi luminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels. [0054] In additional embodiments, the invention includes compositions comprising the above-described antibodies or fragments thereof, and a carrier.
[0055] Certain embodiments of the invention include an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the amino acid sequence of the VH polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and where an antibody or antigen binding fragment thereof comprising the VH polypeptide specifically binds to α6β4 integrin. In further embodiments, the amino acid sequence of the VH polypeptide is selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
[0056] In certain embodiments, the nucleotide sequence encoding the VH polypeptide is optimized for increased expression without changing the amino acid sequence of the VH polypeptide. In further embodiments, the optimization comprises identification and removal of splice donor and splice acceptor sites and/or optimization of codon usage for the cells expressing the polynucleotide. In further embodiments, the nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 66, SEQ ID NO: 76, and SEQ ID NO: 86.
[0057] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, where the amino acid sequence of the VL polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ED NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82, and SEQ ID NO: 92; and where an antibody or antigen binding fragment thereof comprising the VL polypeptide specifically binds to α6β4 integrin. In further embodiments, the amino acid sequence of the VL polypeptide is selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82, and SEQ ID NO: 92.
[0058] In certain embodiments, the nucleotide sequence encoding the VL polypeptide is optimized for increased expression without changing the amino acid sequence of said VL polypeptide. In further embodiments, the optimization comprises identification and removal of splice donor and splice acceptor sites and/or optimization of codon usage for the cells expressing the polynucleotide, hi further embodiments, the nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ DD NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 51, SEQ ID NO: 61, SEQ ID NO: 71, SEQ ID NO: 81, and SEQ ID NO: 91.
[0059] In certain other embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the amino acid sequence of the VH polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ E) NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and where an antibody or antigen binding fragment thereof comprising said VH polypeptide specifically binds to α6β4 integrin.
[0060] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, where the amino acid sequence of the VL polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO: 92; and wherein an antibody or antigen binding fragment thereof comprising said VL polypeptide specifically binds to α6β4 integrin.
[0061] hi some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDRl amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ ID NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88; and where an antibody or antigen binding fragment thereof comprising the VH-CDRl specifically binds to α6β4 integrin. hi further embodiments, the VH- CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 48, SEQ ID NO: 58, SEQ BD NO: 68, SEQ ID NO: 78, and SEQ ID NO: 88.
[0062] hi some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR2 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, and SEQ ID NO: 89; and where an antibody or antigen binding fragment thereof comprising the VH-CDR2 specifically binds to α6β4 integrin. In further embodiments, the VH- CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79, and SEQ ID NO: 89.
[0063] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to α6β4 integrin. In further embodiments, the VH- CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90.
[0064] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDRl amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93; and where an antibody or antigen binding fragment thereof comprising the VL-CDRl specifically binds to α6β4 integrin. In further embodiments, the VL-CDRl amino acid sequence is selected from the group consisting of: SEQ ED NO: 23, SEQ ID NO: 33, SEQ ED NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93.
[0065] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR2 amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ED NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ED NO: 74, SEQ ED NO: 84 and SEQ ED NO: 94; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR2 specifically binds to α6β4 integrin. Ln further embodiments, the VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ED NO: 24, SEQ ED NO: 34, SEQ ID NO: 44, SEQ ED NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84 and SEQ ID NO: 94.
[0066] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ED NO: 25, SEQ ED NO: 35, SEQ ED NO: 45, SEQ ED NO: 55, SEQ ED NO: 65, SEQ ED NO: 75, SEQ ED NO: 85, and SEQ ED NO: 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to α6β4 integrin. In further embodiments, the VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ED NO: 25, SEQ ED NO: 35, SEQ ED NO: 45, SEQ ED NO: 55, SEQ ED NO: 65, SEQ ED NO: 75, SEQ ED NO: 85, and SEQ ED NO: 95.
[0067] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the VH polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ED NOs: 18, 19, and 20; SEQ ED NOs: 28, 29; and 30; SEQ ED NOs: 38, 39, and 40; SEQ ED NOs: 48, 49, and 50; SEQ ED NOs: 58, 59, and 60; SEQ ED NOs: 68, 69, and 70; SEQ ED NOs: 78, 79, and 80; and SEQ ID NOs: 88, 89 and 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to α6β4 integrin.
[0068] Ln some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ED NOs: 23, 24, and 25; SEQ ED NOs: 33, 34, and 35; SEQ ED NOs: 43, 44, and 45; SEQ ED NOs: 53, 54, and 55; SEQ ED NOs: 63, 64, and 65; SEQ ED NOs: 73, 74, and 75; SEQ ED NOs: 83, 84, and 85; and SEQ ID NO: 93, 94, and 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to α6β4 integrin.
[0069] Ln some embodiments, the above-described polynucleotides further comprise a nucleic acid encoding a signal peptide fused to the antibody VH polypeptide or the antibody VL polypeptide.
[0070] Ln certain other embodiments, the above-described polynucleotides further comprise a nucleic acid encoding a heavy chain constant region CHl domain fused to the VH polypeptide, encoding a heavy chain constant region CH2 domain fused to the VH polypeptide, encoding a heavy chain constant region CH3 domain fused to the VH polypeptide, or encoding a heavy chain hinge region fused to said VH polypeptide. In further embodiments, the heavy chain constant region is human IgG4.
[0071] In some embodiments, the above-described polynucleotides comprise a nucleic acid encoding a light chain constant region domain fused to said VL polypeptide. In further embodiments, the light chain constant region is human kappa.
[0072] In various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising a polypeptide encoded by the nucleic acid specifically binds the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
[0073] In various other embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising a polypeptide encoded by the nucleic acid competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
[0074] In various embodiments of the above-describe polynucleotides, the framework regions of the VH polypeptide or VL polypeptide are human, except for five or fewer amino acid substitutions.
[0075] In various embodiments of the above-described polynucleotides, the invention provides an antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid, that binds to a linear epitope or a non-linear conformational epitope.
[0076] In various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is multivalent, and comprises at least two heavy chains and at least two light chains.
[0077] In certain embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is multispecific. In further embodiments, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is bispecific.
[0078] In various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid comprises heavy and light chain variable domains which are fully human. In further embodiments, the heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05.
[0079] In various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid comprises heavy and light chain variable domains which are murine. In further embodiments, the heavy and light chain variable domains are identical to those of a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
[0080] hi various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is humanized.
[0081] In various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is primatized.
[0082] In various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is chimeric.
[0083] hi some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is fully human.
[0084] hi various embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is an Fab fragment, an Fab' fragment, an F(ab)2 fragment, or an Fv fragment, hi certain embodiments of the above-described polynucleotides, the antibody or antigen- binding fragment thereof comprising the polypeptide encoded by the nucleic acid is a single chain antibody.
[0085] hi some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid specifically binds to an α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10'2 M, 10~2 M, 5 x 10"3 M, 10'3 M, 5 x 10"4 M, 10"4 M, 5 x 10"5 M, 10"5 M, 5 x 10"6 M, 10"6 M, 5 x 10"7 M, 10"7 M, 5 x 10"8 M, 10"8 M, 5 x 10"9 M, 1(T9 M, 5 x 10-10 M, 10-10 M, 5 x 1(T" M, 1(T1 M, 5 x 10"12 M, 1(T12 M, 5 x 10'13 M, lO"13
M, 5 x 10"14 M, 10'14 M, 5 x 10"15 M, or 10"15 M. [0086] In some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid preferentially binds to a human α6β4 integrin polypeptide or fragment thereof, relative to a murine α6β4 integrin polypeptide or fragment thereof. [0087] In some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid binds to α6β4 integrin expressed on the surface of a cell. In further embodiments, the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell. [0088] In some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by said nucleic acid blocks laminin from binding to α6β4 integrin. [0089] In some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits α6β4 integrin -mediated activation of PI3-K. [0090] In some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits activation of the Ras/MAPK signaling pathway. [0091] hi some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits α6β4 integrin association with growth factor receptors. In certain embodiments, the growth factor receptor is selected from the group consisting of erbB2,
Met, and Ron. [0092] In some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits α6β4 integrin-mediated cell proliferation or tumor cell growth. [0093] hi some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid induces apoptosis. [0094] hi some embodiments, the above-described polynucleotides further comprise a nucleic acid encoding a heterologous polypeptide. [0095] In some embodiments of the above-described polynucleotides, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents. In further embodiments, the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents. In certain other embodiments, the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
[0096] In some embodiments, the invention provides compositions comprising the above-described polynucleotides.
[0097] In certain other embodiments, the invention provides vectors comprising the above-described polynucleotides, hi further embodiments, the polynucleotides are operably associated with a promoter. In additional embodiments, the invention provides host cells comprising such vectors. In further embodiments, the invention provides vectors where the polynucleotide is operably associated with a promoter.
[0098] hi additional embodiments, the invention provides a method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising culturing a host cell containing a vector comprising the above-described polynucleotides, and recovering said antibody, or fragment thereof. In further embodiments, the invention provides an isolated polypeptide produced by the above-described method.
[0099] hi some embodiments, the invention provides isolated polypeptides encoded by the above-described polynucleotides.
[0100] In further embodiments of the above-described polypeptides, the antibody or fragment thereof comprising the polypeptide specifically binds to α6β4 integrin. Other embodiments include the isolated antibody or fragment thereof comprising the above- described polypeptides.
[0101] In some embodiments, the invention provides a composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, where the VH encoding polynucleotide and the VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ED NO: 37 and SEQ ID NO: 42; SEQ ED NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ED NO: 62; SEQ ID NO: 67 and 72; SEQ ID NO: 77 and 82; and SEQ ED NO: 87 and 92; and where an antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds α6β4 integrin. In further embodiments, the VH encoding polynucleotide and said VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and 72; SEQ DD NO: 77 and 82; and SEQ DD NO: 87 and 92.
[0102] In certain other embodiments, the invention provides a composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, where the VH encoding polynucleotide and the VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences identical, except for less than 20 conservative amino acid substitutions, to reference amino acid sequences selected from the group consisting of: SEQ DD NO: 17 and SEQ DD NO: 22; SEQ DD NO: 27 and SEQ DD NO: 32; SEQ DD NO: 37 and SEQ DD NO: 42; SEQ DD NO: 47 and SEQ DD NO: 52; SEQ DD NO: 57 and SEQ DD NO: 62; SEQ DD NO: 67 and 72; SEQ DD NO: 77 and 82; and SEQ DD NO: 87 and 92; and where an antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds α6β4 integrin.
[0103] In further embodiments, the VH encoding polynucleotide encodes a VH polypeptide comprising VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ DD NOs: 18, 19, and 20; SEQ DD NOs: 28, 29, and 30; SEQ ED NOs: 38, 39, and 40; SEQ DD NOs: 48, 49, and 50; SEQ BD NOs: 58, 59, and 60; SEQ ED NOs: 68, 69, and 70; SEQ DD NOs: 78, 79 and 80; and SEQ DD NOs: 88, 89, and 90; where the VL encoding polynucleotide encodes a VL polypeptide comprising VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ DD NOs: 23, 24, and 25; SEQ DD NOs: 33, 34, and 35; SEQ DD NOs: 43, 44, and 45; SEQ BD NOs: 53, 54, and 55; SEQ DD NOs: 63, 64, and 65; SEQ DD NOs: 73, 74, and 75; SEQ DD NOs: 83, 84, and 85; and SEQ DD NOs: 93, 94 and 95; and where an antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds α6β4 integrin. [0104] In various embodiments of the above-described compositions, the VH encoding polynucleotide further comprises a nucleic acid encoding a signal peptide fused to the antibody VH polypeptide.
[0105] hi various embodiments of the above-described compositions, the VL encoding polynucleotide further comprises a nucleic acid encoding a signal peptide fused to the antibody VL polypeptide.
[0106] hi some embodiments of the above-described compositions, the VH encoding polynucleotide further comprises a nucleic acid encoding a heavy chain constant region CHl domain fused to the VH polypeptide, further comprises a nucleic acid encoding a heavy chain constant region CH2 domain fused to the VH polypeptide, further comprises a nucleic acid encoding a heavy chain constant region CH3 domain fused to the VH polypeptide, or further comprises a nucleic acid encoding a heavy chain hinge region fused to the VH polypeptide, hi further embodiments, the heavy chain constant region is human IgG4.
[0107] In some embodiments of the above-described compositions, the VL encoding polynucleotide further comprises a nucleic acid encoding a light chain constant region domain fused to the VL polypeptide. In further embodiments, the light chain constant region is human kappa.
[0108] In some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides specifically binds the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
[0109] hi some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to α6β4 integrin.
[0110] hi some embodiments of the above-described compositions, the framework regions of the VH and VL polypeptides are human, except for five or fewer amino acid substitutions. [0111] In some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides binds to a linear epitope or a non-linear conformational epitope. [0112] hi some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is multivalent, and comprises at least two heavy chains and at least two light chains. [0113] In some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is multispecific. hi further embodiments, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides is bispecific. [0114] hi some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are fully human. In further embodiments, the heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03,
M65-A11, M66-H09 and M67-F05. [0115] hi some embodiments of the above-described compositions, the antibody or fragment thereof encoded by the VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are murine, hi further embodiments, the heavy and light chain variable domains are identical to those of a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and
1.P5B10.2. [0116] hi various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is humanized. [0117] hi various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is primatized. [0118] hi various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is chimeric. [0119] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is fully human. [0120] In various embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is an Fab fragment, an Fab1 fragment, an F(ab)2 fragment, or an Fv fragment. In certain embodiments of the above-described compositions, the antibody or antigen- binding fragment thereof comprising the polypeptide encoded by the nucleic acid is a single chain antibody.
[0121] In some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid specifically binds to an α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10"2 M, 10"2 M, 5 x 10"3 M, 10"3 M, 5 x 10"4 M, 10"4 M, 5 x 10"5 M, 10"5 M, 5 x 10"6 M, 10"6 M, 5 x 10"7 M, 10"7 M, 5 x 10"8 M, 10"8 M, 5 x 1(T9 M, 10"9 M, 5 x 10"10 M, 10"10 M, 5 x 10"11 M, 10"11 M, 5 x 10"12 M, 10"12 M, 5 x 10"13 M, 10"13 M, 5 x 10"14 M, 10"14 M, 5 x 10"15 M, or 10"15-M.
[0122] In some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid preferentially binds to a human α6β4 integrin polypeptide or fragment thereof, relative to a murine α6β4 integrin polypeptide or fragment thereof.
[0123] In some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid binds to α6β4 integrin expressed on the surface of a cell. In further embodiments, the cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
[0124] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by said nucleic acid blocks laminin from binding to α6β4 integrin.
[0125] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits α6β4 integrin-mediated activation of PI3-K.
[0126] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits activation of the Ras/MAPK signaling pathway.
[0127] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits α6β4 integrin association with growth factor receptors. In certain embodiments, the growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
[0128] In some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid inhibits α6β4 integrin-mediated cell proliferation or tumor cell growth.
[0129] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid induces apoptosis.
[0130] hi some embodiments, the above-described compositions, the VH encoding polynucleotide, the VL encoding polynucleotide, or both the VH and the VL encoding polynucleotides further comprise a nucleic acid encoding a heterologous polypeptide.
[0131] hi some embodiments of the above-described compositions, the antibody or antigen-binding fragment thereof comprising the polypeptide encoded by the nucleic acid is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents. In further embodiments, the cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents. In certain other embodiments, the detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
[0132] In some embodiments of the above-described compositions, the VH encoding polynucleotide is contained on a first vector and the VL encoding polynucleotide is contained on a second vector, hi further embodiments, the VH encoding polynucleotide is operably associated with a first promoter and the VL encoding polynucleotide is operably associated with a second promoter, hi certain other embodiments, the first and second promoters are copies of the same promoter. In further embodiments, the first and second promoters are non-identical.
[0133] hi various embodiments of the above-described compositions, the first vector and the second vector are contained in a single host cell. [0134] In certain other embodiments of the above-described compositions, the first vector and the second vector are contained in separate host cells.
[0135] In some embodiments, the invention provides a method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising culturing the above-described host cells, and recovering the antibody, or fragment thereof.
[0136] In other embodiments, the invention provides a method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising co-culturing separate host cells, and recovering the antibody, or fragment thereof. In further embodiments of the above-described method, the invention provides combining the VH and VL encoding polypeptides, and recovering the antibody, or fragment thereof.
[0137] In some embodiments, the invention provides an antibody or fragment thereof which specifically binds α6β4 integrin, produced by the above-described methods.
[0138] In some embodiments, the invention provides compositions, where the VH encoding polynucleotide and the VL encoding polynucleotide are on the same vector, as well as the vectors therein.
[0139] In various embodiments of the above described vectors, the VH encoding polynucleotide and the VL encoding polynucleotide are each operably associated with a promoter.
[0140] In various embodiments of the above described vectors, the VH encoding polynucleotide and the VL encoding polynucleotide are fused in frame, are co- transcribed from a single promoter operably associated therewith, and are cotranslated into a single chain antibody or antigen-binding fragment thereof.
[0141] In various embodiments of the above described vectors, the VH encoding polynucleotide and said VL encoding polynucleotide are co-transcribed from a single promoter operably associated therewith, but are separately translated. In further embodiments, the vectors further comprise an IRES sequence disposed between the VH encoding polynucleotide and the VL encoding polynucleotide. In certain other embodiments, the polynucleotide encoding a VH and the polynucleotide encoding a VL are separately transcribed, each being operably associated with a separate promoter. In further embodiments, the separate promoters are copies of the same promoter or the separate promoters are non-identical.
[0142] In some embodiments, the invention provides host cells comprising the above- described vectors. [0143] In other embodiments, the invention provides a method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising culturing the above-described host cells, and recovering the antibody, or fragment thereof.
[0144] In some embodiments, the invention provides an antibody or fragment thereof which specifically binds α6β4 integrin, produced by the above-described methods.
[0145] In some embodiments, the invention provides a method for treating a hyperproliferative disorder in an animal, comprising administering to an animal in need of treatment a composition comprising: a) an isolated antibody or fragment as described above; and b) a pharmaceutically acceptable carrier, hi further embodiments, the hyperproliferative disease or disorder is selected from the group consisting of cancer, a neoplasm, a tumor, a malignancy, or a metastasis thereof.
[0146] hi various embodiments of the above-described methods, the antibody or fragment thereof specifically binds to α6β4 integrin expressed on the surface of a malignant cell, hi further embodiments, the binding of the antibody or fragment thereof to the malignant cell results in growth inhibition of the malignant cell.
[0147] hi various embodiments of the above-described methods, the antibody or fragment thereof inhibits α6β4 integrin phosphorylation or inhibits tumor cell proliferation, hi further embodiments, the tumor cell proliferation is inhibited through the prevention or retardation of metastatic growth.
[0148] hi various embodiments of the above-described methods, the antibody or fragment thereof inhibits tumor cell migration, hi further embodiments, the tumor cell proliferation is inhibited through the prevention or retardation of tumor spread to adjacent tissues.
[0149] hi various embodiments of the above-described methods, the hyperproliferative disease or disorder is a neoplasm located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, adrenal gland, parathyroid gland, pituitary gland, testicles, ovary, thymus, thyroid, eye, head, neck, central nervous system, peripheral nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, or urogenital tract.
[0150] hi various embodiments of the above-described methods, the hyperproliferative disease is cancer, said cancer selected from the group consisting of: epithelial squamous cell cancer, melanoma, leukemia, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, renal cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer. In further embodiments, the cancer is selected from the group consisting of stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer. [0151] In various embodiments of the above-described methods, the animal is a mammal. In further embodiments, the mammal is a human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0152] Figure 1 shows the purification of soluble α6Fcβ4 protein.
[0153] Figure 2 shows the titration of murine monoclonal antibodies against CHO- human-α6β4.
[0154] Figure 3 shows inhibition of murine antibody binding with α6Fcβ4 protein.
[0155] Figure 4 shows titration of murine antibodies against colorectal and breast tumor cell lines. [0156] Figure 5 shows the titration of IgG4P human antibodies against CHO-human- α6β4. [0157] Figure 6 shows the titration of IgG4P human antibodies against CHO-murine- α6β4.
[0158] Figure 7 shows specificity of human antibody 59B05.
[0159] Figure 8 shows the inhibition of human antibody binding with α6Fcβ4.
[0160] Figure 9 shows titration of human antibody 59B05 binding to breast and colorectal tumor cell lines. [0161] Figure 10 shows the inhibition of anchorage independent growth in SW620 colorectal cancer cells. [0162] Figure 11 shows the inhibition of anchorage independent growth in MDA-MB-
231 breast cancer cells. [0163] Figure 12 shows the inhibition of adhesion in colorectal cancer cells to SCC25
Deposited Matrix.
[0164] Figure 13 shows induction of apoptosis in human tumor cell lines.
[0165] Figure 14 shows inhibition of Phospho-AKT in breast tumor cells with human α6β4 antibodies. [0166] Figure 15 shows inhibition of SW480 colorectal tumor cell line adhesion on purified rat laminin-5 using full-length IgG4 versus Fab versions of human antibodies
59B05 and 61C03. [0167] Figure 16 shows inhibition of anchorage independent growth in BT474 breat cancer cells using full-length IgG4 versus Fab versions of human antibodies 59B05 and 61C03.
[0168] Figure 17 shows the induction of apoptosis in MCF7 breast cancer cells by human antibody 59B05.
[0169] Figure 18 shows a western blot of CNBr cleavage products from β4 digests.
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
[0170] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an α6β4 integrin antibody," is understood to represent one or more α6β4 integrin antibodies. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.
[0171] As used herein, the term "polypeptide" is intended to encompass a singular
"polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post- expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.
[0172] A polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. As used herein, the term glycoprotein refers to a protein coupled to at least one carbohydrate moiety that is attached to the protein via an oxygen-containing or a nitrogen-containing side chain of an amino acid residue, e.g., a serine residue or an asparagine residue.
[0173] By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
[0174] Also included as polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "fragment," "variant," "derivative" and "analog" when referring to α6β4 integrin antibodies or antibody polypeptides of the present invention include any polypeptides which retain at least some of the antigen-binding properties of the corresponding native antibody or polypeptide. Fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of α6β4 integrin antibodies and antibody polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring Non- naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of α6β4 integrin antibodies and antibody polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein a "derivative" of an α6β4 integrin antibody or antibody polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
[0175] The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding an α6β4 integrin antibody contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
[0176] As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g., a single vector may separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid of the invention may encode heterologous coding regions, either fused or unfused to a nucleic acid encoding an α6β4 integrin antibody or fragment, variant, or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain. [0177] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.
[0178] A variety of transcription control regions are known to those skilled in the art.
These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
[0179] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).
[0180] In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA).
[0181] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild- type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.
[0182] The present invention is directed to certain α6β4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof. Unless specifically referring to full- sized antibodies such as naturally-occurring antibodies, the term " α6β4 integrin antibodies" encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
[0183] As used herein, the term "antigen binding molecule" ("ABM") refers in its broadest sense to a molecule that specifically binds an antigenic determinant. It is understood by those of skill in the art that fragments of mature antibodies can bind specifically to an antigen. Accordingly, an antigen binding molecule, as the term is used herein, includes, but is not limited to, fragments of mature antibodies that bind specifically to a target antigen. An ABM of the present invention need not contain a constant region. If one or more constant region(s) is present, in particular embodiments, the constant region is substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. The ABMs of the present invention may be glycoengineered to enhance antibody dependent cellular cytotoxicity.
[0184] The terms "antibody" and "immunoglobulin" are used interchangeably herein.
An antibody or immunoglobulin comprises at least the variable domain of a heavy chain, and normally comprises at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
[0185] As will be discussed in more detail below, the term "immunoglobulin" comprises various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with some subclasses among them (e.g., γl-γ4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgAl, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention. All immunoglobulin classes are clearly within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000- 70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region.
[0186] Light chains are classified as either kappa or lambda (K, λ). Each heavy chain class may be bound with either a kappa or lambda light chain, hi general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
[0187] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally, hi this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHl, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CL domains actually comprise the carboxy- terminus of the heavy and light chain, respectively.
[0188] As indicated above, the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains, hi some instances, e.g., certain immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of heavy chains only, with no light chains. See, e.g., Hamers-Casterman et ai, Nature 563:446-448 (1993).
[0189] In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen binding domains, referred to as "framework" regions, show less inter-molecular variability. The framework regions largely adopt a β- sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for any given heavy or light chain variable region by one of ordinary skill in the art, since they have been precisely defined (see, "Sequences of Proteins of Immunological Interest," Kabat, E., et al, U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol, /95:901-917 (1987), which are incorporated herein by reference in their entireties). hi the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al, J. MoI Biol. 796:901-917 (1987), which are incorporated herein by reference, where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table I as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.
TABLE l. CDR Definitions1
Figure imgf000034_0001
'Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below). [0191] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al, U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an α6β4 integrin antibody or antigen-binding fragment, variant, or derivative thereof of the present invention are according to the Kabat numbering system.
[0192] In camelid species, the heavy chain variable region, referred to as VHH, forms the entire antigen-binding domain. The main differences between camelid VHH variable regions and those derived from conventional antibodies (VH) include (a) more hydrophobic amino acids in the light chain contact surface of VH as compared to the corresponding region in VHH, (b) a longer CDR3 in VHH, and (c) the frequent occurrence of a disulfide bond between CDRl and CDR3 in VHH.
[0193] In one embodiment, an antigen binding molecule of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an antigen binding molecule of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an antigen binding molecule of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject antigen binding molecules are known in the art and exemplary molecules are described herein.
[0194] Antibodies or antigen-binding fragments, variants, or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to α6β4 integrin antibodies disclosed herein). ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019. Immunoglobulin or antibody molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
[0195] Antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHl, CH2, and CH3 domains. Also included in the invention are antigen- binding fragments also comprising any combination of variable region(s) with a hinge region, CHl, CH2, and CH3 domains. Antibodies or immunospecific fragments thereof of the present invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies, hi another embodiment, the variable region may be condricthoid in origin (e.g., from sharks). As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al. A human antibody is still "human" even if amino acid substitutions are made in the antibody.
[0196] As used herein, the term "heavy chain portion" includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CHl domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, a binding polypeptide for use in the invention may comprise a polypeptide chain comprising a CHl domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHl domain and a CH3 domain; a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHl domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain, hi another embodiment, a polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. Further, a binding polypeptide for use in the invention may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that these domains (e.g., the heavy chain portions) may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.
[0197] hi certain α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein, the heavy chain portions of one polypeptide chain of a multimer are identical to those on a second polypeptide chain of the multimer. Alternatively, heavy chain portion-containing monomers of the invention are not identical. For example, each monomer may comprise a different target binding site, forming, for example, a bispecific antibody.
[0198] The heavy chain portions of a binding polypeptide for use in the diagnostic and treatment methods disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CHl domain derived from an IgGl molecule and a hinge region derived from an IgG3 molecule, hi another example, a heavy chain portion can comprise a hinge region derived, in part, from an IgGl molecule and, in part, from an IgG3 molecule, hi another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGl molecule and, in part, from an IgG4 molecule.
[0199] As used herein, the term "light chain portion" includes amino acid sequences derived from an immunoglobulin light chain. Preferably, the light chain portion comprises at least one of a VL or CL domain.
[0200] α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof disclosed herein may be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide (α6β4 integrin) that they recognize or specifically bind. The portion of a target polypeptide which specifically interacts with the antigen binding domain of an antibody is an "epitope," or an "antigenic determinant." A target polypeptide may comprise a single epitope, but typically comprises at least two epitopes, and can include any number of epitopes, depending on the size, conformation, and type of antigen. The epitope may include a portion of α6 and a portion of β4 together, i.e., the epitope can comprise portions of both components in the heterodimer. Furthermore, it should be noted that an "epitope" on a target polypeptide may be or include non-polypeptide elements, e.g., an "epitope may include a carbohydrate side chain.
[0201] The minimum size of a peptide or polypeptide epitope for an antibody is thought to be about four to five amino acids. Peptide or polypeptide epitopes preferably contain at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids. Since a CDR can recognize an antigenic peptide or polypeptide in its tertiary form, the amino acids comprising an epitope need not be contiguous, and in some cases, may not even be on the same peptide chain. In the present invention, peptide or polypeptide epitope recognized by α6β4 integrin antibodies of the present invention contains a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 15 to about 30 contiguous or non-contiguous amino acids of α6β4 integrin.
[0202] By "specifically binds," it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" may be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D."
[0203] By "preferentially binds," it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.
[0204] By way of non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds said first epitope with a dissociation constant (KD) that is less than the antibody's KD for the second epitope. In another non-limiting example, an antibody may be considered to bind a first antigen preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's KD for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's KD for the second epitope.
[0205] In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an off rate (k(off)) that is less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least one order of magnitude less than the antibody's k(off) for the second epitope. In another non-limiting example, an antibody may be considered to bind a first epitope preferentially if it binds the first epitope with an affinity that is at least two orders of magnitude less than the antibody's k(off) for the second epitope.
[0206] An antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 5 X 10"2 sec'1, 10~2 sec"1, 5 X 10'3 sec'1 or 10~3 sec"1. More preferably, an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an off rate (k(off)) less than or equal to 5 X 10"4 sec"1, 10"4 sec"1, 5 X 10"5 sec'1, or 10'5 sec'1 5 X 10"6 sec"1, 10"6 sec"1, 5 X 10"7 sec"1 or 10"7 sec"1.
[0207] An antibody or antigen-binding fragment, variant, or derivative disclosed herein may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) of greater than or equal to 103 M"1 sec"1, 5 X 103 M"1 sec"1, 104 M"1 sec"1 or 5 X 104 M"1 sec"1. More preferably, an antibody of the invention may be said to bind a target polypeptide disclosed herein or a fragment or variant thereof with an on rate (k(on)) greater than or equal to 105 M"1 sec"1, 5 X 105 M"1 sec"1, 106 M"1 sec"1, or 5 X 106 M"1 sec"1 or 107 M"1 sec"1.
[0208] An antibody is said to competitively inhibit binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
[0209] As used herein, the term "affinity" refers to a measure of the strength of the binding of an individual epitope with the CDR of an immunoglobulin molecule. See, e.g., Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) at pages 27-28. As used herein, the term "avidity" refers to the overall stability of the complex between a population of immunoglobulins and an antigen, that is, the functional combining strength of an immunoglobulin mixture with the antigen. See, e.g. , Harlow at pages 29-34. Avidity is related to both the affinity of individual immunoglobulin molecules in the population with specific epitopes, and also the valencies of the immunoglobulins and the antigen. For example, the interaction between a bivalent monoclonal antibody and an antigen with a highly repeating epitope structure, such as a polymer, would be one of high avidity.
[0210] α6β4 integrin antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their cross- reactivity. As used herein, the term "cross-reactivity" refers to the ability of an antibody, specific for one antigen, to react with a second antigen; a measure of relatedness between two different antigenic substances. Thus, an antibody is cross reactive if it binds to an epitope other than the one that induced its formation. The cross reactive epitope generally contains many of the same complementary structural features as the inducing epitope, and in some cases, may actually fit better than the original.
[0211] For example, certain antibodies have some degree of cross-reactivity, in that they bind related, but non-identical epitopes, e.g., epitopes with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be said to have little or no cross-reactivity if it does not bind epitopes with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a reference epitope. An antibody may be deemed "highly specific" for a certain epitope, if it does not bind any other analog, ortholog, or homolog of that epitope.
[0212] α6β4 integrin antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5 x 10"2 M, 10"2 M, 5 x 10"3 M, 10"3 M, 5 x 10"4 M, 10"4 M, 5 x 10"5 M, 10"5 M, 5 x 10"6 M, 10"6M, 5 x 10"7 M, 10"7 M, 5 x 10"8 M, 10"8 M, 5 x 10"9 M, 10"9 M, 5 x 10"10 M, 10"10 M, 5 x 10"11 M, 10"11 M, 5 x 10"12 M, 10"12 M, 5 x 10"13 M, 10"13M, 5 x 10"14M, 10"14M, 5 x 10"15 M, or 10"15 M.
[0213] α6β4 integrin antibodies or antigen-binding fragments, variants or derivatives thereof of the invention may be "multispecifϊc," e.g., bispecific, trispecific or of greater multispecificity, meaning that it recognizes and binds to two or more different epitopes present on one or more different antigens (e.g., proteins) at the same time. Thus, whether an α6β4 integrin antibody is "monospecific" or "multispecifϊc," e.g., "bispecific," refers to the number of different epitopes with which a binding polypeptide reacts. Multispecific antibodies may be specific for different epitopes of a target polypeptide described herein or may be specific for a target polypeptide as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material.
[0214] As used herein the term "valency" refers to the number of potential binding domains, e.g., antigen binding domains, present in an α6β4 integrin antibody, binding polypeptide or antibody. Each binding domain specifically binds one epitope. When an α6β4 integrin antibody, binding polypeptide or antibody comprises more than one binding domain, each binding domain may specifically bind the same epitope, for an antibody with two binding domains, termed "bivalent monospecific," or to different epitopes, for an antibody with two binding domains, termed "bivalent bispecific." An antibody may also be bispecific and bivalent for each specificity (termed "bispecific tetravalent antibodies"). In another embodiment, tetravalent minibodies or domain deleted antibodies can be made.
[0215] Bispecific bivalent antibodies, and methods of making them, are described, for instance in U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, the disclosures of all of which are incorporated by reference herein. Bispecific tetravalent antibodies, and methods of making them are described, for instance, in WO 02/096948 and WO 00/44788, the disclosures of both of which are incorporated by reference herein. See generally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60- 69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0216] As previously indicated, the subunit structures and three dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term "VH domain" includes the amino terminal variable domain of an immunoglobulin heavy chain and the term "CHl domain" includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHl domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.
[0217] As used herein the term "CH2 domain" includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat EA et al. op. cit. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 residues.
[0218] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that joins the CHl domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al, J. Immunol. 757:4083 (1998)).
[0219] As used herein the term "disulfide bond" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CHl and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).
[0220] As used herein, the term "chimeric antibody" will be held to mean any antibody wherein the immunoreactive region or site is obtained or derived from a first species and the constant region (which may be intact, partial or modified in accordance with the instant invention) is obtained from a second species. In preferred embodiments, the target binding region or site will be from a non-human source (e.g. mouse or primate) and the constant region is human.
[0221] As used herein, the term "engineered antibody" refers to an antibody in which the variable domain in either the heavy and light chain or both is altered by at least partial replacement of one or more CDRs from an antibody of known specificity and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and preferably from an antibody from a different species. An engineered antibody in which one or more "donor" CDRs from a non-human antibody of known specificity is grafted into a human heavy or light chain framework region is referred to herein as a "humanized antibody." It may not be necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the target binding site. Given the explanations set forth in, e.g., U. S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional engineered or humanized antibody.
[0222] As used herein the term "properly folded polypeptide" includes polypeptides
(e.g., α6β4 integrin antibodies) in which all of the functional domains comprising the polypeptide are distinctly active. As used herein, the term "improperly folded polypeptide" includes polypeptides in which at least one of the functional domains of the polypeptide is not active. In one embodiment, a properly folded polypeptide comprises polypeptide chains linked by at least one disulfide bond and, conversely, an improperly folded polypeptide comprises polypeptide chains not linked by at least one disulfide bond.
[0223] As used herein the term "engineered" includes manipulation of nucleic acid or polypeptide molecules by synthetic means (e.g. by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).
[0224] As used herein, the terms "linked," "fused" or "fusion" are used interchangeably.
These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An "in-frame fusion" refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two ore more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.) Although the reading frame is thus made continuous throughout the fused segments, the segments may be physically or spatially separated by, for example, in-frame linker sequence. For example, polynucleotides encoding the CDRs of an immunoglobulin variable region may be fused, in-frame, but be separated by a polynucleotide encoding at least one immunoglobulin framework region or additional CDR regions, as long as the "fused" CDRs are co-translated as part of a continuous polypeptide.
[0225] In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
[0226] The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA or polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
[0227] As used herein, the term "additive effect" is one in which the effect of two molecules acting simultaneously is the simple sum of the effects that they would have if acting alone. As used herein, the term "synergistic effect" is one in which the effect of two molecules is greater than the effect of each molecule individually, or the sum of the individual effects.
[0228] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented. [0229] By "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.
[0230] As used herein, phrases such as "a subject that would benefit from administration of a binding molecule" and "an animal in need of treatment" includes subjects, such as mammalian subjects, that would benefit from administration of a binding molecule used, e.g., for detection of an antigen recognized by a binding molecule (e.g., for a diagnostic procedure) and/or from treatment, i.e., palliation or prevention of a disease such as cancer, with a binding molecule which specifically binds a given target protein. As described in more detail herein, the binding molecule can be used in unconjugated form or can be conjugated, e.g., to a drug, prodrug, or an isotope.
[0231] By "hyperproliferative disease or disorder" is meant all neoplastic cell growth and proliferation, whether malignant or benign, including all transformed cells and tissues and all cancerous cells and tissues. Hyperproliferative diseases or disorders include, but are not limited to, precancerous lesions, abnormal cell growths, benign tumors, malignant tumors, and "cancer." In certain embodiments of the present invention, the hyperproliferative disease or disorder, e.g., the precancerous lesion, abnormal cell growth, benign tumor, malignant tumor, or "cancer" comprises cells which express, over-express, or abnormally express α6β4 integrin.
[0232] Additional examples of hyperproliferative diseases, disorders, and/or conditions include, but are not limited to neoplasms, whether benign or malignant, located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital tract. Such neoplasms, in certain embodiments, express, over-express, or abnormally express α6β4 integrin.
[0233] Other hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above. In certain embodiments of the present invention the diseases involve cells which express, over-express, or abnormally express α6β4 integrin. [0234] As used herein, the terms "tumor" or "tumor tissue" refer to an abnormal mass of tissue that results from excessive cell division, in certain cases tissue comprising cells which express, over-express, or abnormally express α6β4 integrin. A tumor or tumor tissue comprises "tumor cells" which are neoplastic cells with abnormal growth properties and no useful bodily function. Tumors, tumor tissue and tumor cells may be benign or malignant. A tumor or tumor tissue may also comprise "tumor-associated non- tumor cells", e.g., vascular cells which form blood vessels to supply the tumor or tumor tissue. Non-tumor cells may be induced to replicate and develop by tumor cells, for example, the induction of angiogenesis in a tumor or tumor tissue.
[0235] As used herein, the term "malignancy" refers to a non-benign tumor or a cancer.
As used herein, the term "cancer" connotes a type of hyperproliferative disease which includes a malignancy characterized by deregulated or uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers are noted below and include: squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. The term "cancer" includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor). Cancers conducive to treatment methods of the present invention involves cells which express, over-express, or abnormally express α6β4 integrin.
[0236] Other examples of cancers or malignancies include, but are not limited to: Acute
Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non- Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T- CeIl Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-CeIl Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
[0237] The method of the present invention may be used to treat premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where nonneoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976). Such conditions in which cells begin to express, over-express, or abnormally express α6β4 integrin, are particularly treatable by the methods of the present invention.
[0238] Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Hyperplastic disorders which can be treated by the method of the invention include, but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocyte hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate, nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.
[0239] Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplastic disorders which can be treated by the method of the invention include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
[0240] Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be treated by the method of the invention include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
[0241] Additional pre-neoplastic disorders which can be treated by the method of the invention include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.
[0242] In preferred embodiments, the method of the invention is used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.
[0243] Additional hyperproliferative diseases, disorders, and/or conditions include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythro leukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. II. cc6β4 integrin
[0244] Naturally occurring alpha6/beta4 integrin (α6β4 integrin) is a heterodimer single- pass type I membrane protein composed of one alpha and one beta subunit that are non- covalently linked together.
A. α6
[0245] Full-length α6 consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. An integrin α6 heavy chain and an integrin α6 light chain are also present in the protein. α6 is also known in the art by the names ITGA6, VLA-6 and CD49f antigen. Six isoforms of human α6 have been reported, however, two of the isoforms are dominant.
[0246] The following polypeptide sequence was reported as the human α6 polypeptide
(isoform A) sequence and has the accession number NP 000201 in Genbank.
[0247] Full-Length Human α6 (isoform A) (SEQ ID NO: 1):
1 MAAAGQLCLL YLSAGLLSRL GAAFNLDTRE DNVIRKYGDP GSLFGFSLAM
HWQLQPEDKR 61 LLLVGAPRAE ALPLQRANRT GGLYSCDITA RGPCTRIEFD NDADPTSESK
EDQWMGVTVQ 121 SQGPGGKWT CAHRYEKRQH VNTKQESRDI FGRC YVLSQN LRIEDDMDGG
DWSFCDGRLR 181 GHEKFGSCQQ GVAATFTKDF HYΓVFGAPGT YNWKGΓVRVE QKNNTFFDMN
IFEDGPYEVG
241 GETEHDESLV PVPANSYLGF SLDSGKGΓVS KDEITFVSGA PRANHSGAW
LLKRDMKSAH
301 LLPEHiFDGE GLASSFGYDV AWDLNKDGW QDΓVIGAPQY FDRDGEVGGA
VYVYMNQQGR 361 WNNVKPiRLN GTKDSMFGIA VKNIGDINQD GYPDIAVGAP YDDLGKVFΓY
HGSANGINTK
421 PTQVLKGISP YFGYSIAGNM DLDRNSYPDV AVGSLSDSVT IFRSRPVDSfI QKTITVTPNR
481 iDLRQKTACG APSGICLQVK SCFEYTANPA GYNPSISΓVG TLEAEKERRK SGLSSRVQFR
541 NQGSEPKYTQ ELTLKRQKQK VCMEETLWLQ DNIRDKLRPI PITASVEIQE
PSSRRRVNSL 601 PEVLPILNSD EPKTAHIDVH FLKEGCGDDN VCNSNLKLEY KFCTREGNQD
KFSYLPIQKG 661 VPELVLKDQK DLALEITVTN SPSNPRNPTK DGDDAHEAKL IATFPDTLTY
SAYRELRAFP 721 EKQLSCVANQ NGSQADCELG NPFKRNSNVT FYLVLSTTEV TFDTPDLDIN
LKLETTSNQD 781 NLAPITAKAK WIELLLSVS GVAKPSQVYF GGTWGEQAM KSEDEVGSLI
EYEFRVINLG 841 KPLTNLGTAT LNIQWPKEIS NGKWLLYLVK VESKGLEKVT CEPQKELNSL
NLTESHNSRK 901 KREITEKQID DNRKFSLFAE RKYQTLNCSV NVNCVNIRCP LRGLDSKASL
ILRSRLWNST 961 FLEEYSKLNY LDILMRAFID VTAAAENIRL PNAGTQVRVT VFPSKTVAQY
SGVPWWΠLV
1021 AILAGILMLA LLVFILWKCG FFKRNKKDHY DATYHKAEIH AQPSDKERLT SDA
[0248] The following polypeptide sequence was reported as the human α6 polypeptide
(isoform B) sequence and has the accession number NP_001073286 in Genbank. [0249] Full-Length Human α6 (isoform B) (SEQ ID NO:2):
1 MAAAGQLCLL YLSAGLLSRL GAAFNLDTRE DNVIRKYGDP GSLFGFSLAM
HWQLQPEDKR 61 LLLVGAPRAE ALPLQRANRT GGLYSCDITA RGPCTRIEFD NDADPTSESK
EDQWMGVTVQ 121 SQGPGGKWT C AHRYEKRQH VNTKQESRDI FGRC YVLSQN LRIEDDMDGG
DWSFCDGRLR 181 GHEKFGSCQQ GVAATFTKDF HYΓVFGAPGT YNWKGΓVRVE QKNNTFFDMN
IFEDGPYEVG
241 GETEHDESLV PVPANSYLGF SLDSGKGΓVS KDEITFVSGA PRANHSGAVV
LLKRDMKSAH
301 LLPEHiFDGE GLASSFGYDV AVVDLNKDGW QDΓVIGAPQY FDRDGEVGGA
VYVYMNQQGR 361 WNNVKPiRLN GTKDSMFGIA VKNIGDINQD GYPDIAVGAP YDDLGKVFΓY
HGSANGINTK 421 PTQVLKGISP YFGYSIAGNM DLDRNSYPDV AVGSLSDSVT IFRSRPVINI
QKTITVTPNR 481 IDLRQKT ACG APSGICLQVK SCFEYTANPA GYNPSISIVG TLEAEKERRK
SGLSSRVQFR 541 NQGSEPKYTQ ELTLKRQKQK VCMEETLWLQ DNIRDKLRPI PITASVEIQE
PSSRRRVNSL 601 PEVLPILNSD EPKTAHIDVH FLKEGCGDDN VCNSNLKLEY KFCTREGNQD
KFSYLPIQKG 661 VPELVLKDQK DIALEITVTN SPSNPRNPTK DGDDAHEAKL IATFPDTLTY
SAYRELRAFP 721 EKQLSCVANQ NGSQADCELG NPFKRNSNVT FYLVLSTTEV TFDTPDLDDSf
LKLETTSNQD
781 NLAPITAKAK VVIELLLSVS GVAKPSQVYF GGTWGEQAM KSEDEVGSLI
EYEFRVINLG 841 KPLTNLGTAT LNIQWPKEIS NGKWLLYLVK VESKGLEKVT CEPQKEINSL
NLTESHNSRK 901 KREITEKQID DNRKFSLFAE RKYQTLNCSV NVNCVNIRCP LRGLDSKASL
ILRSRLWNST 961 FLEEYSKLNY LDILMRAFID VT AAAENIRL PNAGTQVRVT VFPSKTVAQY
SGVPWWIILV 1021 AILAGILMLA LLVFILWKCG FFKRSRYDDS VPRYHAVRIR KEEREKDEK
YIDNLEKKQW 1081 ITKWNENESY S [0250] The other four isoforms have been reported with the following sequences in
FASTA:.
P23229|ITA6_HUMAN Integrin alpha-6 - Homo sapiens (Human).
MAAAGQLCLLYLSAGLLSRLGAAFNLDTREDNVIRKYGDPGSLFGFSLAMHWQLQPED KRLLLVGAPRGEALPLQRANRTGGLYSCDIT ARGPCTRIEFDND ADPTSESKEDQWMG VTVQSQGPGGKVVTCAHRYEKRQHVNTKQESRDIFGRCYVLSQNLRIEDDMDGGDWS FCDGRLRGHEK-FGSCQQGVAATFTKDFHYIVFGAPGTYNWKGIVRVEQKNNTFFDMNI
FEDGPYEVGGETEHDESLVPVPANSYLGLLFLTSVSYTDPDQFVYKTRPPREQPDTFPDV MMNSYLGFSLDSGKGIVSKDEITFVSGAPRANHSGA WLLKRDMKS AHLLPEHIFDGEG LASSFGYDVAWDLNKDGWQDIVIGAPQYFDRDGEVGGAVYVYMNQQGRWNNVKPI RLNGTKDSMFGIAVKNIGDINQDGYPDIAVGAPYDDLGKVFIYHGSANGINTKPTQVLK GISPYFGYSIAGNMDLDRNSYPDVAVGSLSDSVTIFRSRPVINIQKTITVTPNRIDLRQKT ACGAPSGICLQVKSCFEYT ANP AGYNPSISIVGTLEAEKERRKSGLSSRVQFRNQGSEPK YTQELTLKRQKQKVCMEETLWLQDNIRDKLRPIPIT ASVEIQEPSSRRRVNSLPEVLPILN SDEPKTAHID VHFLKEGCGDDNVCNSNLKLEYKFCTREGNQDKFSYLPIQKGVPELVLK DQKDIALEITVTNSPSNPRNPTKDGDDAHEAKLIATFPDTLTYSAYRELRAFPEKQLSCV ANQNGSQADCELGNPFKRNSNVTFYLVLSTTEVTFDTPDLDINLKLETTSNQDNLAPITA KAKWIELLLSVSGVAKPSQVYFGGTVVGEQAMKSEDEVGSLIEYEFRVINLGKPLTNL GTATLNIQWPKEISNGKWLLYLVKVESKGLEKVTCEPQKEINSLNLTESHNSRKKREITE KQIDDNRKFSLF AERKYQTLNCSVNVNCVNIRCPLRGLDSKASLILRSRLWNSTFLEEYS KLNYLDILMRAFIDVTAAAENIRLPNAGTQVRVTVFPSKTVAQYSGVPWWIILVAILAGI LMLALLVFILWKCGFFKRSRYDDSVPRYHAVRIRKEEREIKDEKYIDNLEKKQWITKWN RNESYS (SEQ ID NO:3) sp_vs|P23229-4|ITA6 JHUMAN Isoform Alpha-6X2A of P23229 - Homo sapiens (Human) MAAAGQLCLLYLSAGLLSRLGAAFNLDTREDNVIRKYGDPGSLFGFSLAMHWQLQPED KRLLLVGAPRGEALPLQRANRTGGLYSCDITARGPCTRIEFDND ADPTSESKEDQWMG VTVQSQGPGGKWTCAHRYEKRQHVNTKQESRDIFGRCYVLSQNLRIEDDMDGGDWS
FCDGRLRGHEKFGSCQQGv AATFTKDFHYΓVFGAPGTYNWKGLLFLTSVSYTDPDQFV YKTRPPREQPDTFPDVMMNSYLGFSLDSGKGΓVSKDEITFVSGAPRANHSGAWLLKRD MKSAHLLPEHΓFDGEGLASSFGYDVAWDLNKDGWQDΓVIGAPQYFDRDGEVGGAVY VYMNQQGRWNNVKPIRLNGTKDSMFGIA VKNIGDINQDGYPDIAVGAP YDDLGKVFIY HGSANGiNTKPTQVLKGisPYFGYSiAGNMDLDRNSYPDv AVGSLSDSVTIFRSRPVΓNIQ KTiTVTPNRiDLRQKTACGAPSGiCLQ VKSCFEYT ANP AGYNPSISΓVGTLEAEKERRKSG
LSSRVQFRNQGSEPKYTQELTLKRQKQKVCMEETLWLQDNIRDKLRPIPITASVEIQEPS SRRRVNSLPEVLPILNSDEPKTAHID VHFLKEGCGDDNVCNSNLKLEYKFCTREGNQDK
FSYLPIQKGVPELVLKDQKDLALEITVTNSPSNPRNPTKDGDDAHEAKLIATFPDTLTYSA YRELRAFPEKQLSCV ANQNGSQADCELGNPFKRNSNVTFYLVLSTTEVTFDTPDLDINL KLETTSNQDNLAPITAKAKVVIELLLSVSGVAKPSQVYFGGTVVGEQAMKSEDEVGSLI EYEFRVΓNLGKPLTNLGTATLNIQWPKEISNGKWLLYLVKVESKGLEKVTCEPQKEINSL NLTESHNSRKKREITEKQIDDNRKFSLF AERKYQTLNCSVNVNCVNIRCPLRGLDSKASL ILRSRLWNSTFLEEYSKLNYLDILMRAFIDVTAAAENIRLPNAGTQVRVTVFPSKTVAQY SGVP WWΠLV AILAGILMLALLVFILWKCGFFKRNKKDHYDATYHKAEIHAQPSDKERL
TSDA (SEQ ID NO:4)
>sp_vs|P23229-5|ITA6_HUMAN Isoform Alpha-6X2B of P23229 - Homo sapiens (Human) MAAAGQLCLLYLSAGLLSRLGAAFNLDTREDNVIRKYGDPGSLFGFSLAMHWQLQPED KRLLLVGAPRGEALPLQRANRTGGLYSCDIT ARGPCTRIEFDND ADPTSESKEDQWMG VTVQSQGPGGKWTCAHRYEKRQHVNTKQESRDIFGRCYVLSQNLRIEDDMDGGDWS FCDGRLRGHEKFGSCQQGV AATFTKDFHYIVFGAPGTYNWKGLLFLTSVSYTDPDQFV YKTRPPREQPDTFPDVMMNSYLGFSLDSGKGIVSKDEITFVSGAPRANHSGAWLLKRD MKS AHLLPEHIFDGEGLASSFGYDV AVVDLNKDGWQDIVIGAPQYFDRDGEVGGAVY
VYMNQQGRWNNVK-PIRLNGTKIDSMFGIAVKNIGDINQDGYPDIAVGAPYDDLGKVFIY
HGSANGINTKPTQVLKGISPYFGYSIAGNMDLDRNSYPDVAVGSLSDSVTIFRSRPVINIQ
KTiTVTPNRiDLRQKTACGAPSGiCLQVKSCFEYTANP AGYNPSISΓVGTLEAEKERRKSG
LSSRVQFRNQGSEPKYTQELTLKRQKQKVCMEETLWLQDNIRDKLRPIPIT ASVEIQEPS
SRRRVNSLPEVLPILNSDEPKTAHED VHFLKEGCGDDNVCNSNLKLEYKFCTREGNQDK
FSYLPIQKGVPELVLKDQKDIALEITVTNSPSNPRNPTKDGDDAHEAKLIATFPDTLTYSA
YRELRAFPEKQLSCVANQNGSQADCELGNPFKRNSNVTFYLVLSTTEVTFDTPDLDINL
KLETTSNQDNLAPITAKAKWIELLLSVSGVAKPSQVYFGGTWGEQAMKSEDEVGSLI
EYEFRVINLGKPLTNLGTATLMQWPKEISNGKWLLYLVKVESKGLEKVTCEPQKEINSL
NLTESHNSRKKREITEKQIDDNRKFSLF AERKYQTLNCSVNVNCVNIRCPLRGLDSKASL
ILRSRLWNSTFLEEYSKLNYLDILMRAFIDVTAAAENIRLPNAGTQVRVTVFPSKTVAQY
SGVPWWΠLVAΓLAGILMLALLVFILWKCGFFKRSRYDDSVPRYHAVRIRKEEREIKDEK
YIDNLEKKQWITKWNRNESYS (SEQ ID NO:5) sp_vs|P23229-6|ITA6_HUMAN Isoform Alpha-6X1X2A of P23229 - Homo sapiens (Human)
MAAAGQLCLLYLSAGLLSRLGAAFNLDTREDNVIRKYGDPGSLFGFSLAMHWQLQPED
KRLLLVGAPRGEALPLQRANRTGGLYSCDIT ARGPCTRIEFDND ADPTSESKEDQWMG
VTVQSQGPGGKVVTCAHRYEKRQHVNTKQESRDIFGRCYVLSQNLRIEDDMDGGDWS
FCDGRLRGHEKFGSCQQGV AATFTKDFHYIVFGAPGTYNWKGIVRVEQKNNTFFDMNI
FEDGPYEVGGETEHDESLVPVPANSYLGLLFLTSVSYTDPDQFVYKTRPPREQPDTFPDV
MMNSYLGFSLDSGKGIVSKDEITFVSGAPRANHSGAWLLKRDMKSAHLLPEHIFDGEG
LASSFGYDVAWDLNKDGWQDΓVIGAPQYFDRDGEVGGAVYVYMNQQGRWNNVKPI
RLNGTKDSMFGIAVKNIGDINQDGYPDIAVGAPYDDLGKVFIYHGSANGINTKPTQVLK
GISPYFGYSIAGNMDLDRNSYPDVAVGSLSDSVTIFRSRPVINIQKTITVTPNRIDLRQKT
ACGAPSGICLQ VKSCFEYTANP AGYNPSISIVGTLEAEKERRKSGLSSRVQFRNQGSEPK
YTQELTLKRQKQKVCMEETLWLQDNIRDKLRPIPIT ASVEIQEPSSRRRVNSLPEVLPILN
SDEPKTAHID VHFLKEGCGDDNVCNSNLKLEYKFCTREGNQDKFSYLPIQKGVPELVLK
DQKDIALEITVTNSPSNPRNPTKDGDDAHEAKLLATFPDTLTYSAYRELRAFPEKQLSCV
ANQNGSQADCELGNPFKRNSNVTFYLVLSTTEVTFDTPDLDINLKLETTSNQDNLAPITA
KAKWIELLLSVSGVAKPSQVYFGGTWGEQAMKSEDEVGSLIEYEFRVINLGKPLTNL
GTATLNIQWPKEISNGKWLLYLVKVESKGLEKVTCEPQKEINSLNLTESHNSRKKREITE
KQIDDNRKFSLF AERKYQTLNCSVNVNCVNIRCPLRGLDSKASLILRSRLWNSTFLEEYS KLNYLDILMRAFIDVTAAAENIRLPNAGTQVRVTVFPSKTV AQYSGVP WWnLV AILAGI LMLALLVFILWKCGFFKRNKKDHYDATYHKAEIHAQPSDKERLTSDA (SEQ ED NO:6)
[0251] The mouse α6 polypeptide has been reported with the following sequences in
FASTA: Q61739|ITA6_MOUSE Integrin alpha-6 - Mus musculus (Mouse).
MAVAGQLCLLYLSAGLLARLGTAFNLDTREDNVΓRKSGDPGSLFGFSLAMHWQLQPED KRLLLVGAPRAE ALPLQRANRTGGLYSCDITSRGPCTRTEFDND ADPMSESKEDQWMG VTVQSQGPGGKVVTCAHRYEKRQHVNTKQESRDΓFGRCYVLSQNLRIEDDMDGGDWS
FCDGRLRGHEKFGSCQQGV AATFTKDFHYIVFGAPGTYNWKGΓVRVEQKNNTFFDMNI
FEDGPYEVGGETDHDESLVP vp ANSYLGFSLDSGKGΓVSKDDITFVSGAPRANHSGAW LLKRDMKS AHLLPEYIFDGEGLASSFGYDV AWDLNADGWQDIVIGAPQYFDRDGEVG GAVYVYΓNQQGKWSNVKPIRLNGTKDSMFGISVKNIGDINQDGYPDIAVGAPYDDLGK VFΓYHGSPTGΠTKPTQVLEGTSPYFGYSIAGNMDLDRNSYPDLAVGSLSDSVTIFRSRPVI NILKTITVTPNRIDLRQKSMCGSPSGICLKVKACFEYT AKPSGYNPPISILGILEAEKERRK SGLSSRVQFRNQGSEPKYTQELTLNRQKQRACMEETLWLQENIRDKLRPIPITASVEIQE PTSRRR VNSLPEVLPILNSNE AKTVQTD VHFLKEGCGDDNVCNSNLKLEYKFGTREGNQ DKFSYLPIQKGIPELVLKDQKDIALEITVTNSPSDPRNPRKDGDDAHEAKLIATFPDTLTY SAYRELRAFPEKQLSCVANQNGSQADCELGNPFKRNSSVTFYLILSTTEVTFDTTDLDIN LKLETTSNQDKLAPITAKAKWIELLLSLSGVAKPSQVYFGGTVVGEQAMKSEDEVGSL ffiYEFRVINLGKPLKNLGTATLNIQWPKEISNGKWLLYLMKVESKGLEQIVCEPHNEINY LKLKESHNSRKKRELPEKQIDDSRKFSLFPERKYQTLNCSVNVRCVNπtCPLRGLDTKAS LVLCSRLWNSTFLEEYSKLNYLDILVRASIDVTAAAQNIKLPHAGTQVRVTVFPSKTVA
QYSGV AWWΠLLAVLAGILMLALLVFLLWKCGFFKRSRYDDSIPRYHAVRIRKEEREIK
DEKHMDNLEKKQWITKWNENESYS (SEQ ID NO:7)
[0252] The chick α6 polypeptide has been reported with the following sequences in
FASTA:
P26007|ITA6_CHICK Integrin alpha-6 - Gallus gallus (Chicken).
MAAALLLYLPLLPGLAGAFNLDAENVIGRRGEPGSLFGFSLAMHRQLQPQEKRLLLVG APREKAFPSQQANRTGGLYSCDITSSDTRCTRVVFDEDTDPKMESKEDQWMGVTVQSQ GPGGNVVTCAHRYEKRQYVNTVQETRDIIGRCYVLSQDLTIKDDMDNGVWSFCDGRL
RGHEKFGSCQQGv AATFTRD YHYΓVFGAPGTYNWKGWRAEQKNQTFYDLGIFDDGP
YEVGDESRQDKNLVPVPANSYLGFSLDSGKGΓVSQDEMTFVSGAPRANHSGAVVLLKK
EKNQRALSLEHMFEGEGLASSFGYDVAWDLNSDGWQDIVVGAPQYFDRSGDIGGAV
YIYESFQRGKWEGIKPIRLNGTADSMFGLAVENVGDINQDGYPDIAVGAP YDGFGKVYIY
HGSKNGINTEPAQILDGEKTGTNFFGYSIAGNMDLDKNSYPDIAVGSLSDSVSVFRSRPV
ISITKSITVQPDKLDLKKKNPEDPSEIWMDVKACFQYTANPR]SN^NPRIKINYTFEAENER
RQLGLPSRVRFKD YLSDQFT ASTTLIGQNSKRCVTAKLVLQEKIKDKLRPIPIAVSVNIAG LESGSSSTRKERALPDLIPILNSNESETKITKVEFLKEGCGEDNECHSNLKLQYRFCTREG NEDRFTYLPIENGIPVLVLKDQKDIALEITVTNNPSDARNPQKDGEDAYEAKLIATFPDS LTYSAFREMRGYPEKQLTCGANQNGSQAECELGNPFKRNSNVTFYLILSTTKVNVDTT DLDINLKLETTSTQVNSTAITASAKWLELLLSLTGVAKPSQVYFGGNIVGESAMKSED NIGNLIE YEFRVTNLGRPLKTFGTASLDIQWPKEISNGKWLLYLMKIESKGLEKVSCQPQ NEINVLHV AESHNSRRKREIAEKQLTDSKTFSLFSERKYKTLDCKVNAQCVDIRCPLKGF DSKASILLRSRLWNSTFLEEFSKMNYLDILVRASISVPAAAKNVKLTNEAAQVRVTVFP AKPV ALYTGVP WWIIAV AIF AGVLMLALLVFLLWKCGFFKRSKKDHYDATYHKAEIHA QPSDKERLTSDA (SEQ ID NO:8)
[0253] The α6 polypeptide domain designations used herein are defined as follows:
Table 2. Example α6 polypeptide domains
Figure imgf000056_0001
[0254] As one of skill in the art will appreciate, the beginning and ending residues of the domains listed below may vary depending upon the computer modeling program used or the method used for determining the domain.
B. β4
[0255] Full-length β4 consists of a signal sequence, an extracellular domain, a transmembrane domain and a cytoplasmic domain. A von Willebrand factor type A (VWFA) domain exists in the extracellular domain and four fibronectin type-Ill domains and one Calx-beta domain exist in the cytoplasmic domain. β4 is also known in the art by the names ITGB4, GP150 and CD104 antigen. Five isoforms of human β4 exist, however 3 isoforms are dominant.
[0256] The following polypeptide sequence was reported as the human β4 polypeptide
(isoform 4 A or isoform 3) sequence and has the accession number AAC51632 in Genbank.
[0257] Full-Length Human β4 (isoform 4A or isoform 3) (SEQ ID NO:9):
1 MAGPRPSPWA RLLLAALISV SLSGTLANRC KKAPVKSCTE CVRVDKDCAY CTDEMFRDRR
6i CNTQAELLAA GCQRESΓVVM ESSFQITEET QIDTTLRRSQ MSPQGLRVRL
RPGEERHFEL 121 EVFEPLESPV DLYILMDFSN SMSDDLDNLK KMGQNLARVL SQLTSDYTIG
FGKFVDKVSV 181 PQTDMRPEKL KEPWPNSDPP FSFKNVISLT EDVDEFRNKL QGERISGNLD
APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
DTTGTYTQYR
301 TQDYPSVPTL VRLLAKHNII PIFAVTNYSY SYYEKLHTYF PVSSLGVLQE
DSSNΓVELLE
361 EAFNRIRSNL DIRALDSPRG LRTEVTSKMF QKTRTGSFHI RRGEVGIYQV
QLRALEHVDG
421 THVCQLPEDQ KGNIHLKPSF SDGLKMDAGI ICDVCTCELQ KEVRSARCSF
NGDFVCGQCV 481 CSEGWSGQTC NCSTGSLSDI QPCLREGEDK PCSGRGECQC GHCVCYGEGR
YEGQFCEYDN 541 FQCPRTSGFL CNDRGRCSMG QCVCEPGWTG PSCDCPLSNA TCIDSNGGIC
NGRGHCECGR 601 CHCHQQSLYT DTICEINYSA IHPGLCEDLR SCVQCQAWGT GEKKGRTCEE
CNFKVKMVDE 661 LKRAEEVWR CSFRDEDDDC T YS YTMEGDG APGPNSTVLV HKKKDCPPGS
FWWLIPLLLL 721 LLPLLALLLL LCWKYCACCK ACLALLPCCN RGHMVGFKED HYMLRENLMA
SDHLDTPMLR 781 SGNLKGRDVV RWKVTNNMQR PGFATHAASI NPTELVPYGL SLRLARLCTE
NLLKPDTREC 841 AQLRQEVEEN LNEVYRQISG VHKLQQTKFR QQPNAGKKQD HTIVDTVLMA
PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
RPEDDDEKQL
961 LVEAIDVPAG TATLGRRLVN ITIIKEQARD VVSFEQPEFS VSRGDQVARI
PVIRRVLDGG 1021 KSQVSYRTQD GTAQGNRDYI PVEGELLFQP GEAWKELQVK LLELQEVDSL
LRGRQVRRFH 1081 VQLSNPKFGA HLGQPHSTTI IIRDPDELDR SFTSQMLSSQ PPPHGDLGAP
QNPNAKAAGS 1141 RKIHFNWLPP SGKPMGYRVK YWIQGDSESE AHLLDSKVPS VELTNLYPYC
DYEMKVCAYG 1201 AQGEGPYSSL VSCRTHQEVP SEPGRLAFNV VSSTVTQLSW AEPAETNGEI
TAYEVCYGLV 1261 NDDNRPIGPM KKVLVDNPKN RMLLIENLRE SQPYRYTVKA RNGAGWGPER
EAIINLATQP
1321 KRPMSIPIIP DIPIVD AQSG ED YDSFLMYS DD VLRSPSGS QRPS VSDDTE HLVNGRMDFA
1381 FPGSTNSLHR MTTTSAAAYG THLSPHVPHR VLSTSSTLTR DYNSLTRSEH
SHSTTLPRDY 1441 STLTSVSSHD SRLTAGVPDT PTRLVFSALG PTSLRVSWQE PRCERPLQGY
SVEYQLLNGG
1501 ELHRLNIPNP AQTSWVEDL LPNHSYVFRV RAQSQEGWGR EREGVITIES
QVHPQSPLCP 1561 LPGSAFTLST PSAPGPLVFT ALSPDSLQLS WERPRRPNGD IVGYLVTCEM
AQGGGPATAF
1621 RVDGDSPESR LTVPGLSENV PYKFKVQART TEGFGPEREG IITIESQDGG
PFPQLGSRAG
1681 LFQHPLQSEY SSISTTHTSA TEPFLVDGPT LGAQHLEAGG SLTRHVTQEF
VSRTLTTSGT 1741 LSTHMDQQFF QT [0258] The following polypeptide sequence was reported as the human β4 polypeptide
(isoform 4B or isoform 2) sequence and has the accession number AAC51634 in Genbank.
[0259] Full-Length Human β4 (isoform 4B or isoform 2) (SEQ ID NO: 10):
1 MAGPRPSPWA RLLLAALISV SLSGTLANRC KKAPVKSCTE CVRVDKDCAY
CTDEMFRDRR 61 CNTQAELLAA GCQRESIWM ESSFQITEET QIDTTLRRSQ MSPQGLRVRL
RPGEERHFEL 121 EVFEPLESPV DLYILMDFSN SMSDDLDNLK KMGQNLARVL SQLTSDYTIG
FGKFVDKVSV 181 PQTDMRPEKL KEPWPNSDPP FSFKNVISLT EDVDEFRNKL QGERISGNLD
APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
DTTGTYTQYR
301 TQDYPSVPTL VRLLAKHNII PIFAVTNYSY SYYEKLHTYF PVSSLGVLQE
DSSNΓVELLE
361 EAFNRiRSNL DIRALDSPRG LRTEVTSKMF QKTRTGSFHI RRGEVGΓYQV QLRALEHVDG
421 THVCQLPEDQ KGNIHLKPSF SDGLKMDAGI ICDVCTCELQ KEVRSARCSF
NGDFVCGQCV 481 CSEGWSGQTC NCSTGSLSDI QPCLREGEDK PCSGRGECQC GHCVCYGEGR
YEGQFCEYDN 541 FQCPRTSGFL CNDRGRCSMG QCVCEPGWTG PSCDCPLSNA TCIDSNGGIC
NGRGHCECGR 601 CHCHQQSLYT DTICEINYSA mPGLCEDLR SC VQCQAWGT GEKKGRTCEE
CNFKVKMVDE 661 LKRAEEWVR CSFRDEDDDC TYS YTMEGDG APGPNSTVLV HKKKDCPPGS
FWWLIPLLLL 721 LLPLLALLLL LCWKYCACCK ACLALLPCCN RGHMVGFKED HYMLRENLMA
SDHLDTPMLR 781 SGNLKGRDW RWKVTNNMQR PGF ATHAASI NPTELVPYGL SLRLARLCTE
NLLKPDTREC
841 AQLRQEVEEN LNEVYRQISG VHKLQQTKFR QQPNAGKKQD HTΓVDTVLMA
PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
RPEDDDEKQL 961 LVEAiDVPAG TATLGRRLVN ITIΠCEQARD WSFEQPEFS VSRGDQVARI
PVBRRVLDGG 1021 KSQVSYRTQD GTAQGNRDYI PVEGELLFQP GEAWKELQVK LLELQEVDSL
LRGRQVRRFH
1081 VQLSNPKFGA HLGQPHSTTI IIRDPDELDR SFTSQMLSSQ PPPHGDLGAP
QNPNAKAAGS
1141 RKIHFNWLPP SGKPMGYRVK YWIQGDSESE AHLLDSKVPS VELTNLYPYC
DYEMKVCAYG 1201 AQGEGPYSSL VSCRTHQEVP SEPGRLAFNV VSSTVTQLSW AEPAETNGEI
TAYEVCYGLV 1261 NDDNRPIGPM KKVLVDNPKN RMLLIENLRE SQPYRYTVKA RNGAGWGPER
EAIINLATQP 1321 KRPMSIPIIP DIPIVDAQSG EDYDSFLMYS DDVLRSPSGS QRPSVSDDTE
HLVNGRMDFA 1381 FPGSTNSLHR MTTTSAAAYG THLSPHVPHR VLSTSSTLTR DYNSLTRSEH
SHSTTLPRDY
1441 STLTSvssHG LPPIWEHGRS RLPLSWALGS RSRAQMKGFP PSRGPRDSII
LAGRPAAPSW 1501 GPDSRLTAGV PDTPTRLVFS ALGPTSLRVS WQEPRCERPL QGYSVEYQLL
NGGELHRLNI 1561 PNPAQTSVW EDLLPNHSYV FRVRAQSQEG WGREREGVIT IESQVHPQSP
LCPLPGSAFT
1621 LSTPSAPGPL VFTALSPDSL QLSWERPRRP NGDIVGYLVT CEMAQGGGPA TAFRVDGDSP
1681 ESRLTVPGLS ENVPYKFKVQ ARTTEGFGPE REGIITIESQ DGGPFPQLGS
RAGLFQHPLQ 1741 SEYSSISTTH TSATEPFLVD GPTLGAQHLE AGGSLTRHVT QEFVSRTLTT
SGTLSTHMDQ
1801 QFFQT
[0260] The following polypeptide sequence was reported as the human β4 polypeptide
(isoform 4C or isoform 1) sequence and has the accession number AAC51633 in Genbank.
[0261] Full-Length Human β4 (isoform 4C or isoform 1) (SEQ ID NO: 11):
1 MAGPRPSPWA RLLLAALISV SLSGTLANRC KKAPVKSCTE CVRVDKDCAY
CTDEMFRDRR 61 CNTQAELLAA GCQRESIWM ESSFQITEET QIDTTLRRSQ MSPQGLRVRL
RPGEERHFEL 121 EVFEPLESPV DLYILMDFSN SMSDDLDNLK KMGQNLARVL SQLTSDYTIG
FGKFVDKVSV 181 PQTDMRPEKL KEPWPNSDPP FSFKNVISLT EDVDEFRNKL QGERISGNLD
APEGGFDAIL 241 QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI MSRNDERCHL
DTTGTYTQYR
301 TQDYPSVPTL VRLLAKHNII PIFAVTNYSY SYYEKLHTYF PVSSLGVLQE
DSSNΓVELLE
361 EAFNRIRSNL DIRALDSPRG LRTEVTSKMF QKTRTGSFHI RRGEVGIYQV
QLRALEHVDG
421 THVCQLPEDQ KGNIHLKPSF SDGLKMDAGI ICDVCTCELQ KEVRSARCSF
NGDFVCGQCV 481 CSEGWSGQTC NCSTGSLSDI QPCLREGEDK PCSGRGECQC GHCVCYGEGR
YEGQFCEYDN 541 FQCPRTSGFL CNDRGRCSMG QCVCEPGWTG PSCDCPLSNA TCIDSNGGIC
NGRGHCECGR 601 CHCHQQSLYT DTICEINYSA IHPGLCEDLR SCVQCQAWGT GEKKGRTCEE
CNFKVKMVDE 661 LKRAEEVWR CSFRDEDDDC TYS YTMEGDG APGPNST VLV HKKKDCPPGS
FWWLIPLLLL 721 LLPLLALLLL LCWKYCACCK ACLALLPCCN RGHMVGFKED HYMLRENLMA
SDHLDTPMLR 781 SGNLKGRDVV RWKVTNNMQR PGFATHAASI NPTELVPYGL SLRLARLCTE
NLLKPDTREC 841 AQLRQEVEEN LNEVYRQISG VHKLQQTKFR QQPNAGKKQD HTIVDTVLMA
PRSAKPALLK 901 LTEKQVEQRA FHDLKVAPGY YTLTADQDAR GMVEFQEGVE LVDVRVPLFI
RPEDDDEKQL 961 LVEAiDVPAG TATLGRRLVN ΓTIIKEQARD WSFEQPEFS VSRGDQVARI
PVIRRVLDGG 1021 KSQVSYRTQD GTAQGNRDYI PVEGELLFQP GEAWKELQVK LLELQEVDSL
LRGRQVRRFH
1081 VQLSNPKFGA HLGQPHSTTI IIRDPDELDR SFTSQMLSSQ PPPHGDLGAP
QNPNAKAAGS 1141 RKIHFNWLPP SGKPMGYRVK YWIQGDSESE AHLLDSKVPS VELTNLYPYC
DYEMKVCAYG 1201 AQGEGPYSSL VSCRTHQEVP SEPGRLAFNV VSSTVTQLSW AEPAETNGEI
TAYEVCYGLV 1261 NDDNRPIGPM KKVLVDNPKN RMLLIENLRE SQPYRYTVKA RNGAGWGPER
EAIINLATQP
1321 KRPMSIPIIP DIPIVDAQSG EDYDSFLMYS DDVLRSPSGS QRPSVSDDTG
CGWKFEPLLG 1381 EELDLRRVTW RLPPELIPRL SASSGRSSDA EAPHGPPDDG GAGGKGGSLP
RSATPGPPGE 1441 HLVNGRMDFA FPGSTNSLHR MTTTSAAAYG THLSPHVPHR VLSTSSTLTR
DYNSLTRSEH 1501 SHSTTLPRDY STLTSVSSHD SRLTAGVPDT PTRLVFSALG PTSLRVSWQE
PRCERPLQGY 1561 SVEYQLLNGG ELHRLNIPNP AQTSVWEDL LPNHSYVFRV RAQSQEGWGR
EREGVITIES 1621 QVHPQSPLCP LPGSAFTLST PSAPGPLVFT ALSPDSLQLS WERPRRPNGD
IVGYLVTCEM 1681 AQGGGPATAF RVDGDSPESR LTVPGLSENV PYKFKVQART TEGFGPEREG
IΓΠESQDGG
1741 PFPQLGSRAG LFQHPLQSEY SSISTTHTSA TEPFLVDGPT LGAQHLEAGG
SLTRHVTQEF 1801 VSRTLTTSGT LSTHMDQQFF QT
[0262] The other two isoforms have been reported with the following sequences in
FASTA: sp_vs|P16144-4|ITB4_HUMAN Isoform Beta-4D of Pl 6144 - Homo sapiens (Human)
MAGPRPSPWARLLLAALISVSLSGTLANRCKKAPVKSCTECVRVDKDCAYCTDEMFRD
RRCNTQAELLAAGCQRESΓWMESSFQITEETQIDTTLRRSQMSPQGLRVRLRPGEERHF
ELEVFEPLESPVDLYILMDFSNSMSDDLDNLKKMGQNLARVLSQLTSDYTIGFGKFVDK
VSVPQTDMRPEKLKEPWPNSDPPFSFKNVISLTEDVDEFRNKLQGERISGNLDAPEGGFD
AILQTAVCTRDIGWRPDSTHLLVFSTES AFHYE ADGANVLAGIMSRNDERCHLDTTGTY
TQYRTQDYPSVPTLVRLLAKHNIIPIF AVTNYSYSYYEKLHTYFPVSSLGVLQEDSSNIVE
LLEEAFNRIRSNLDIRALDSPRGLRTEVTSKMFQKTRTGSFHIRRGEVGΓYQVQLRALEH
VDGTHVCQLPEDQKGNΓHLKPSFSDGLKMDAGIICDVCTCELQKE VRS ARCSFNGDFVC GQCVCSEGWSGQTCNCSTGSLSDIQPCLREGEDKPCSGRGECQCGHCVCYGEGRYEGQ FCEYDNFQCPRTSGFLCNDRGRCSMGQCVCEPGWTGPSCDCPLSNATCIDSNGGICNGR GHCECGRCHCHQQSLYTDTICEINYSAIHPGLCEDLRSCVQCQA WGTGEKKGRTCEEC NFKVKMVDELKRAEEVVVRCSFRDEDDDCTYSYTMEGDGAPGPNSTVLVHKKKDCPP GSFWWLIPLLLLLLPLLALLLLLCWKYCACCKACLALLPCCNRGHMVGFKEDHYMLRE NLMASDHLDTPMLRSGNLKGRDVVRWKVTNNMQRPGFATHAASINPTELVPYGLSLR LARLCTENLLKPDTRECAQLRQEVEENLNE VYRQISGVHKLQQTKFRQQPNAGKKQDH TIVDTVLMAPRSAKPALLKLTEKQVEQRAFHDLKVAPGYYTLTADQDARGMVEFQEG
VELVD VRVPLFIRPEDDDEKQLLVEAID vp AGTATLGRRLVNITΠKEQARDWSFEQPEF
SVSRGDQVARIPVIRRVLDGGKSQVSYRTQDGTAQGNRDYIPVEGELLFQPGEAWKEL QVKLLELQEVDSLLRGRQVRRFHVQLSNPKFGAHLGQPHSTTHIRDPDELDRSFTSQML SSQPPPHGDLGAPQNPNAKAAGSRKIHFNWLPPSGKPMGYRVKYWIQGDSESEAHLLD SKVPSVELTNLYPYCDYEMKVCAYGAQGEGPYSSLVSCRTHQEVPSEPGRLAFNWSS TVTQLSWAEP AETNGEITAYEVCYGLVNDDNRPIGPMKKVLVDNPKNRMLLIENLRES
QPYRYTVKARNGAGWGPERE AIINLATQPKRPMSIPNPDIPIVDAQSGED YDSFLMYSD
DVLRSPSGSQRPSVSDDTEHLVNGRMDFAFPGSTNSLHRMTTTSAAAYGTHLSPHVPHR
VLSTSSTLTRDYNSLTRSEHSHSTTLPRDYSTLTSVSSHDSRLTAGVPDTPTRLVFSALGP
TSLRVSWQEPRCERPLQGYSVEYQLLNGGELHRLNIPNPAQTSWVEDLLPNHSYVFRV
RAQSQEGWGREREGVITIESQVHPQSPLCPLPGSAFTLSTPSAPGPLVFTALSPDSLQLSW
ERPRRPNGDΓVGYLVTWPATAFRVDGDSPESRLTVPGLSENVPYKFKVQARTTEGFGPE
REGIITIESQDGGPFPQLGSRAGLFQHPLQSEYSSITTTHTSATEPFLVDGPTLGAQHLEAG
GSLTRHVTQEFVSRTLTTSGTLSTHMDQQFFQT (SEQ ID NO: 12)
>sp_vs|P 16144-5 |ITB4_HUMAN Isoform Beta-4E of P16144 - Homo sapiens (Human)
MAGPRPSPWARLLLAALISVSLSGTLANRCKKAPVKSCTECVRVDKDCAYCTDEMFRD
RRCNTQAELLAAGCQRESIWMESSFQITEETQIDTTLRRSQMSPQGLRVRLRPGEERHF
ELEVFEPLESPVDLYILMDFSNSMSDDLDNLKKMGQNLARVLSQLTSDYTIGFGKFVDK
VSVPQTDMRPEKLKEPWPNSDPPFSFKNVISLTEDVDEFRNKLQGERISGNLDAPEGGFD
AILQTAVCTRDIGWRPDSTHLLVFSTESAFHYE ADGANVLAGIMSRNDERCHLDTTGTY
TQYRTQDYPSVPTLVRLLAKHNIIPIFAVTNYSYSYYEKLHTYFPVSSLGVLQEDSSNΓVE
LLEEAFNRIRSNLDIRALDSPRGLRTEVTSKMFQKTRTGSFHIRRGEVGIYQVQLRALEH
VDGTHVCQLPEDQKGNIHLKPSFSDGLKMDAGIICDVCTCELQKEVRSARCSFNGDFVC
GQCVCSEGWSGQTCNCSTGSLSDIQPCLREGEDKPCSGRGECQCGHCVCYGEGRYEGQ
FCEYDNFQCPRTSGFLCNDRGRCSMGQCVCEPGWTGPSCDCPLSNATCIDSNGGICNGR
GHCECGRCHCHQQSLYTDTICEINYSAIHPGLCEDLRSCVQCQAWGTGEKKGRTCEEC
NFKVKMVDELKRAEEVVVRCSFRDEDDDCTYSYTMEGDGAPGPNSTVLVHKKKDCPP
GSFWWLIPLLLLLLPLLALLLLLCWKYCACCKACLALLPCCNRGHMVGFKEDHYMLRE
NLMASDHLDTPMLRSGNLKGRDVVRWKVTNNMQRPGFATHAASINPTELVPYGLSLR
LARLCTENLLKPDTRECAQLRQEVEENVRTQELGLAGDV AERGLQ ADLRCTQAP ADQ
VP AAAQCREKARPHHCGHSADGAPLGQAGP AEAYREAGGTEGLPRPQGGPRLLHPHC
RPGRPGHGGVPGGRGAGGRTGAPLYPA (SEQ ID NO: 13)
[0263] The rat β4 polypeptide has been reported with the following sequences in
FASTA:
Q64632|ITB4_RAT Integrin beta-4 - Rattus norvegicus (Rat).
MAGLCSSPWVKLLLAWLSAGLPGNMANRCKKAQVKSCTECIRVDKSCAYCTDELFK
ERRCNTQAD VLAAGCRGESVLVMESSLEITENIQIDTSLHRSQVSPQGLQ VRLRPGEERN
FVFKVFEPLESPVDLYILMDFSNSMSDDLDNLKQMGQNLAKILRQLTSDYTIGFGKFVD
KVSVPQTDMRPEKLKEPWPNSDPPFSFKNVISLTENVEEFWDKLQGERISGNLDAPEGG
FD AILQTAVCTRDIGWRADSTHLLVFSTES AFHYE ADGANVLAGIMNRNDEKCHLDAT
GAYTQYKTQDYPSVPTLVRLLAKHNIIPIFAVTNYSYSYYEKLHKYFPVSSLGVLQEDSS
NIVELLEEAFYRIRSNLDIRALDSPRGLRTEVTSDTLQKTETGSFHIKRGEVGTYNVHLR
AVEDIDGTHVCQLAKEDQRGNIHLKPSFSDGLRMDASVICDMCACELQKEVQSARCHY
RGDFMCGHCVCNEGWSGKTCNCSTGSLSDTQPCLREGEDKPCSGHGECQCGRCVCYG
EGRYEGHFCEYDNFQCPRTSGFLCNDRGRCSMGECVCEPGWTGRSCDCPLSNATCIDS NGGICNGLGFCECGRCHCNQRSSLYTDTTCEINYSAIRLGLCEDLRSCVQCQAWGTGEK
KGRTCEECNFKVKMVDELKKAEEWEYCSFRDEDDDCTYSYTVEGDGSPGPNSTVLV
HKKKDCLPAPSWWLIPLLIFLLLLLVLLLLLCWKYCACCKACLGLLPCCNQGHMVGFK
EDHYMLRENLMASDHLDTPMLRSGNLKGRDTVRWKITNNVQRPGFATHAASISPTELV
PYGLSLRLGRLCTENLMKPGTRECDQLRQEVEENLNEVYRQVNGVHKLQQTKFRQQP
NAGKKQDHTIVDTVLLAPRSAKQSLLKLTEKQVEQGSFHELKVAPGYYTLTAEQDARG
MVEFQEGVELVD VRVPLFIRPEDDDEKQLLVEAID VPVGTATLGRRLVNITΠKEQASGI
VSFEQPEYSVSRGDQVARIPVIRHILDNGKSQVSYSTQDNTAHGHRDYVPVEGELLFYP
GETWKELQ VKLLELQEVDSLLRGRQVRRFQVQLSNPKFGARLGQPNTATVΠGEQDETD
RSLINEISASPPLPRGDLGAPQNPNAKAAGSRKIHFNWLPPPGKPMGYRVKYWVQGDSE
SEAHLLDSKVPSVELTNLYPYCD YEMKVCAYGAHGEGPYSSLVSCRTHQEVPSEPGRL
AFNVVSSTVTQLSWAEPAETNGEITAYEVCYGLVNEDNRPIGPMKKVLVDNPKNRMLL
IENLRESQPYRYTVKARNGAGWGPEREAIINLATQPKRPMSIPΠPDIPIVDAQGGEDYEN
FLMYSDDVLRSPASSQRPSVSDDTEHLVNGRMDFAYPGSANSLHRMTAANVAYGTHL
SPHQTHRMLSTSSTLTRDYHSLTRTDHSQSGTLPRDYSTLTSLSSQGLPPIWEDGRSRLPL
SWTLGSWSRAQMKGVPASRGSPDSIILAGQSAAPSWGTDSRGAMGVPDTPTRLVFSAL
GPTSLKVSWQEPQCDRALLGYSVEYQLLNGGEMHRLNIPNPGQTSVWEDLLPNHSYV
FRVRAQSQEGWGREREGVITIESQVHPQSPLCPLPGSAFTLSTPSAPGPLVFT ALSPDSLQ
LSWERPRRPNGDILGYLVTCEMAQGGGP ARTFRVDGDNPESRLTVPGLSENVP YKFKV QARTTEGFGPEREGIITIESQDGGPFPQLGSHSGLFQNPLQSEYSTVTSTHSTTTTEPFLID GLTLGTQRLEAGGSLTRHVTQEFVSRTLTTSGSLSTHMDQQFFQT (SEQ ID NO: 14)
[0264] The mouse β4 polypeptide (isoform 1) has been reported with the following and has the accession number NP_001005608 in Genbank. [0265] Full-Length mouse β4 (isoform 1) (SEQ ID NO: 15):
1 MAGPCCSPWV KLLLLAAMLS ASLPGDLANR CKKAQVKSCT ECIRVDKSCA YCTDELFKER
6i RCNTQAELLA AGCRGESILV MESSLEITEN TQΠDTSLHRS QVSPQGLQVR
LRPGEERSFV 121 FQVFEPLESP VDLYILMDFS NSMSDDLDNL KQMGQNLAKI LRQLTSDYTI
GFGKFVDKVS 181 VPQTDMRPEK LKEPWPNSDP PFSFKNVISL TENVEEFWNK LQGERISGNL
DAPEGGFDAI 241 LQTAVCTRDI GWRADSTHLL VFSTESAFHY EADGANVLAG IMNRNDEKCH
LDASGAYTQY
301 KTQDYPSVPT LVRLLAKHNI IPIFAVTNYS YSYYEKLHKY FPVSSLGVLQ
EDSSNΓVELL
361 EEAFYRIRSN LDIRALDSPR GLRTEVTSDT LQKTETGSFH IKRGEVGTYN
VHLRAVEDID 421 GTHVCQLAKE DQGGNIHLKP SFSDGLRMDA SVICDVCPCE LQKEVRSARC
HFRGDFMCGH 481 CVCNEGWSGK TCNCSTGSLS DTQPCLREGE DKPCSGHGEC QCGRCVCYGE
GRYEGHFCEY 541 DNFQCPRTSG FLCNDRGRCS MGECVCEPGW TGRSCDCPLS NATCIDSNGG
ICNGRGYCEC 601 GRCHCNQQSL YTDTTCEINY SAIRLGLCED LRSCVQCQAW GTGEKKGRAC
DDCPFKVKMV 661 DELKKEEWE YCSFRDEDDD CTYS YNVEGD GSPGPNST VL VHKKKDCPPG
SFWWLIPLLI 721 FLLLLLALLL LLCWKYCACC KACLCiLLFCC NRGHMVGFKE DHYMLRENLM
ASDHLDTPML 781 RSGNLKGRDT VRWKITNNVQ RPGFATHAAS TSPTELVPYG LSLRLGRLCT
ENLMKPGTRE 841 CDQLRQEVEE NLNEVYRQVS GAHKLQQTKF RQQPNTGKKQ DHTIVDTVLL
APRSAKQMLL
901 KLTEKQVEQG SFHELKVAPG YYTVTAEQDA RGMVEFQEGV ELVDVRVPLF
IRPEDDDEKQ 961 LLVE AID VP V GT ATLGRRLV NITIIKEQ AS GWSFEQPEY S VSRGDQ VAR
IPVIRHILDN 1021 GKSQVSYSTQ DNTAHGHRDY VPVEGELLFH PGETWKELQV KLLELQEVDS
LLRGRQVRRF 1081 QVQLSNPKFG ARLGQPSTTT VILDETDRSL INQTLSSPPP PHGDLGAPQN
PNAKAAGSRK 1141 IHFNWLPPPG KPMGYRVKYW IQGDSESEAH LLDSKVPSVE LTNLYPYCDY
EMKVCAYGAQ 1201 GEGPYSSLVS CRTHQEVPSE PGRLAFNWS STVTQLSWAE PAETNGEITA
YEVCYGLVNE 1261 DNRPIGPMKK VLVDNPKNRM LLIENLRESQ PYRYTVKARN GAGWGPEREA
IINLATQPKR 1321 PMSIPIIPDI PIVDAQGGED YENFLMYSDD VLRSPASSQR PSVSDDTEHL
VNGRMDFAYP 1381 GSANSLHRMT AANVAYGTHL SPHLSHRVLS TSSTLTRDYH SLTRTEHSHS
GTLPRDYSTL
1441 TSLSSQGLPP IWEDGRSRLP LSWTLGSLSR AHMKGVPASR GSPDSIILAG QSAAPSWGTD
1501 SRGAVGVPDT PTRLVFSALG PTSLKVSWQE PQCDRMLLGY SVEYQLLNGG
EMHRLNIPNP 1561 GQTSVWEDL LPNHSYVFRV RAQSQEGWGR EREGVITIES QVHPQSPLCP
LPGSAFTLST 1621 PSAPGPLVFT ALSPDSLQLS WERPRRPNGD ILGYLVTCEM AQGGAPARTF
RVDGDNPESR 1681 LTVPGLSENV PYKFKVQART TEGFGPEREG IITIESQVGG PFPQLGSHSG
LFQNPVQSEF
1741 SSVTSTHSTT TEPFLMDGLT lgtqrleagg sltrhvtqef vtrtltasgs lsthmdqqff 1801 qt
[0266] The β4 polypeptide domain designations used herein are defined as follows:
Table 3. Example β4 polypeptide domains
Figure imgf000064_0001
[0267] As one of skill in the art will appreciate, the beginning and ending residues of the domains listed below may vary depending upon the computer modeling program used or the method used for determining the domain.
[0268] The present invention is also directed to α6β4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof which bind specifically, preferentially, or competitively to non-human α6β4 integrin proteins, e.g., α6β4 integrin from rodents or non-human primates.
[0269] α6β4 integrin is expressed in a large number of tumor cells, including, but not limited to certain of the following: bladder tumors (Oncol. Rep. 7:13-16 (2000)); breast tumors (Breast Cancer Res. 9:203 (2007) and Exp. Cell Res. 312:3822-3834 (2006)); colon tumors, (Exp. Cell Res. 266:1-10 (2001)); gastric tumors (Am. J. Pathol. 149:781- 793 (1996)); lung tumors (J. Cell Biochem. 55:409-418 (1994)); ovarian tumors (Hum. Pathol. 34:803-808 (2003)); pancreatic tumors,(J. Cell Sci. 116:4373-4390 (2003)); skin tumors (Cancer Cell 3:201-202 (2003); head and neck tumors (Laryngoscope 112:2025- 2032 (2002); brain tumors (Glia. 26:55-63 (1999)) and prostate tumors (Prostate 46:240- 248 (2001)). HI. α6β4 INTEGRIN ANTIBODIES
[0270] In one embodiment, the present invention is directed to α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof. For example, the present invention includes at least the antigen-binding domains of certain monoclonal antibodies, and fragments, variants, and derivatives thereof shown in Tables 4 and 5 . Table 4 lists human anti-human α6β4 integrin Fab regions identified from a phage display library.
Table 4: α6β4 integrin specific Fabs.
Figure imgf000065_0002
[0271] Table 5 lists murine anti-human α6β4 integrin monoclonal antibodies identified by hybridoma technology.
Table 5: Murine Monoclonal Antibodies
Figure imgf000065_0001
[0272] As used herein, the term "antigen binding domain" includes a site that specifically binds an epitope on an antigen (e.g., an epitope of α6β4 integrin). The antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
[0273] The present invention is more specifically directed to an α6β4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the α6β4 integrin antibody specifically binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61- C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to α6β4 integrin.
[0274] The invention is further drawn to an α6β4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the α6β4 integrin antibody competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to α6β4 integrin from binding to α6β4 integrin.
[0275] The invention is also drawn to an α6β4 integrin antibody, or antigen-binding fragment, variant or derivatives thereof, where the α6β4 integrin antibody comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to α6β4 integrin.
[0276] Methods of making antibodies are well known in the art and described herein.
Once antibodies to various fragments of, or to the full-length α6β4 integrin without the signal sequence, have been produced, determining which amino acids, or epitope, of α6β4 integrin to which the antibody or antigen binding fragment binds can be determined by epitope mapping protocols as described herein as well as methods known in the art (e.g. double antibody-sandwich ELISA as described in "Chapter 11 - Immunology," Current Protocols in Molecular Biology, Ed. Ausubel et ai, v.2, John Wiley & Sons, Inc. (1996)). Additional epitope mapping protocols may be found in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press (1996), which are both incorporated herein by reference in their entireties. Epitope mapping can also be performed by commercially available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wisconsin)).
[0277] Additionally, antibodies produced which bind to any portion of α6β4 integrin can then be screened for their ability to act as an antagonist of α6β4 integrin for example, to inhibit binding of laminin to α6β4 integrin, to inhibit activation of the PI3-K/Akt pathway including but not limited to Rac, mTOR and Akt, to inhibit activation of the Ras/MAPK signaling pathway, to inhibit the interaction of α6β4 with growth factor receptors including but not limited to erbB2, Met, and Ron, to inhibit integrin clustering in lipid rafts, to inhibit the interaction with cytoskeletal components, to induce apoptosis, to inhibit receptor tyrosine kinase (RTK) activity, to promote caspase cleavage, to induce integrin internalization or to inhibit tumor cell growth, proliferation, motility or metastasis. Antibodies can be screened for these and other properties according to methods described in detail in the Examples. Other functions of antibodies of the present invention can be tested using other assays as described in the Examples herein.
[0278] In other embodiments, the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of α6β4 integrin, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids of SEQ ID NOs: 1-15, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ID NO:1-15. The amino acids of a given epitope of SEQ ID NOs:l-15 as described may be, but need not be contiguous or linear, hi certain embodiments, at least one epitope of α6β4 integrin comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of α6 or β4 integrin as expressed on the surface of a cell or as a soluble fragment, e.g., fused to an IgG Fc region. Thus, in certain embodiments at least one epitope of α6β4 integrin comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ID NOs: 1-15, where non-contiguous amino acids form an epitope through protein folding.
[0279] hi certain embodiments, the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of α6β4 integrin, where the epitope comprises, consists essentially of, or consists of a portion of α6 and a portion of β4. Thus, in certain embodiments at least one epitope of α6β4 integrin comprises, consists essentially of, or consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of an α6 sequence selected from the group consisting of SEQ ID NOs: 1-8 and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of a β4 sequence selected from the group consisting of SEQ ED NOs:9-15, where non-contiguous amino acids form an epitope through protein folding.
[0280] In other embodiments, the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of α6β4 integrin, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or noncontiguous amino acids of SEQ ID NOs:l-15 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety may be included such that the α6β4 integrin antibody binds with higher affinity to modified target protein than it does to an unmodified version of the protein. Alternatively, the oc6β4 integrin antibody does not bind the unmodified version of the target protein at all.
[0281] hi certain aspects, the present invention is directed to an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically binds to a α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) which is less than the KD for a given reference monoclonal antibody.
[0282] hi certain embodiments, an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds specifically to at least one epitope of α6β4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of α6β4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of α6β4 integrin or fragment or variant described above; or binds to at least one epitope of α6β4 integrin or fragment or variant described above with an affinity characterized by a dissociation constant KD of less than about 5 x 10"2 M, about 10'2 M, about 5 x 10"3 M, about 10"3 M, about 5 x 10^ M, about 10"4 M, about 5 x 10"5 M, about 10"5 M, about 5 x 10"6 M, about 10"6 M, about 5 x 10"7 M, about 10"7 M, about 5 x 10"8 M, about 10"8 M, about 5 x 10"9 M, about 10"9 M, about 5 x 10"10 M, about 10"10 M, about 5 x 10"11 M, about 10"" M, about 5 x 10"12 M, about 10"12 M, about 5 x 10"13 M, about 10"13 M, about 5 x 10"14 M, about 10"14 M, about 5 x 10"15 M, or about 10"15 M. hi a particular aspect, the antibody or fragment thereof preferentially binds to a human α6β4 integrin polypeptide or fragment thereof, relative to a murine α6β4 integrin polypeptide or fragment thereof, hi another particular aspect, the antibody or fragment thereof preterentially binds to one or more α6β4 integrin polypeptides or fragments thereof, e.g., one or more mammalian α6β4 integrin polypeptides.
[0283] As used in the context of antibody binding dissociation constants, the term
"about" allows for the degree of variation inherent in the methods utilized for measuring antibody affinity. For example, depending on the level of precision of the instrumentation used, standard error based on the number of samples measured, and rounding error, the term "about 10"2 M" might include, for example, from 0.05 M to 0.005 M.
[0284] In specific embodiments, an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds α6β4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10"2 sec"1, 10"2 sec"1, 5 X 10"3 sec"1 or 10'3 sec"1. Alternatively, an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds α6β4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10"4 sec"1, 10"4 sec"1, 5 X 10"5 sec"1, or 10"5 sec"1 5 X 10"6 sec"1, 10"6 sec"1, 5 X 10"7 sec"1 or 10"7 sec"1.
[0285] In other embodiments, an antibody, or antigen-binding fragment, variant, or derivative thereof of the invention binds α6β4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 103 M"1 sec"1, 5 X 103 M"1 sec"1, 104 M"1 sec"1 or 5 X 104 M"1 sec"1. Alternatively, an antibody, or antigen- binding fragment, variant, or derivative thereof of the invention binds α6β4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 105 M"1 sec"1, 5 X 105 M"1 sec"1, 106 M"1 sec"1, or 5 X 106 M"1 sec"1 or 107 M"1 sec"1.
[0286] In various embodiments, an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof as described herein is an antagonist of α6β4 integrin activity. In certain embodiments, for example, binding of an antagonist α6β4 integrin antibody to α6β4 integrin as expressed on a tumor cell inhibits binding of laminin to α6β4 integrin, inhibits activation of the PB-K/ Akt pathway, or inhibits tumor cell proliferation, motility or metastasis.
[0287] Unless it is specifically noted, as used herein a "fragment thereof in reference to an antibody refers to an antigen-binding fragment, i.e., a portion of the antibody which specifically binds to the antigen. In one embodiment, an α6β4 integrin antibody, e.g., an antibody of the invention is a bispecific α6β4 integrin antibody, e.g., a bispecific antibody, minibody, domain deleted antiDody, or fusion protein having binding specificity for more than one epitope, e.g., more than one antigen or more than one epitope on the same antigen. In one embodiment, a bispecific α6β4 integrin antibody has at least one binding domain specific for at least one epitope on a target polypeptide disclosed herein, e.g., α6β4 integrin. In another embodiment, a bispecific α6β4 integrin antibody has at least one binding domain specific for an epitope on a target polypeptide and at least one target binding domain specific for a drug or toxin. In yet another embodiment, a bispecific α6β4 integrin antibody has at least one binding domain specific for an epitope on a target polypeptide disclosed herein, and at least one binding domain specific for a prodrug. A bispecific α6β4 integrin antibody may be a tetravalent antibody that has two target binding domains specific for an epitope of a target polypeptide disclosed herein and two target binding domains specific for a second target. Thus, a tetravalent bispecific α6β4 integrin antibody may be bivalent for each specificity.
[0288] α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention, as known by those of ordinary skill in the art, can comprise a constant region which mediates one or more effector functions. For example, binding of the Cl component of complement to an antibody constant region may activate the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. Further, antibodies bind to receptors on various cells via the Fc region, with a Fc receptor binding site on the antibody Fc region binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
[0289] Accordingly, certain embodiments of the invention include an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof, in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain .domains. For instance, in certain antibodies, one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted. In other embodiments, certain antibodies for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG4 heavy chain constant region, which is altered to eliminate glycosylation, referred to elsewhere herein as "agly" antibodies. While not being bound by theory, it is believed that "agly" antibodies may have an improved safety and stability profile in vivo. Methods of producing aglycosylated antibodies, having desired effector function are found for example in WO 2005018572, which is incorporated by reference in its entirety.
[0290] In certain α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof described herein, the Fc portion may be mutated to decrease effector function using techniques known in the art. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases it may be that constant region modifications consistent with the instant invention moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability and other biochemical effects of the modifications, such as tumor localization, biodistribution and serum half- life, may easily be measured and quantified using well know immunological techniques without undue experimentation.
[0291] Modified forms of α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made from whole precursor or parent antibodies using techniques known in the art. Exemplary techniques are discussed in more detail herein.
[0292] In certain embodiments both the variable and constant regions of α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof are fully human. Fully human antibodies can be made using techniques that are known in the art and as described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in US patents: 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human antibodies can likewise be produced by various display technologies, e.g., phage display or other viral display systems, as described in more detail elsewhere herein.
[0293] α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be made or manufactured using techniques that are known in the art. In certain embodiments, antibody molecules or fragments thereof are "recombinantly produced," i.e., are produced using recombinant DNA technology. Exemplary techniques for making antibody molecules or fragments thereof are discussed in more detail elsewhere herein.
[0294] α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention also include derivatives that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
[0295] In certain embodiments, α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention will not elicit a deleterious immune response in the animal to be treated, e.g., in a human. In one embodiment, α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, that retains or substantially retains the antigen- binding properties of the parent antibody, but which is less immunogenic in humans. This may be achieved by various methods, including (a) grafting the entire non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a part of one or more of the non-human complementarity determining regions (CDRs) into a human framework and constant regions with or without retention of critical framework residues; or (c) transplanting the entire non-human variable domains, but "cloaking" them with a human-like section by replacement of surface residues. Such methods are disclosed in Morrison et al., Proc. Natl. Acad. Sd. 5i:6851- 6855 (1984); Morrison et al, Adv. Immunol. 44:65-92 (1988); Verhoeyen et al, Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 57:169-217 (1994), and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are hereby incorporated by reference in their entirety.
[0296] De-immunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term "de-immunization" includes alteration of an antibody to modify T cell epitopes {see, e.g., WO9852976A1, WO0034317A2). For example, VH and VL sequences from the starting antibody are analyzed and a human T cell epitope "map" from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody. A range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein, which are then tested for function. Typically, between 12 and 24 variant antibodies are generated and tested. Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
[0297] α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art. For example, an α6β4 integrin antibody, e.g., a binding polypeptide, e.g., an α6β4 integrin-specific antibody or immunospecific fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, chickens, hamsters, goats, donkeys, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
[0298] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et al, in: Monoclonal Antibodies and T-CeIl Hybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporated by reference in their entireties). The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using α6β4 integrin knockout mice to increase the regions of epitope recognition. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology as described elsewhere herein.
[0299] Using art recognized protocols, in one example, antibodies are raised in mammals by multiple subcutaneous or intraperitoneal injections of the relevant antigen {e.g., purified α6β4 integrin or cells or cellular extracts comprising α6β4 integrin) and an adjuvant. This immunization typically elicits an immune response that comprises production of antigen-reactive antibodies from activated splenocytes or lymphocytes. While the resulting antibodies may be harvested from the serum of the animal to provide polyclonal preparations, it is often desirable to isolate individual lymphocytes from the spleen, lymph nodes or peripheral blood to provide homogenous preparations of monoclonal antibodies (MAbs). Preferably, the lymphocytes are obtained from the spleen.
[0300] In this well known process (Kohler et al, Nature 256:495 (1975)) the relatively short-lived, or mortal, lymphocytes from a mammal which has been injected with antigen are fused with an immortal tumor cell line (e.g., a myeloma cell line), thus, producing hybrid cells or "hybridomas" which are both immortal and capable of producing the genetically coded antibody of the B cell. The resulting hybrids are segregated into single genetic strains by selection, dilution, and regrowth with each individual strain comprising specific genes for the formation of a single antibody. They produce antibodies, which are homogeneous against a desired antigen and, in reference to their pure genetic parentage, are termed "monoclonal."
[0301] Hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. Those skilled in the art will appreciate that reagents, cell lines and media for the formation, selection and growth of hybridomas are commercially available from a number of sources and standardized protocols are well established. Generally, culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies against the desired antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by in vitro assays such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). After hybridoma cells are identified that produce antibodies of the desired specificity, affinity and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp 59-103 (1986)). It will further be appreciated that the monoclonal antibodies secreted by the subclones may be separated from culture medium, ascites fluid or serum by conventional purification procedures such as, for example, protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
[0302] Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments may be produced recombinantly or by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain. [0303] Those skilled in the art will also appreciate that DNA encoding antibodies or antibody fragments (e.g., antigen binding sites) may also be derived from antibody libraries, such as phage display libraries. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M 13 binding domains expressed from phage with Fab, Fv OE DAB (individual Fv region from light or heavy chains) or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Exemplary methods are set forth, for example, in EP 368 684 Bl ; U.S. patent. 5,969,108, Hoogenboom, H.R. and Chames, Immunol. Today 27:371 (2000); Nagy et al Nat. Med. <S:801 (2002); Huie et al, Proc. Natl. Acad. Sci. USA 98:2682 (2001); Lui et al., J. MoI Biol. 575:1063 (2002), each of which is incorporated herein by reference. Several publications (e.g., Marks et al, Bio/Technology 70:779-783 (1992)) have described the production of high affinity human antibodies by chain shuffling, as well as combinatorial infection and in vivo recombination as a strategy for constructing large phage libraries. In another embodiment, Ribosomal display can be used to replace bacteriophage as the display platform (see, e.g., Hanes et al, Nat. Biotechnol. 75:1287 (2000); Wilson et al, Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al, J. Immunol. Methods 248:31 (2001)). In yet another embodiment, cell surface libraries can be screened for antibodies (Boder et al, Proc. Natl. Acad. Sci. USA 97: 10701 (2000); Daugherty et al, J. Immunol Methods 243:211 (2000)). Such procedures provide alternatives to traditional hybridoma techniques for the isolation and subsequent cloning of monoclonal antibodies.
[0304] In phage display methods, functional antibody domains are displayed on the surface of phage particles, which carry the polynucleotide sequences encoding them. For example, DNA sequences encoding VH and VL regions are amplified or otherwise isolated from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. In certain embodiments, the DNA encoding the VH and VL regions are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M 13 and the VH or VL regions are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest (i.e., an α6β4 integrin polypeptide or a fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
[0305] Additional examples of phage display methods that can be used to make the antibodies include those disclosed in Brinkman et al, J. Immunol. Methods 752:41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol 24:952-958 (1994); Persic et al, Gene 187:9-18 (1997); Burton et al, Advances in Immunology 57:191-280 (1994); PCT Application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.
[0306] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 12 (6) :864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).
[0307] Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al, Methods in Enzymology 203:46-88 (1991); Shu et al, PNAS 90:7995-7999 (1993); and Skerra et al, Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, J. Immunol. Methods /25:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entireties. In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al, Proc. Natl. Acad. Sci. 57:851-855 (1984); Neuberger et al., Nature 372:604-608 (1984); Takeda et al, Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
[0308] Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Pat. No. 5,585,089; Riechmann et al, Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR- grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5) :489-498 (1991); Studnicka et al, Protein Engineering 7^:805-814 (1994); Roguska. et al, PNAS 97:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).
[0309] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. [0310] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. Ln particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a desired target polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety, hi addition, companies such as Abgenix, hie. (Freemont, Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.
[0311] Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." hi this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al, Bio/Technology 72:899-903 (1988). See also, U.S. Patent No. 5,565,332.) [0312] Further, antibodies to target polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" target polypeptides using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):A31- 444 (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate antiidiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a desired target polypeptide and/or to bind its ligands/receptors, and thereby block its biological activity.
[0313] In another embodiment, DNA encoding desired monoclonal antibodies may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The isolated and subcloned hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as, but not limited to, E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which may be synthetic as described herein) may be used to clone constant and variable region sequences for the manufacture antibodies as described in Newman et al, U.S. Pat. No. 5,658,570, filed January 25, 1995, which is incorporated by reference herein. Essentially, this entails extraction of RNA from the selected cells, conversion to cDNA, and amplification by PCR using Ig specific primers. Suitable primers for this purpose are also described in U.S. Pat. No. 5,658,570. As will be discussed in more detail below, transformed cells expressing the desired antibody may be grown up in relatively large quantities to provide clinical and commercial supplies of the immunoglobulin.
[0314] In one embodiment, an α6β4 integrin antibody of the invention comprises at least one heavy or light chain CDR of an antibody molecule. In another embodiment, an α6β4 integrin antibody of the invention comprises at least two CDRs from one or more antibody molecules. In another embodiment, an α6β4 integrin antibody of the invention comprises at least three CDRs from one or more antibody molecules. In another embodiment, an α6β4 integrin antibody of the invention comprises at least four CDRs from one or more antibody molecules. In another embodiment, an α6β4 integrin antibody of the invention comprises at least five CDRs from one or more antibody molecules. In another embodiment, an α6β4 integrin antibody of the invention comprises at least six CDRs from one or more antibody molecules. Exemplary antibody molecules comprising at least one CDR that can be included in the subject α6β4 integrin antibodies are described herein.
[0315] In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. MoI. Biol. 278:457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, e.g., α6β4 integrin. Preferably, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
[0316] Alternatively, techniques described for the production of single chain antibodies
(U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. ScL USA 55:5879-5883 (1988); and Ward et al, Nature 554:544-554 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain antibody. Techniques for the assembly of functional Fv fragments in E coli may also be used (Skerra et al., Science 242:1038-1041 (1988)). [0317] Yet other embodiments of the present invention comprise the generation of human or substantially human antibodies in transgenic animals (e.g., mice) that are incapable of endogenous immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which is incorporated herein by reference). For example, it has been described that the homozygous deletion of the antibody heavy- chain joining region in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of a human immunoglobulin gene array to such germ line mutant mice will result in the production of human antibodies upon antigen challenge. Another preferred means of generating human antibodies using SCDD mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated herein by reference. It will be appreciated that the genetic material associated with these human antibodies may also be isolated and manipulated as described herein.
[0318] Yet another highly efficient means for generating recombinant antibodies is disclosed by Newman, Biotechnology 10: 1455-1460 (1992). Specifically, this technique results in the generation of primatized antibodies that contain monkey variable domains and human constant sequences. This reference is incorporated by reference in its entirety herein. Moreover, this technique is also described in commonly assigned U.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which is incorporated herein by reference.
[0319] In another embodiment, lymphocytes can be selected by micromanipulation and the variable genes isolated. For example, peripheral blood mononuclear cells can be isolated from an immunized mammal and cultured for about 7 days in vitro. The cultures can be screened for specific IgGs that meet the screening criteria. Cells from positive wells can be isolated. Individual Ig-producing B cells can be isolated by FACS or by identifying them in a complement-mediated hemolytic plaque assay. Ig-producing B cells can be micromanipulated into a tube and the VH and VL genes can be amplified using, e.g. , RT-PCR. The VH and VL genes can be cloned into an antibody expression vector and transfected into cells (e.g. , eukaryotic or prokaryotic cells) for expression.
[0320] Alternatively, antibody-producing cell lines may be selected and cultured using techniques well known to the skilled artisan. Such techniques are described in a variety of laboratory manuals and primary publications. In this respect, techniques suitable for use in the invention as described below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991) which is herein incorporated by reference in its entirety, including supplements.
[0321] Antibodies of the present invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques as described herein.
[0322] In one embodiment, an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises a synthetic constant region wherein one or more domains are partially or entirely deleted ("domain-deleted antibodies"). In certain embodiments compatible modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ΔCH2 constructs). For other embodiments a short connecting peptide may be substituted for the deleted domain to provide flexibility and freedom of movement for the variable region. Those skilled in the art will appreciate that such constructs are particularly preferred due to the regulatory properties of the CH2 domain on the catabolic rate of the antibody. Domain deleted constructs can be derived using a vector encoding an IgGi human constant domain (see, e.g., WO 02/060955 A2 and WO02/096948A2). This vector is engineered to delete the CH2 domain and provide a synthetic vector expressing a domain deleted IgGj constant region.
[0323] In certain embodiments, α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention are minibodies. Minibodies can be made using methods described in the art (see, e.g., US patent 5,837,821 or WO 94/09817A1).
[0324] In one embodiment, an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof of the invention comprises an immunoglobulin heavy chain having deletion or substitution of a few or even a single amino acid as long as it permits association between the monomelic subunits. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase tumor localization. Similarly, it may be desirable to simply delete that part of one or more constant region domains that control the effector function (e.g. complement binding) to be modulated. Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies may be synthetic through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Yet other embodiments comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it may be desirable to insert or replicate specific sequences derived from selected constant region domains. The present invention also provides antibodies that comprise, consist essentially of, or consist of, variants (including derivatives) of antibody molecules (e.g., the VH regions and/or VL regions) described herein, which antibodies or fragments thereof immunospecifically bind to an α6β4 integrin polypeptide or fragment or variant thereof. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding an α6β4 integrin antibody, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH region, VH-CDRl, VH-CDR2, VH-CDR3, VL region, VL-CDRl, VL-CDR2, or VL-CDR3. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains ( e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind an α6β4 integrin polypeptide). [0326] For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations may be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen, indeed some such mutations do not alter the amino acid sequence whatsoever. These types of mutations may be useful to optimize codon usage, or improve a hybridoma's antibody production. Codon-optimized coding regions encoding α6β4 integrin antibodies of the present invention are disclosed elsewhere herein. Alternatively, non-neutral missense mutations may alter an antibody's ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement. One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e.g., improvements in antigen binding activity or change in antibody specificity). Following mutagenesis, the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to immunospecifϊcally bind at least one epitope of an α6β4 integrin polypeptide) can be determined using techniques described herein or by routinely modifying techniques known in the art.
IV. POLYNUCLEOTIDES ENCODING α6β4 INTEGRIN ANTIBODIES
[0327] The present invention also provides for nucleic acid molecules encoding α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention.
[0328] In one embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH), where at least one of the CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2, or VH-CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Alternatively, the VH-CDRl, VH-CDR2, and VH- CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Thus, according to this embodiment a heavy chain variable region of the invention has VH-CDRl, VH-CDR2, or VH-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 6. In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2, or VL-CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Alternatively, the VL-CDRl, VL-CDR2, and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Thus, according to this embodiment a light chain variable region of the invention has VL-CDRl, VL-CDR2, or VL-CDR3 polypeptide sequences related to the polypeptide sequences shown in Table 6.
TABLE 6: Reference VH-CDRl, VH-CDR2, and VH-CDR3 and VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences*
OO
Figure imgf000087_0001
06
Figure imgf000088_0001
00 00
Figure imgf000089_0001
00
Figure imgf000090_0001
O
Figure imgf000091_0001
^O
Figure imgf000092_0001
Kt
Figure imgf000093_0001
*Determined by the Kabat system (see supra). N=nucleotide sequence, P=polypeptide sequence.
[0330] As known in the art, "sequence identity" between two polypeptides or two polynucleotides is determined by comparing the amino acid or nucleic acid sequence of one polypeptide or polynucleotide to the sequence of a second polypeptide or polynucleotide. When discussed herein, whether any particular polypeptide is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.
[0331] hi certain embodiments, an antibody or antigen-binding fragment comprising the
VH encoded by the polynucleotide specifically or preferentially binds to α6β4 integrin. hi certain embodiments the nucleotide sequence encoding the VH polypeptide is altered without altering the amino acid sequence encoded thereby. For instance, the sequence may be altered for improved codon usage in a given species, to remove splice sites, or the remove restriction enzyme sites. Sequence optimizations such as these are described in the examples and are well known and routinely carried out by those of ordinary skill in the art.
[0332] In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2, and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2, and VH-CDR3 groups shown in Table 6. hi certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to α6β4 integrin. [0333] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, where the VH polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ED NOs: 78, 79, and 80; and SEQ ID NOs: 88, 89 and 90; and where an antibody or antigen binding fragment thereof comprising the VH-CDR3 specifically binds to α6β4 integrin.
[0334] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to α6β4 integrin.
[0335] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to an α6β4 integrin polypeptide or fragment thereof, or a α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10"2 M, 10"2 M, 5 x 10"3 M, 10"3 M, 5 x 10"4 M, 10"4 M, 5 x 10"5 M, 10"5 M, 5 x 10"6 M, 10"6 M, 5 x 10"7 M, 10"7 M, 5 x 10"8 M, 10"8 M, 5 x 10"9 M, 10"9 M, 5 x 10"10 M, 10"10 M, 5 x 10"11 M, 10'11 M, 5 x 10-12 M, 10-12 M, 5 x 10"13 M, 10"13 M, 5 x 10"14 M, 10"14 M, 5 x 10"15 M, or 10'15 M.
[0336] hi certain embodiments, an antibody or antigen-binding fragment comprising the
VL encoded by the polynucleotide specifically or preferentially binds to α6β4 integrin.
[0337] In another embodiment, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2, and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL- CDR2, and VL-CDR3 groups shown in Table 6. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to α6β4 integrin.
[0338] In a further aspect, the present invention provides an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2, and VL-CDR3 regions are encoded by nucleotide sequences which are identical to the nucleotide sequences which encode the VL-CDRl, VL-CDR2, and VL-CDR3 groups shown in Table 6. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by the polynucleotide specifically or preferentially binds to α6β4 integrin.
[0339] In some embodiments, the invention provides an isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs:83, 84, and 85; and SEQ ID NOs:93, 94 and 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to α6β4 integrin.
[0340] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to α6β4 integrin.
[0341] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an α6β4 integrin polypeptide or fragment thereof, or a α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10"2 M, 10"2 M, 5 x 10"3 M, 103 M, 5 x 10"4 M, lO"4 M, 5 x 10"5 M, W5 M, 5 x 10"6 M, 10"6 M, 5 x 10~7 M, 10"7 M, 5 x Kr8 M, 1(T8 M, 5 x ICT9 M, 10'9 M, 5 x 10"10 M, lO'10 M, 5 x lO'11 M, 1(T11 M, 5 x 10' 12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10"14 M, 10"14 M, 5 x 10'15 M, or 10"15 M.
[0342] In a further embodiment, the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VH at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to α6β4 integrin.
[0343] In another aspect, the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by the polynucleotide specifically or preferentially binds to α6β4 integrin.
[0344] In a further embodiment, the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a VH-encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76 and 86. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to α6β4 integrin.
[0345] In another aspect, the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH of the invention, where the amino acid sequence of the VH is selected from the group consisting of SEQ ED NOs: 17, 27, 37, 47, 57, 67, 77 and 87. The present invention further includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VH of the invention, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76 and 86. In certain embodiments, an antibody or antigen-binding fragment comprising the VH encoded by such polynucleotides specifically or preferentially binds to α6β4 integrin.
[0346] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to α6β4 integrin.
[0347] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VH encoded by one or more of the polynucleotides described above specifically or preferentially binds to an α6β4 integrin polypeptide or fragment thereof, or a α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10"2 M, 10"2 M, 5 x 10"3 M, 1(T3 M, 5 x 10"4 M, 10"4 M, 5 x 10"5 M, 10"5 M, 5 x 10"6 M, 10"6 M, 5 x 1(T7 M, 10"7 M, 5 x 10"8 M, 10"8 M, 5 x 10"9 M, 1(T9 M, 5 x 10"10 M, 10"10 M, 5 x 10"11 M, 1(T11 M, 5 x 10"12 M, 10"12 M, 5 x 1(T13 M, 10"13 M, 5 x 10"14 M, 10'14 M, 5 x 10"15 M, or 10"15 M.
[0348] In a further embodiment, the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid encoding a VL at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 32, 42, 52, 62, 72, 82 and 92. In a further embodiment, the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a VL- encoding nucleic acid at least 80%, 85%, 90% 95% or 100% identical to a reference nucleic acid sequence selected from the group consisting of SEQ ID NOs: 21, 31, 41, 51, 61, 71, 81 and 91. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to α6β4 integrin.
[0349] In another aspect, the present invention includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL having a polypeptide sequence selected from the group consisting of SEQ ID NOs: 22, 32, 42, 52, 62, 72, 82 and 92. The present invention further includes an isolated polynucleotide comprising, consisting essentially of, or consisting of a nucleic acid sequence encoding a VL of the invention, where the sequence of the nucleic acid is selected from the group consisting of SEQ ID NOs: 21, 31, 41, 51, 61, 71, 81 and 91. In certain embodiments, an antibody or antigen-binding fragment comprising the VL encoded by such polynucleotides specifically or preferentially binds to α6β4 integrin.
[0350] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to α6β4 integrin.
[0351] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a VL encoded by one or more of the polynucleotides described above specifically or preferentially binds to an α6β4 integrin polypeptide or fragment thereof, or a α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10"2 M, 10"2 M, 5 x 10"3 M, 10'3 M, 5 x 10"4 M, 10"4 M, 5 x 10"5 M, 10"5 M, 5 x l(ϊ6 M, 10"6 M, 5 x 10"7 M, 10"7 M, 5 x 10"8 M, 10"8 M, 5 x 10"9 M, 10"9 M, 5 x 10"10 M, 10"10 M, 5 x 10"11 M, 10"11 M, 5 x 10" 12 M, 10"12 M, 5 x 10"13 M, 10"13 M, 5 x 10"14 M, 10"14 M, 5 x 10"15 M, or 10~15 M.
[0352] Any of the polynucleotides described above may further include additional nucleic acids, encoding, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein.
[0353] Also, as described in more detail elsewhere herein, the present invention includes compositions comprising the polynucleotides comprising one or more of the polynucleotides described above. In one embodiment, the invention includes compositions comprising a first polynucleotide and second polynucleotide wherein said first polynucleotide encodes a VH polypeptide as described herein and wherein said second polynucleotide encodes a VL polypeptide as described herein. Specifically a composition which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide, wherein the VH polynucleotide and the VL polynucleotide encode polypeptides, respectively at least 80%, 85%, 90% 95% or 100% identical to reference VH and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, and 87 and 92. Or alternatively, a composition which comprises, consists essentially of, or consists of a VH polynucleotide, and a VL polynucleotide at least 80%, 85%, 90% 95% or 100% identical, respectively, to reference VL and VL nucleic acid sequences selected from the group consisting of SEQ ID NOs: 16 and 21, 26 and 31, 36 and 41, 46 and 51, 56 and 61, 66 and 71, 76 and 81, and 86 and 91. In certain embodiments, an antibody or antigen-binding fragment comprising the VH and VL encoded by the polynucleotides in such compositions specifically or preferentially binds to α6β4 integrin.
[0354] The present invention also includes fragments of the polynucleotides of the invention, as described elsewhere. Additionally polynucleotides which encode fusion polynucleotides, Fab fragments, and other derivatives, as described herein, are also contemplated by the invention.
[0355] The polynucleotides may be produced or manufactured by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 77:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
[0356] Alternatively, a polynucleotide encoding an α6β4 integrin antibody, or antigen- binding fragment, variant, or derivative thereof may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the antibody may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+RNA, isolated from, any tissue or cells expressing the antibody or other α6β4 integrin antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g. , a cDNA clone from a cDNA library that encodes the antibody or other α6β4 integrin antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
[0357] Once the nucleotide sequence and corresponding amino acid sequence of the α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof is determined, its nucleotide sequence may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1990) and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1998), which are both incorporated by reference herein in their entireties ), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
[0358] A polynucleotide encoding an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide encoding α6β4 integrin antibody, or antigen- binding fragment, variant, or derivative thereof can be composed of single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, a polynucleotide encoding an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide encoding an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.
[0359] An isolated polynucleotide encoding a non-natural variant of a polypeptide derived from an immunoglobulin {e.g., an immunoglobulin heavy chain portion or light chain portion) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the immunoglobulin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more non-essential amino acid residues.
V. α6β4 INTEGRIN ANTIBODY POLYPEPTIDES
[0360] The present invention is further directed to isolated polypeptides which make up α6β4 integrin antibodies, and polynucleotides encoding such polypeptides. α6β4 integrin antibodies of the present invention comprise polypeptides, e.g., amino acid sequences encoding α6β4 integrin-specific antigen binding regions derived from immunoglobulin molecules. A polypeptide or amino acid sequence "derived from" a designated protein refers to the origin of the polypeptide having a certain amino acid sequence. In certain cases, the polypeptide or amino acid sequence which is derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
[0361] hi one embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH), where at least one of VH-CDRs of the heavy chain variable region or at least two of the VH-CDRs of the heavy chain variable region are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2 or VH-CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Alternatively, the VH-CDRl, VH-CDR2 and VH-CDR3 regions of the VH are at least 80%, 85%, 90% or 95% identical to reference heavy chain VH-CDRl, VH-CDR2 and VH-CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Thus, according to this embodiment a heavy chain variable region of the invention has VH-CDRl, VH-CDR2 and VH-CDR3 polypeptide sequences related to the groups shown in Table 6, supra. While Table 6 shows VH-CDRs defined by the Kabat system, other CDR definitions, e.g., VH-CDRs defined by the Chothia system, are also included in the present invention. In certain embodiments, an antibody or antigen- binding fragment comprising the VH specifically or preferentially binds to α6β4 integrin.
[0362] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and VH-CDR3 groups shown in Table 6. In certain embodiments, an antibody or antigen-binding fragment comprising the VH specifically or preferentially binds to α6β4 integrin.
[0363] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences which are identical to the VH-CDRl, VH-CDR2 and VH-CDR3 groups shown in Table 6, except for one, two, three, four, five, or six amino acid substitutions in any one VH-CDR. In larger CDRs, e.g., VH-CDR-3, additional substitutions may be made in the CDR, as long as the a VH comprising the VH-CDR specifically or preferentially binds to α6β4 integrin. In certain embodiments the amino acid substitutions are conservative. In certain embodiments, an antibody or antigen- binding fragment comprising the VH specifically or preferentially binds to α6β4 integrin.
[0364] In some embodiments, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90, except for one, two, three, four, five or six amino acid substitutions in at least one of said VH-CDRs.
[0365] In some embodiments, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VH) in which the VH-CDRl, VH-CDR2 and VH-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90.
[0366] In a further embodiment, the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VH polypeptide amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77 and 87. In certain embodiments, an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to α6β4 integrin.
[0367] In another aspect, the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VH polypeptide selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57 67, 77 and 87. In certain embodiments, an antibody or antigen-binding fragment comprising the VH polypeptide specifically or preferentially binds to α6β4 integrin.
[0368] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VH polypeptides described above specifically or preferentially binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to α6β4 integrin.
[0369] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of one or more of the VH polypeptides described above specifically or preferentially binds to an α6β4 integrin polypeptide or fragment thereof, or a α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10'2 M, 10"2 M, 5 x 10"3 M, 10"3 M, 5 x 10"4 M, 10"4 M, 5 x 10'5 M, 10"5 M, 5 x 10'6 M, 10"6 M, 5 x 10"7 M, 10"7 M, 5 x 10"8 M, 10" 8 M, 5 x 10'9 M, 10"9 M, 5 x 10"10 M, 10"10 M, 5 x 10"11 M, 10"11 M, 5 x 10"12 M, 10"12 M, 5 x 10'13 M, 10"13 M, 5 x 10"14 M, 10"14 M, 5 x 10"15 M, or 10"15 M.
[0370] In another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL), where at least one of the VL-CDRs of the light chain variable region or at least two of the VL-CDRs of the light chain variable region are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2 or VL-CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Alternatively, the VL-CDRl, VL-CDR2 and VL-CDR3 regions of the VL are at least 80%, 85%, 90% or 95% identical to reference light chain VL-CDRl, VL-CDR2 and VL- CDR3 amino acid sequences from monoclonal α6β4 integrin antibodies disclosed herein. Thus, according to this embodiment a light chain variable region of the invention has VL- CDRl, VL-CDR2 and VL-CDR3 polypeptide sequences related to the polypeptides shown in Table 6, supra. While Table 6 shows VL-CDRs defined by the Kabat system, other CDR definitions, e.g., VL-CDRs defined by the Chothia system, are also included in the present invention. In certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to α6β4 integrin.
[0371] hi another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin light chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL-CDR2 and VL-CDR3 groups shown in Table 6. hi certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to α6β4 integrin.
[0372] hi another embodiment, the present invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences which are identical to the VL-CDRl, VL-CDR2 and VL-CDR3 groups shown in Table 6, except for one, two, three, four, five, or six amino acid substitutions in any one VL-CDR. hi larger CDRs, additional substitutions may be made in the VL-CDR, as long as the a VL comprising the VL-CDR specifically or preferentially binds to α6β4 integrin. In certain embodiments the amino acid substitutions are conservative, hi certain embodiments, an antibody or antigen-binding fragment comprising the VL specifically or preferentially binds to α6β4 integrin.
[0373] hi some embodiments, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ED NOs: 83, 84 and 85; and SEQ ID NOs: 93, 94 and 95, except for one, two, three, four, five or six amino acid substitutions in at least one of said VL-CDRs.
[0374] hi some embodiments, the invention provides an isolated polypeptide comprising, consisting essentially of, or consisting of an immunoglobulin heavy chain variable region (VL) in which the VL-CDRl, VL-CDR2 and VL-CDR3 regions have polypeptide sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ED NOs: 33, 34, and 35; SEQ ED NOs: 43, 44, and 45; SEQ ED NOs: 53, 54, and 55; SEQ ED NOs: 63, 64, and 65; SEQ ED NOs: 73, 74, and 75; SEQ ED NOs: 83, 84 and 85; and SEQ ED NOs: 93, 94 and 95.
[0375] In a further embodiment, the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide at least 80%, 85%, 90% 95% or 100% identical to a reference VL polypeptide sequence selected from the group consisting of SEQ ED NOs: 22, 32, 42, 52, 62, 72, 82 and 92. In certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to α6β4 integrin.
[0376] En another aspect, the present invention includes an isolated polypeptide comprising, consisting essentially of, or consisting of a VL polypeptide selected from the group consisting of SEQ ED NOs: 22, 32, 42, 52, 62, 72, 82 and 92. In certain embodiments, an antibody or antigen-binding fragment comprising the VL polypeptide specifically or preferentially binds to α6β4 integrin.
[0377] En certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, one or more of the VL polypeptides described above specifically or preferentially binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61- C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, or will competitively inhibit such a monoclonal antibody or fragment from binding to α6β4 integrin. [0378] In certain embodiments, an antibody or antigen-binding fragment thereof comprising, consisting essentially of, or consisting of a one or more of the VL polypeptides described above specifically or preferentially binds to an α6β4 integrin polypeptide or fragment thereof, or a α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10"2 M, 10"2 M, 5 x 10~3 M, 10"3 M, 5 x 10"4 M, 10"4 M, 5 x 10"5 M, 10"5 M, 5 x 106 M, 10"6 M, 5 x 10"7 M, 10'7 M, 5 x 10'8 M, 10"8 M, 5 x 10"9 M, 10'9 M, 5 x 10'10 M, 10"10 M, 5 x lO"11 M, 10"π M, 5 x 10" 12 M, 10"12 M, 5 x 10 13 M, 10"13 M, 5 x 10"14 M, 10"14 M, 5 x 10"15 M, or 10"15 M.
[0379] In other embodiments, an antibody or antigen-binding fragment thereof comprises, consists essentially of or consists of a VH polypeptide, and a VL polypeptide, where the VH polypeptide and the VL polypeptide, respectively are at least 80%, 85%, 90% 95% or 100% identical to reference VL and VL polypeptide amino acid sequences selected from the group consisting of SEQ ID NOs: 17 and 22, 27 and 32, 37 and 42, 47 and 52, 57 and 62, 67 and 72, 77 and 82, and 87 and 92. In certain embodiments, an antibody or antigen-binding fragment comprising these VH and VL polypeptides specifically or preferentially binds to α6β4 integrin.
[0380] Any of the polypeptides described above may further include additional polypeptides, e.g., a signal peptide to direct secretion of the encoded polypeptide, antibody constant regions as described herein, or other heterologous polypeptides as described herein. Additionally, polypeptides of the invention include polypeptide fragments as described elsewhere. Additionally polypeptides of the invention include fusion polypeptide, Fab fragments, and other derivatives, as described herein.
[0381] Also, as described in more detail elsewhere herein, the present invention includes compositions comprising the polypeptides described above.
[0382] It will also be understood by one of ordinary skill in the art that α6β4 integrin antibody polypeptides as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g., have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the starting sequence.
[0383] Furthermore, nucleotide or amino acid substitutions, deletions, or insertions leading to conservative substitutions or changes at "non-essential" amino acid regions may be made. For example, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions, or deletions, e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty or more individual amino acid substitutions, insertions, or deletions. In other embodiments, a polypeptide or amino acid sequence derived from a designated protein may be identical to the starting sequence except for two or fewer, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, fifteen or fewer, or twenty or fewer individual amino acid substitutions, insertions, or deletions. In certain embodiments, a polypeptide or amino acid sequence derived from a designated protein has one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions relative to the starting sequence.
[0384] Certain α6β4 integrin antibody polypeptides of the present invention comprise, consist essentially of, or consist of an amino acid sequence derived from a human amino acid sequence. However, certain α6β4 integrin antibody polypeptides comprise one or more contiguous amino acids derived from another mammalian species. For example, an α6β4 integrin antibody of the present invention may include a primate heavy chain portion, hinge portion, or antigen binding region, hi another example, one or more murine-derived amino acids may be present in a non-murine antibody polypeptide, e.g. , in an antigen binding site of an α6β4 integrin antibody, hi another example, the antigen binding site of an α6β4 integrin antibody is fully murine. In certain therapeutic applications, α6β4 integrin-specific antibodies, or antigen-binding fragments, variants, or analogs thereof are designed so as to not be immunogenic in the animal to which the antibody is administered.
[0385] In certain embodiments, an α6β4 integrin antibody polypeptide comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, a single-chain fv antibody fragment of the invention may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).
[0386] An α6β4 integrin antibody polypeptide of the invention may comprise, consist essentially of, or consist of a fusion protein. Fusion proteins are chimeric molecules which comprise, for example, an immunoglobulin antigen-binding domain with at least one target binding site, and at least one heterologous portion, i.e., a portion with which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought together in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. Fusion proteins may be created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.
[0387] The term "heterologous" as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the rest of the entity to which it is being compared. For instance, as used herein, a "heterologous polypeptide" to be fused to an α6β4 integrin antibody, or an antigen- binding fragment, variant, or analog thereof is derived from a non-immunoglobulin polypeptide of the same species, or an immunoglobulin or non-immunoglobulin polypeptide of a different species.
[0388] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. [0389] Alternatively, in another embodiment, mutations may be introduced randomly along all or part of the immunoglobulin coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into α6β4 integrin antibodies for use in the diagnostic and treatment methods disclosed herein and screened for their ability to bind to the desired antigen, e.g., α6β4 integrin.
VI. FUSION PROTEINS AND ANTIBODY CONJUGATES
[0390] As discussed in more detail elsewhere herein, α6β4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, α6β4 integrin-specifϊc α6β4 integrin antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
[0391] α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody binding α6β4 integrin. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
[0392] α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. α6β4 integrin-specfic antibodies may be modified by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in the α6β4 integrin-specific antibody, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, or on moieties such as carbohydrates. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given α6β4 integrin-specific antibody. Also, a given α6β4 integrin-specific antibody may contain many types of modifications. α6β4 integrin-specific antibodies may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic α6β4 integrin-specific antibodies may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, e.g., Proteins - Structure And Molecular Properties, T. E. Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993); Posttranslational Covalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al, Ann NY Acad Sci 663:48-62 (1992)). The present invention also provides for fusion proteins comprising an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof, and a heterologous polypeptide. The heterologous polypeptide to which the antibody is fused may be useful for function or is useful to target the α6β4 integrin polypeptide expressing cells. In one embodiment, a fusion protein of the invention comprises, consists essentially of, or consists of, a polypeptide having the amino acid sequence of any one or more of the VH regions of an antibody of the invention or the amino acid sequence of any one or more of the VL regions of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises, consists essentially of, or consists of a polypeptide having the amino acid sequence of any one, two, three of the VH-CDRs of an α6β4 integrin-specifϊc antibody, or fragments, variants, or derivatives thereof, or the amino acid sequence of any one, two, three of the VL-CDRs of an α6β4 integrin-specific antibody, or fragments, variants, or derivatives thereof, and a heterologous polypeptide sequence. In one embodiment, the fusion protein comprises a polypeptide having the amino acid sequence of a VH-CDR3 of an α6β4 integrin-specific antibody of the present invention, or fragment, derivative, or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to at least one epitope of α6β4 integrin. In another embodiment, a fusion protein comprises a polypeptide having the amino acid sequence of at least one VH region of an α6β4 integrin-specific antibody of the invention and the amino acid sequence of at least one VL region of an α6β4 integrin-specific antibody of the invention or fragments, derivatives or variants thereof, and a heterologous polypeptide sequence. Preferably, the VH and VL regions of the fusion protein correspond to a single source antibody (or scFv or Fab fragment) which specifically binds at least one epitope of α6β4 integrin. In yet another embodiment, a fusion protein for use in the diagnostic and treatment methods disclosed herein comprises a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of an α6β4 integrin-specific antibody and the amino acid sequence of any one, two, three or more of the VL CDRs of an α6β4 integrin-specific antibody, or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, two, three, four, five, six, or more of the VH-CDR(s) or VL-CDR(s) correspond to single source antibody (or scFv or Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. Exemplary fusion proteins reported in the literature include fusions of the T cell receptor (Gascoigne et al, Proc. Natl. Acad. Sci. USA 54:2936-2940 (1987)); CD4 (Capon et al., Nature 537:525-531 (1989); Traunecker et al, Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA 9:347-353 (1990); and Byrn et al., Nature 344:661- 670 (1990)); L-selectin (homing receptor) (Watson et al., J. Cell. Biol. 110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991)); CD44 (Aruffo et al., Cell 67:1303-1313 (1990)); CD28 and B7 (Linsley et al., J. Exp. Med. 773:721-730 (1991)); CTLA-4 (Lisley et al, J. Exp. Med. J 74:561-569 (1991)); CD22 (Stamenkovic et al, Cell 66: 1133-1144 (1991)); TNF receptor (Ashkenazi et al, Proc. Natl. Acad. Sci. USA 55:10535-10539 (1991); Lesslauer et al, Eur. J. Immunol. 27:2883-2886 (1991); and Peppel et al, J. Exp. Med. 774:1483-1489 (1991)); and IgE receptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No. 1448 (1991)).
[0395] As discussed elsewhere herein, α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be fused to heterologous polypeptides to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. For example, in one embodiment, PEG can be conjugated to the α6β4 integrin antibodies of the invention to increase their half- life in vivo. Leong, S.R., et al, Cytokine /(5:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weir et al, Biochem. Soc. Transactions 30:512 (2002).
[0396] Moreover, α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be fused to marker sequences, such as a peptide to facilitate their purification or detection. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 86:S2l- 824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, Cell 37:161 (1984)) and the "flag" tag.
[0397] Fusion proteins can be prepared using methods that are well known in the art (see for example US Patent Nos. 5,116,964 and 5,225,538). The precise site at which the fusion is made may be selected empirically to optimize the secretion or binding characteristics of the fusion protein. DNA encoding the fusion protein is then transfected into a host cell for expression.
[0398] α6β4 integrin antibodies of the present invention may be used in non-conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the patient. α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can be labeled or conjugated either before or after purification, when purification is performed.
[0399] In particular, α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
[0400] Those skilled in the art will appreciate that conjugates may also be assembled using a variety of techniques depending on the selected agent to be conjugated. For example, conjugates with biotin are prepared e.g. by reacting a binding polypeptide with an activated ester of biotin such as the biotin N-hydroxysuccinimide ester. Similarly, conjugates with a fluorescent marker may be prepared in the presence of a coupling agent, e.g. those listed herein, or by reaction with an isothiocyanate, preferably fluorescein-isothiocyanate. Conjugates of the α6β4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention are prepared in an analogous manner.
[0401] The present invention further encompasses α6β4 integrin antibodies, or antigen- binding fragments, variants, or derivatives thereof of the invention conjugated to a diagnostic or therapeutic agent. The α6β4 integrin antibodies can be used diagnostically to, for example, monitor the development or progression of a neurological disease as part of a clinical testing procedure to, e.g. , determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, e.g., U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 1311, 111In or 99Tc.
[0402] An α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged α6β4 integrin antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
[0403] One of the ways in which an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol. 37:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, FIa., (1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme, which is bound to the α6β4 integrin antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
[0404] Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof, it is possible to detect the antibody through the use of a radioimmunoassay (RIA) {see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
[0405] An α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0406] Techniques for conjugating various moieties to an α6β4 integrin antibody, or antigen-binding fragment, variant, or derivative thereof are well known, see, e.g., Arnon et ah, "Monoclonal Antibodies For Immunotargeting Of Drugs hi Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents hi Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).
[0407] hi particular, binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin- specific antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG. hi another embodiment, a binding molecule, e.g., a binding polypeptide, e.g., an α6β4 integrin-specific antibody or immunospecific fragment thereof for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that decreases vascularization of tumors. In other embodiments, the disclosed compositions may comprise binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof coupled to drugs or prodrugs. Still other embodiments of the present invention comprise the use of binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin or diphtheria toxin. The selection of which conjugated or unconjugated binding molecule to use will depend on the type and stage of cancer, use of adjunct treatment (e.g., chemotherapy or external radiation) and patient condition. It will be appreciated that one skilled in the art could readily make such a selection in view of the teachings herein.
[0408] It will be appreciated that, in previous studies, anti-tumor antibodies labeled with isotopes have been used successfully to destroy cells in solid tumors as well as lymphomas/leukemias in animal models, and in some cases in humans. Exemplary radioisotopes include: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. The radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death. The isotopes used to produce therapeutic conjugates typically produce high energy α- or β-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non-immunogenic.
[0409] With respect to the use of radiolabeled conjugates in conjunction with the present invention, binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof may be directly labeled (such as through iodination) or may be labeled indirectly through the use of a chelating agent. As used herein, the phrases "indirect labeling" and "indirect labeling approach" both mean that a chelating agent is covalently attached to a binding molecule and at least one radionuclide is associated with the chelating agent. Such chelating agents are typically referred to as bifunctional chelating agents as they bind both the polypeptide and the radioisotope. Particularly preferred chelating agents comprise l-isothiocycmatobenzyl-3- methyldiothelene triaminepentaacetic acid ("MX-DTPA") and cyclohexyl diethylenetriamine pentaacetic acid ("CHX-DTPA") derivatives. Other chelating agents comprise P-DOTA and EDTA derivatives. Particularly preferred radionuclides for indirect labeling include 111In and 90Y.
[0410] As used herein, the phrases "direct labeling" and "direct labeling approach" both mean that a radionuclide is covalently attached directly to a polypeptide (typically via an amino acid residue). More specifically, these linking technologies include random labeling and site-directed labeling. In the latter case, the labeling is directed at specific sites on the polypeptide, such as the N-linked sugar residues present only on the Fc portion of the conjugates. Further, various direct labeling techniques and protocols are compatible with the instant invention. For example, Technetium-99 labeled polypeptides may be prepared by ligand exchange processes, by reducing pertechnate (TcO4 ") with stannous ion solution, chelating the reduced technetium onto a Sephadex column and applying the binding polypeptides to this column, or by batch labeling techniques, e.g. by incubating pertechnate, a reducing agent such as SnCl2, a buffer solution such as a sodium-potassium phthalate-solution, and the antibodies. In any event, preferred radionuclides for directly labeling antibodies are well known in the art and a particularly preferred radionuclide for direct labeling is 131I covalently attached via tyrosine residues. Binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecifϊc fragments thereof for use in the diagnostic and treatment methods disclosed herein may be derived, for example, with radioactive sodium or potassium iodide and a chemical oxidizing agent, such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidizing agent, such as lactoperoxidase, glucose oxidase and glucose.
[0411] Patents relating to chelators and chelator conjugates are known in the art. For instance, U.S. Patent No. 4,831,175 of Gansow is directed to polysubstituted diethylenetriaminepentaacetic acid chelates and protein conjugates containing the same, and methods for their preparation. U.S. Patent Nos. 5,099,069, 5,246,692, 5,286,850, 5,434,287 and 5,124,471 of Gansow also relate to polysubstituted DTPA chelates. These patents are incorporated herein by reference in their entireties. Other examples of compatible metal chelators are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), 1,4,8,11-tetraazatetradecane, 1,4,8,11- tetraazatetradecane- 1 ,4,8, 11 -tetraacetic acid, 1 -oxa-4,7, 12, 15-tetraazaheptadecane- 4,7,12,15-tetraacetic acid, or the like. Cyclohexyl-DTPA or CHX-DTPA is particularly preferred and is exemplified extensively below. Still other compatible chelators, including those yet to be discovered, may easily be discerned by a skilled artisan and are clearly within the scope of the present invention.
[0412] Compatible chelators, including the specific bifunctional chelator used to facilitate chelation U.S. Patent Nos. 6,682,134, 6,399,061, and 5,843,439, incorporated herein by reference in their entireties, are preferably selected to provide high affinity for trivalent metals, exhibit increased tumor-to-non-tumor ratios and decreased bone uptake as well as greater in vivo retention of radionuclide at target sites, i.e., B-cell lymphoma tumor sites. However, other bifunctional chelators that may or may not possess all of these characteristics are known in the art and may also be beneficial in tumor therapy.
[0413] It will also be appreciated that, in accordance with the teachings herein, binding molecules may be conjugated to different radiolabels for diagnostic and therapeutic purposes. To this end the aforementioned U.S. Patent Nos. 6,682,134, 6,399,061, and 5,843,439 disclose radiolabeled therapeutic conjugates for diagnostic "imaging" of tumors before administration of therapeutic antibody. "In2B8" conjugate comprises a murine monoclonal antibody, 2B8, specific to human CD20 antigen, that is attached to 111In via a bifunctional chelator, i.e., MX-DTPA (diethylenetriaminepentaacetic acid), which comprises a 1 :1 mixture of l-isothiocyanatobenzyl-3-methyl-DTPA and 1-methyl- 3-isothiocyanatobenzyl-DTPA. 111In is particularly preferred as a diagnostic radionuclide because between about 1 to about 10 mCi can be safely administered without detectable toxicity; and the imaging data is generally predictive of subsequent 90Y-labeled antibody distribution. Most imaging studies utilize 5 mCi " 'in-labeled antibody, because this dose is both safe and has increased imaging efficiency compared with lower doses, with optimal imaging occurring at three to six days after antibody administration. See, for example, Murray, J. Nuc. Med. 26: 3328 (1985) and Carraguillo et al, J. Nuc. Med. 26: 67 (1985).
[0414] As indicated above, a variety of radionuclides are applicable to the present invention and those skilled in the can readily determine which radionuclide is most appropriate under various circumstances. For example, 131I is a well known radionuclide used for targeted immunotherapy. However, the clinical usefulness of 131I can be limited by several factors including: eight-day physical half-life; dehalogenation of iodinated antibody both in the blood and at tumor sites; and emission characteristics (e.g., large gamma component) which can be suboptimal for localized dose deposition in tumor. With the advent of superior chelating agents, the opportunity for attaching metal chelating groups to proteins has increased the opportunities to utilize other radionuclides such as 111In and 90Y. 90Y provides several benefits for utilization in radioimmunotherapeutic on applications: the 64 hour half- life of Y is long enough to allow antibody accumulation by tumor and, unlike e.g., 131I, 90Y is a pure beta emitter of high energy with no accompanying gamma irradiation in its decay, with a range in tissue of 100 to 1,000 cell diameters. Furthermore, the minimal amount of penetrating radiation allows for outpatient administration of 90Y-labeled antibodies. Additionally, internalization of labeled antibody is not required for cell killing, and the local emission of ionizing radiation should be lethal for adjacent tumor cells lacking the target molecule.
[0415] Additional preferred agents for conjugation to binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof are cytotoxic drugs, particularly those which are used for cancer therapy. As used herein, "a cytotoxin or cytotoxic agent" means any agent that is detrimental to the growth and proliferation of cells and may act to reduce, inhibit or destroy a cell or malignancy. Exemplary cytotoxins include, but are not limited to, radionuclides, biotoxins, enzymatically active toxins, cytostatic or cytotoxic therapeutic agents, prodrugs, immunologically active ligands and biological response modifiers such as cytokines. Any cytotoxin that acts to retard or slow the growth of immunoreactive cells or malignant cells is within the scope of the present invention.
[0416] Exemplary cytotoxins include, in general, cytostatic agents, alkylating agents, anti-metabolites, antiproliferative agents, tubulin binding agents, hormones and hormone antagonists, and the like. Exemplary cytostatics that are compatible with the present invention include alkylating substances, such as mechlorethamine, triethylenephosphoramide, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan or triaziquone, also nitrosourea compounds, such as carmustine, lomustine, or semustine. Other preferred classes of cytotoxic agents include, for example, the maytansinoid family of drugs. Other preferred classes of cytotoxic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, and the podophyllotoxins. Particularly useful members of those classes include, for example, adriamycin, carminomycin, daunorubicin (daunomycin), doxorubicin, aminopterin, methotrexate, methopterin, mithramycin, streptonigrin, dichloromethotrexate, mitomycin C, actinomycin-D, porfiromycin, 5-fluorouracil, floxuridine, ftorafur, 6-mercaptopurine, cytarabine, cytosine arabinoside, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine and the like. Still other cytotoxins that are compatible with the teachings herein include taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, tenoposide, colchicin, dihydroxy anthracin dione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Hormones and hormone antagonists, such as corticosteroids, e.g. prednisone, progestins, e.g. hydroxyprogesterone or medroprogesterone, estrogens, e.g. diethylstilbestrol, antiestrogens, e.g. tamoxifen, androgens, e.g. testosterone, and aromatase inhibitors, e.g. aminogluthetimide are also compatible with the teachings herein. One skilled in the art may make chemical modifications to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the invention.
[0417] One example of particularly preferred cytotoxins comprise members or derivatives of the enediyne family of anti-tumor antibiotics, including calicheamicin, esperamicins or dynemicins. These toxins are extremely potent and act by cleaving nuclear DNA, leading to cell death. Unlike protein toxins which can be cleaved in vivo to give many inactive but immunogenic polypeptide fragments, toxins such as calicheamicin, esperamicins and other enediynes are small molecules which are essentially non-immunogenic. These non-peptide toxins are chemically-linked to the dimers or tetramers by techniques which have been previously used to label monoclonal antibodies and other molecules. These linking technologies include site-specific linkage via the N-linked sugar residues present only on the Fc portion of the constructs. Such site-directed linking methods have the advantage of reducing the possible effects of linkage on the binding properties of the constructs.
[0418] As previously alluded to, compatible cytotoxins for preparation of conjugates may comprise a prodrug. As used herein, the term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into the more active parent form. Prodrugs compatible with the invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate containing prodrugs, peptide containing prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to the more active cytotoxic free drug. Further examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention comprise those chemotherapeutic agents described above.
[0419] Among other cytotoxins, it will be appreciated that binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein can also be associated with or conjugated to a biotoxin such as ricin subunit A, abrin, diptheria toxin, botulinum, cyanginosins, saxitoxin, shigatoxin, tetanus, tetrodotoxin, trichothecene, verrucologen or a toxic enzyme. Preferably, such constructs will be made using genetic engineering techniques that allow for direct expression of the antibody-toxin construct. Other biological response modifiers that may be associated with the binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin- specific antibodies or immunospecific fragments thereof disclosed herein comprise cytokines such as lymphokines and interferons. In view of the instant disclosure it is submitted that one skilled in the art could readily form such constructs using conventional techniques.
[0420] Another class of compatible cytotoxins that may be used in association with or conjugated to the disclosed binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof, are radiosensitizing drugs that may be effectively directed to tumor or immunoreactive cells. Such drugs enhance the sensitivity to ionizing radiation, thereby increasing the efficacy of radiotherapy. An antibody conjugate internalized by the tumor cell would deliver the radiosensitizer nearer the nucleus where radiosensitization would be maximal. The unbound radiosensitizer linked binding molecules of the invention would be cleared quickly from the blood, localizing the remaining radiosensitization agent in the target tumor and providing minimal uptake in normal tissues. After rapid clearance from the blood, adjunct radiotherapy would be administered in one of three ways: 1.) external beam radiation directed specifically to the tumor, 2.) radioactivity directly implanted in the tumor or 3.) systemic radioimmunotherapy with the same targeting antibody. A potentially attractive variation of this approach would be the attachment of a therapeutic radioisotope to the radiosensitized immunoconjugate, thereby providing the convenience of administering to the patient a single drug.
[0421] In certain embodiments, a moiety that enhances the stability or efficacy of a binding molecule, e.g., a binding polypeptide, e.g., an α6β4 integrin -specific antibody or immunospecific fragment thereof can be conjugated. For example, in one embodiment, PEG can be conjugated to the binding molecules of the invention to increase their half- life in vivo. Leong, S.R., et al., Cytokine 75:106 (2001); Adv. in DrugDeliv. Rev. 54:531 (2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).
[0422] The present invention further encompasses the use of binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments conjugated to a diagnostic or therapeutic agent. The binding molecules can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen. Detection can be facilitated by coupling the binding molecule, e.g., binding polypeptide, e.g., α6β4 integrin-specific antibody or immunospecific fragment thereof to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 1251, 1311, 111In or 99Tc. [0423] A binding molecule, e.g., a binding polypeptide, e.g., an α6β4 integrin-specific antibody or immunospecific fragment thereof also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged binding molecule is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
[0424] One of the ways in which a binding molecule, e.g., a binding polypeptide, e.g., an α6β4 integrin-specific antibody or immunospecific fragment thereof can be detectably labeled is by linking the same to an enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological Associates Quarterly Publication, Walkersville, Md., Diagnostic Horizons 2:1-7 (1978)); Voller et ah, J. Clin. Pathol. 57:507-520 (1978); Butler, J. E., Meth. Enrymol. 75:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, FIa., (1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo (1981). The enzyme, which is bound to the binding molecule will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 -steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose- 6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
[0425] Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the binding molecule, e.g., binding polypeptide, e.g., α6β4 integrin-specific antibody or immunospecific fragment thereof, it is possible to detect cancer antigens through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, (March, 1986)), which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.
[0426] A binding molecule, e.g., a binding polypeptide, e.g., an α6β4 integrin- specific antibody or immunospecific fragment thereof can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0427] Techniques for conjugating various moieties to a binding molecule, e.g., a binding polypeptide, e.g., an α6β4 integrin-specific antibody or immunospecific fragment thereof are well known, see, e.g., Amon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents hi Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody hi Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).
VII. EXPRESSION OF ANTIBODY POLYPEPTIDES
[0428] As is well known, RNA may be isolated from the original hybridoma cells or from other transformed cells by standard techniques, such as guanidinium isothiocyanate extraction and precipitation followed by centrifugation or chromatography. Where desirable, mRNA may be isolated from total RNA by standard techniques such as chromatography on oligo dT cellulose. Suitable techniques are familiar in the art.
[0429] In one embodiment, cDNAs that encode the light and the heavy chains of the antibody may be made, either simultaneously or separately, using reverse transcriptase and DNA polymerase in accordance with well known methods. PCR may be initiated by consensus constant region primers or by more specific primers based on the published heavy and light chain DNA and amino acid sequences. As discussed above, PCR also may be used to isolate DNA clones encoding the antibody light and heavy chains. In this case the libraries may be screened by consensus primers or larger homologous probes, such as mouse constant region probes.
[0430] DNA, typically plasmid DNA, may be isolated from the cells using techniques known in the art, restriction mapped and sequenced in accordance with standard, well known techniques set forth in detail, e.g., in the foregoing references relating to recombinant DNA techniques. Of course, the DNA may be synthetic according to the present invention at any point during the isolation process or subsequent analysis.
[0431] Following manipulation of the isolated genetic material to provide α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention, the polynucleotides encoding the α6β4 integrin antibodies are typically inserted in an expression vector for introduction into host cells that may be used to produce the desired quantity of α6β4 integrin antibody.
[0432] Recombinant expression of an antibody, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody which binds to a target molecule described herein, e.g., α6β4 integrin, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
[0433] The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain is advantageously placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. ScL USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
[0434] The term "vector" or "expression vector" is used herein to mean vectors used in accordance with the present invention as a vehicle for introducing into and expressing a desired gene in a host cell. As known to those skilled in the art, such vectors may easily be selected from the group consisting of plasmids, phages, viruses and retroviruses. Ln general, vectors compatible with the instant invention will comprise a selection marker, appropriate restriction sites to facilitate cloning of the desired gene and the ability to enter and/or replicate in eukaryotic or prokaryotic cells.
[0435] For the purposes of this invention, numerous expression vector systems may be employed. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by cotransformation. Additional elements may also be needed for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. [0436] In particularly preferred embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (preferably human) synthetic as discussed above, hi one embodiment, this is effected using a proprietary expression vector of Biogen EDEC, Inc., referred to as NEOSPLA (disclosed in U.S. patent 6,159,730). This vector contains the cytomegalovirus promoter/enhancer, the mouse beta globin major promoter, the SV40 origin of replication, the bovine growth hormone polyadenylation sequence, neomycin phosphotransferase exon 1 and exon 2, the dihydrofolate reductase gene and leader sequence. This vector has been found to result in very high level expression of antibodies upon incorporation of variable and constant region genes, transfection in CHO cells, followed by selection in G418 containing medium and methotrexate amplification. Of course, any expression vector which is capable of eliciting expression in eukaryotic cells may be used in the present invention. Examples of suitable vectors include, but are not limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, PSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAXl, and pZeoSV2 (available from Invitrogen, San Diego, CA), and plasmid pCI (available from Promega, Madison, WI). In general, screening large numbers of transformed cells for those which express suitably high levels if immunoglobulin heavy and light chains is routine experimentation which can be carried out, for example, by robotic systems. Vector systems are also taught in U.S. Pat. Nos. 5,736,137 and 5,658,570, each of which is incorporated by reference in its entirety herein. This system provides for high expression levels, e.g., > 30 pg/cell/day. Other exemplary vector systems are disclosed e.g., in U.S. Patent 6,413,777.
[0437] In other preferred embodiments the α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention may be expressed using polycistronic constructs such as those disclosed in United States Patent Application Publication No. 2003-0157641 Al, filed November 18, 2002 and incorporated herein in its entirety. In these novel expression systems, multiple gene products of interest such as heavy and light chains of antibodies may be produced from a single polycistronic construct. These systems advantageously use an internal ribosome entry site (IRES) to provide relatively high levels of α6β4 integrin antibodies, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof in eukaryotic host cells. Compatible ERES sequences are disclosed in U.S. Pat. No. 6,193,980 which is also incorporated herein. Those skilled in the art will appreciate that such expression systems may be used to effectively produce the full range of α6β4 integrin antibodies disclosed in the instant application.
[0438] More generally, once the vector or DNA sequence encoding a monomelic subunit of the α6β4 integrin antibody has been prepared, the expression vector may be introduced into an appropriate host cell. Introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, Ridgway, A. A. G. "Mammalian Expression Vectors" Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction into the host is via electroporation. The host cells harboring the expression construct are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy and/or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence-activated cell sorter analysis (FACS), immunohistochemistry and the like.
[0439] The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody for use in the methods described herein. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
[0440] As used herein, "host cells" refers to cells which harbor vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene. In descriptions of processes for isolation of antibodies from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of antibody unless it is clearly specified otherwise. In other words, recovery of polypeptide from the "cells" may mean either from spun down whole cells, or from the cell culture containing both the medium and the suspended cells.
[0441] A variety of host-expression vector systems may be utilized to express antibody molecules for use in the methods described herein. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichiά) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
[0442] The host cell line used for protein expression is often of mammalian origin; those skilled in the art are credited with ability to preferentially determine particular host cell lines which are best suited for the desired gene product to be expressed therein. Exemplary host cell lines include, but are not limited to, CHO (Chinese Hamster Ovary), DG44 and DUXBI l (Chinese Hamster Ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI with S V40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line), SP2/0 (mouse myeloma), P3x63-Ag3.653 (mouse myeloma), BFA-IcIBPT (bovine endothelial cells), RAJI (human lymphocyte) and 293 (human kidney). CHO cells are particularly preferred. Host cell lines are typically available from commercial services, the American Tissue Culture Collection or from published literature.
[0443] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
[0444] For long-term, high- yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which stably express the antibody molecule.
[0445] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22:817 1980) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sd. USA 77:357 (1980); O'Hare et al, Proc. Natl. Acad. Sd. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 72:488- 505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 52:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993);, TIB TECH ll(5):\55-2\5 (May, 1993); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. MoI. Biol. J 50:1 (1981), which are incorporated by reference herein in their entireties.
[0446] The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Academic Press, New York, Vol. 3. (1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, MoI. Cell. Biol. 3:257 (1983)).
[0447] In vitro production allows scale-up to give large amounts of the desired polypeptides. Techniques for mammalian cell cultivation under tissue culture conditions are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary and/or desired, the solutions of polypeptides can be purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or (immuno-)affinity chromatography, e.g., after preferential biosynthesis of a synthetic hinge region polypeptide or prior to or subsequent to the HIC chromatography step described herein.
[0448] Genes encoding α6β4 integrin antibodies, or antigen-binding fragments, variants, or derivatives thereof of the invention can also be expressed non-mammalian cells such as bacteria or insect or yeast or plant cells. Bacteria which readily take up nucleic acids include members of the enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that, when expressed in bacteria, the heterologous polypeptides typically become part of inclusion bodies. The heterologous polypeptides must be isolated, purified and then assembled into functional molecules. Where tetravalent forms of antibodies are desired, the subunits will then self- assemble into tetravalent antibodies (WO02/096948A2).
[0449] In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 75:3101- 3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione- agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
[0450] In addition to prokaryotes, eukaryotic microbes may also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among eukaryotic microorganisms although a number of other strains are commonly available, e.g. , Pichia pastoris.
[0451] For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al, Gene 7:141 (1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. This plasmid already contains the TRPl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
[0452] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is typically used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into nonessential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
[0453] Once an antibody molecule of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography {e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Alternatively, a preferred method for increasing the affinity of antibodies of the invention is disclosed in US 2002 0123057 Al.
VIII. TREATMENT METHODS USING THERAPEUTIC α6β4 INTEGRIN-SPECIFIC ANTIBODIES, OR DVIMUNOSPECIFIC FRAGMENTS THEREOF
[0454] One embodiment of the present invention provides methods for treating a hyperproliferative disease or disorder, e.g. , cancer, a malignancy, a tumor, or a metastasis thereof, in an animal suffering from such disease or predisposed to contract such disease, the method comprising, consisting essentially of, or consisting of administering to the animal an effective amount of an antibody or immunospecific fragment thereof, that binds to α6β4 integrin or a variant of α6β4 integrin. Suitable antibodies include all antibodies and antigen-specific fragments thereof described herein. Examples include, but are not limited to, an isolated antibody or antigen-binding fragment thereof which specifically binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2, an isolated antibody or antigen- binding fragment thereof which specifically binds to α6β4 integrin, where the antibody or fragment thereof competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66- H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to α6β4 integrin, or an isolated antibody or antigen-binding fragment thereof which specifically binds to α6β4 integrin, where the antibody or fragment thereof comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2. In certain embodiments an antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof inhibits laminin from binding to α6β4 integrin. In a further embodiment, an antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof expressed on a cell, in particular a tumor cell inhibits activation of downstream signal transduction molecules involved in cell proliferation, motility and/or metastasis. Such molecules include, but are not limited to PI3 -K, Akt, mTOR and Rac. In a further embodiment, an antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits activation of the Ras/MAPK signaling pathway. In still a further embodiment, an antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits the interaction of α6β4 with growth factor receptors such as erbB2, Met, and Ron. In yet a further embodiment, an antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits integrin clustering in lipid rafts. In still further embodiments, an antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits the interaction with cytoskeletal components, induces apoptosis, and induces integrin internalization. In yet a further embodiment, an antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof expressed on a cell, in particular a tumor cell, inhibits cell proliferation, motility, and/or metastasis.
[0456] An antibody of the present invention which specifically binds to α6β4 integrin or a variant thereof, to be used in treatment methods disclosed herein can be prepared and used as a therapeutic agent that stops, reduces, prevents, or inhibits cellular activities involved in cellular hyperproliferation, e.g., cellular activities that induce the altered or abnormal pattern of vascularization that is often associated with hyperproliferative diseases or disorders.
[0457] Antibodies or immunospecific fragments thereof of the present invention include, but are not limited to monoclonal, chimeric or humanized antibodies, and fragments of antibodies that bind specifically to tumor-associated proteins such as α6β4 integrin. The antibodies may be monovalent, bivalent, polyvalent, or bifunctional antibodies, and the antibody fragments include Fab F(ab')2, and Fv.
[0458] Therapeutic antibodies according to the invention can be used in unlabeled or unconjugated form, or can be coupled or linked to cytotoxic moieties such as radiolabels and biochemical cytotoxins to produce agents that exert therapeutic effects.
[0459] In certain embodiments, an antibody, or immunospecific fragment thereof of the invention includes an antigen binding domain. An antigen binding domain is formed by antibody variable regions that vary from one antibody to another. Naturally occurring antibodies comprise at least two antigen binding domains, i.e., they are at least bivalent. As used herein, the term "antigen binding domain" includes a site that specifically binds an epitope on an antigen (e.g., a cell surface or soluble antigen). The antigen binding domain of an antibody typically includes at least a portion of an immunoglobulin heavy chain variable region and at least a portion of an immunoglobulin light chain variable region. The binding site formed by these variable regions determines the specificity of the antibody.
[0460] The present invention provides methods for treating various hyperproliferative disorders, e.g., by inhibiting tumor growth, in a mammal, comprising, consisting essentially of, or consisting of administering to the mammal an effective amount of a antibody or antigen-binding fragment thereof which specifically or preferentially binds to α6β4 integrin, e.g., human α6β4 integrin.
[0461] The present invention is more specifically directed to a method of treating a hyperproliferative disease, e.g., inhibiting or preventing tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation, in an animal, e.g., a mammal, e.g., a human, comprising, consisting essentially of, or consisting of administering to an animal in need thereof an effective amount of a an antibody or immunospecific fragment thereof, which specifically or preferentially binds to one or more epitopes of α6β4 integrin.
[0462] hi other embodiments, the present invention includes a method for treating a hyperproliferative disease, e.g., inhibiting tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation in an animal, e.g., a human patient, where the method comprises administering to an animal in need of such treatment an effective amount of a composition comprising, consisting essentially of, or consisting of, in addition to a pharmaceutically acceptable carrier, an antibody, or immunospecific fragment thereof, which specifically binds to at least one epitope of α6β4 integrin, where the epitope comprises, consists essentially of, or consists of at least about four to five amino acids amino acids of SEQ ED NOs: 1-15, at least seven, at least nine, or between at least about 15 to about 30 amino acids of SEQ ED NOs:l-15. The amino acids of a given epitope of SEQ ED NOs:l-15 as described may be, but need not be contiguous.
[0463] Ln certain embodiments, the at least one epitope of α6β4 integrin comprises, consists essentially of, or consists of a non-linear epitope formed by the extracellular domain of α6β4. integrin as expressed on the surface of a cell. Thus, in certain embodiments the at least one epitope of α6β4 integrin comprises, consists essentially of, or consists of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of SEQ ED NOs: 1-15, where non-contiguous amino acids form an epitope through protein folding.
[0464] In certain embodiments, the present invention includes an antibody, or antigen- binding fragment, variant, or derivative thereof which specifically or preferentially binds to at least one epitope of α6β4 integrin, where the epitope comprises, consists essentially of, or consists of a portion of α6 and a portion of β4. Thus, in certain embodiments at least one epitope of α6β4 integrin comprises, consists essentially of, or consists of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or noncontiguous amino acids of an α6 sequence selected from the group consisting of SEQ ID NOs: 1-8 and at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, between about 15 to about 30, or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 contiguous or non-contiguous amino acids of a β4 sequence selected from the group consisting of SEQ ED NOs:9-15, where non-contiguous amino acids form an epitope through protein folding.
[0465] In other embodiments, the present invention includes a method for treating a hyperproliferative disease, e.g., inhibiting tumor formation, tumor growth, tumor invasiveness, and/or metastasis formation in an animal, e.g., a human patient, where the method comprises administering to an animal in need of such treatment an effective amount of a composition comprising, consisting essentially of, or consisting of, in addition to a pharmaceutically acceptable carrier, an antibody, or immunospecific fragment thereof, which specifically binds to at least one epitope of α6β4 integrin, where the epitope comprises, consists essentially of, or consists of, in addition to one, two, three, four, five, six or more contiguous or non-contiguous amino acids of SEQ ID NOs:l-15 as described above, and an additional moiety which modifies the protein, e.g., a carbohydrate moiety may be included such that the binding molecule binds with higher affinity to modified target protein than it does to an unmodified version of the protein. Alternatively, the binding molecule does not bind the unmodified version of the target protein at all.
[0466] More specifically, the present invention provides a method of treating cancer in a human, comprising administering to a human in need of treatment a composition comprising an effective amount of an α6β4 integrin-specific antibody or immunospecific fragment thereof, and a pharmaceutically acceptable carrier. Types of cancer to be treated include, but are not limited to, stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer. [0467] In certain embodiments, an antibody or fragment thereof binds specifically to at least one epitope of α6β4 integral or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to an unrelated, or random epitope; binds preferentially to at least one epitope of α6β4 integrin or fragment or variant described above, i.e., binds to such an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope; competitively inhibits binding of a reference antibody which itself binds specifically or preferentially to a certain epitope of α6β4 integrin or fragment or variant described above; or binds to at least one epitope of α6β4 integrin or fragment or variant described above with an affinity characterized by a dissociation constant KD of less than about 5 x 10"2 M, about 10"2 M, about 5 x 10"3 M, about 10"3 M, about 5 x 10"4 M, about 10"4M, about 5 x 10"5 M, about 10"5 M, about 5 x 10"6M, about 10" 6 M, about 5 x 10"7 M, about 10"7 M, about 5 x 10"8 M, about 10"8 M, about 5 x 10"9 M, about 10"9M, about 5 x 10"10M, about 10"10M, about 5 x 10"11 M, about 10"11 M, about 5 x 10"12 M, about 10"12 M, about 5 x 10"13 M, about 10"13 M, about 5 x 10"14M, about 10"14 M, about 5 x 10"15 M, or about 10"15 M. As used in the context of antibody binding dissociation constants, the term "about" allows for the degree of variation inherent in the methods utilized for measuring antibody affinity. For example, depending on the level of precision of the instrumentation used, standard error based on the number of samples measured, and rounding error, the term "about 10"2 M" might include, for example, from 0.05 M to 0.005 M. In certain embodiments, antibodies and fragments thereof of the present invention cross-react with α6β4 integrin proteins of other species from which they were raised, e.g., an antibody or fragment thereof which specifically binds to human α6β4 integrin also binds to murine α6β4 integrin. Other suitable antibodies or fragments thereof of the present invention include those that are highly species specific.
[0468] In specific embodiments, antibodies or immunospecific fragments thereof disclosed herein bind α6β4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10"2 sec"1, 10"2 sec"1, 5 X 10"3 sec"1 or 10"3 sec"1. Other antibodies or immunospecific fragments thereof disclosed herein bind α6β4 integrin polypeptides or fragments or variants thereof with an off rate (k(off)) of less than or equal to 5 X 10"4 sec"1, 10"4 sec"1, 5 X 105 sec"1, or 10"5 sec"1 5 X 10"6 sec"1, 10"6 sec"1, 5 X lO-7 SeC-1 Or IO-7 SeC"1. [0469] In other embodiments, antibodies or immunospecific fragments thereof disclosed herein bind α6β4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) of greater than or equal to 103 M'1 sec"1, 5 X 103 M"1 sec"1, 104 M"1 sec"1 or 5 X 104 M"1 sec"1. Other antibodies or immunospecific fragments thereof for use in the diagnostic and treatment methods disclosed herein bind α6β4 integrin polypeptides or fragments or variants thereof with an on rate (k(on)) greater than or equal to 105 M"1 sec"1, 5 X 105 M"1 sec"1, 106 M"1 sec'1, or 5 X 106 M"1 sec"1 or 107 M"1 sec"1.
[0470] hi various embodiments, one or more binding molecules as described above is an antagonist of α6β4 integrin activity, for example, binding of an antagonist α6β4 integrin antibody to α6β4 integrin as expressed on a tumor cell inhibits binding of laminin, inhibits activation of molecules downstream in the signal transduction pathway, e.g., PI3- K, Akt, mTOR and RAC , or inhibits tumor cell proliferation, motility or metastasis.
IX. DIAGNOSTIC OR PROGNOSTIC METHODS USING α6β4 INTEGRIN-SPECIFIC BINDING MOLECULES AND NUCLEIC ACID AMPLIFICATION ASSAYS
[0471] α6β4 integrin-specific antibodies, or fragments, derivatives, or analogs thereof, can be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and/or conditions associated with the aberrant expression and/or activity of α6β4 integrin. α6β4 integrin expression is increased in tumor tissue and other neoplastic conditions.
[0472] α6β4 integrin-specific antibodies or fragments thereof, are useful for diagnosis, treatment, prevention and/or prognosis of hyperproliferative disorders in mammals, preferably humans. Such disorders include, but are not limited to, cancer, neoplasms, tumors and/or as described under elsewhere herein, especially α6β4 integrin-associated cancers such as stomach cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
[0473] For example, as disclosed herein, α6β4 integrin expression is associated with at least stomach, brain, bladder, colon, lung, breast, pancreatic, ovarian, and prostate tumor tissues. Accordingly, antibodies (and antibody fragments) directed against α6β4 integrin may be used to detect particular tissues expressing increased levels of α6β4 integrin. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.
[0474] Thus, the invention provides a diagnostic method useful during diagnosis of a cancers and other hyperproliferative disorders, which involves measuring the expression level of α6β4 integrin protein or transcript in tissue or other cells or body fluid from an individual and comparing the measured expression level with a standard α6β4 integrin expression levels in normal tissue or body fluid, whereby an increase in the expression level compared to the standard is indicative of a disorder.
[0475] One embodiment provides a method of detecting the presence of abnormal hyperproliferative cells, e.g., precancerous or cancerous cells, in a fluid or tissue sample, comprising assaying for the expression of α6β4 integrin in tissue or body fluid samples of an individual and comparing the presence or level of α6β4 integrin expression in the sample with the presence or level of α6β4 integrin expression in a panel of standard tissue or body fluid samples, where detection of α6β4 integrin expression or an increase in α6β4 integrin expression over the standards is indicative of aberrant hyperproliferative cell growth.
[0476] More specifically, the present invention provides a method of detecting the presence of abnormal hyperproliferative cells in a body fluid or tissue sample, comprising (a) assaying for the expression of α6β4 integrin in tissue or body fluid samples of an individual using α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention, and (b) comparing the presence or level of α6β4 integrin expression in the sample with a the presence or level of α6β4 integrin expression in a panel of standard tissue or body fluid samples, whereby detection of α6β4 integrin expression or an increase in α6β4 integrin expression over the standards is indicative of aberrant hyperproliferative cell growth.
[0477] With respect to cancer, the presence of a relatively high amount of α6β4 integrin protein in biopsied tissue from an individual may indicate the presence of a tumor or other malignant growth, may indicate a predisposition for the development of such malignancies or tumors, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
[0478] α6β4 integrin-specific antibodies of the present invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al, J. Cell Biol. 707:976-985 (1985); Jalkanen, et al, J. Cell Biol. 705:3087-3096 (1987)). Other antibody-based methods useful for detecting protein expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. Suitable assays are described in more detail elsewhere herein.
[0479] One aspect of the invention is a method for the in vivo detection or diagnosis of a hyperproliferative disease or disorder associated with aberrant expression of α6β4 integrin in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled antibody or fragment thereof of the present invention, which specifically binds to α6β4 integrin; b) waiting for a time interval following the administering for permitting the labeled binding molecule to preferentially concentrate at sites in the subject where α6β4 integrin is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of α6β4 integrin. Background level can be determined by various methods including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
[0480] It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of, e.g., 99Tc. The labeled binding molecule, e.g., antibody or antibody fragment, will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982). [0481] Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours, hi another embodiment the time interval following administration is 5 to 20 days or 7 to 10 days.
[0482] Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
[0483] In a specific embodiment, the binding molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). hi another embodiment, the binding molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument, hi another embodiment, the binding molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography, hi yet another embodiment, the binding molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
[0484] Antibody labels or markers for in vivo imaging of α6β4 integrin expression include those detectable by X-radiography, nuclear magnetic resonance imaging (NMR), MRI, CAT-scans or electron spin resonance imaging (ESR). For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR. include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where in vivo imaging is used to detect enhanced levels of α6β4 integrin expression for diagnosis in humans, it may be preferable to use human antibodies or "humanized" chimeric monoclonal antibodies as described elsewhere herein. [0485] In a related embodiment to those described above, monitoring of an already diagnosed disease or disorder is carried out by repeating any one of the methods for diagnosing the disease or disorder, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
[0486] Where a diagnosis of a disorder, including diagnosis of a tumor, has already been made according to conventional methods, detection methods as disclosed herein are useful as a prognostic indicator, whereby patients continuing to exhibiting enhanced α6β4 integrin expression will experience a worse clinical outcome relative to patients whose expression level decreases nearer the standard level.
[0487] By "assaying the expression level of the tumor associated α6β4 integrin polypeptide" is intended qualitatively or quantitatively measuring or estimating the level of α6β4 integrin polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level) or relatively (e.g., by comparing to the cancer associated polypeptide level in a second biological sample). Preferably, α6β4 integrin polypeptide expression level in the first biological sample is measured or estimated and compared to a standard α6β4 integrin polypeptide level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once the "standard" α6β4 integrin polypeptide level is known, it can be used repeatedly as a standard for comparison.
[0488] By "biological sample" is intended any biological sample obtained from an individual, cell line, tissue culture, or other source of cells potentially expressing α6β4 integrin. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid), and other tissue sources which contain cells potentially expressing α6β4 integrin. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art.
[0489] In an additional embodiment, antibodies, or immunospecific fragments of antibodies directed to a conformational epitope of α6β4 integrin may be used to quantitatively or qualitatively detect the presence of α6β4 integrin gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluoresence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection. [0490] Cancers that may be diagnosed, and/or prognosed using the methods described above include but are not limited to, stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
X. IMMUNOASSAYS
[0491] α6β4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and noncompetitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994), which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).
[0492] Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors {e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4.degree. C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4. degree. C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994) at 10.16.1. [0493] Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32p or 1251) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York Vol. 1 (1994) at 10.8.1.
[0494] ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. F or further discussion regarding ELISAs see, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1 (1994) at 11.2.1.
[0495] The binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen {e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.
[0496] α6β4 integrin-specific antibodies may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of cancer antigen gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled α6β4 integrin-specific antibody or fragment thereof, preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of α6β4 integrin protein, or conserved variants or peptide fragments, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
[0497] Immunoassays and non-immunoassays for α6β4 integrin gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding to α6β4 integrin or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.
[0498] The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled α6β4 integrin-specific antibody. The solid phase support may then be washed with the buffer a second time to remove unbound antibody. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.
[0499] By "solid phase support or carrier" is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
[0500] The binding activity of a given lot of α6β4 integrin-specific antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
[0501] There are a variety of methods available for measuring the affinity of an antibody- antigen interaction, but relatively few for determining rate constants. Most of the methods rely on either labeling antibody or antigen, which inevitably complicates routine measurements and introduces uncertainties in the measured quantities.
[0502] Surface plasmon reasonance (SPR) as performed on BIAcore offers a number of advantages over conventional methods of measuring the affinity of antibody-antigen interactions: (i) no requirement to label either antibody or antigen; (ii) antibodies do not need to be purified in advance, cell culture supernatant can be used directly; (iii) real-time measurements, allowing rapid semi-quantitative comparison of different monoclonal antibody interactions, are enabled and are sufficient for many evaluation purposes; (iv) biospecific surface can be regenerated so that a series of different monoclonal antibodies can easily be compared under identical conditions; (v) analytical procedures are fully automated, and extensive series of measurements can be performed without user intervention. BIAapplications Handbook, version AB (reprinted 1998), BIACORE code No. BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), BIACORE code No. BR-1001-84.
[0503] SPR based binding studies require that one member of a binding pair be immobilized on a sensor surface. The binding partner immobilized is referred to as the ligand. The binding partner in solution is referred to as the analyte. In some cases, the ligand is attached indirectly to the surface through binding to another immobilized molecule, which is referred as the capturing molecule. SPR response reflects a change in mass concentration at the detector surface as analytes bind or dissociate.
[0504] Based on SPR, real-time BIAcore measurements monitor interactions directly as they happen. The technique is well suited to determination of kinetic parameters. Comparative affinity ranking is extremely simple to perform, and both kinetic and affinity constants can be derived from the sensorgram data.
[0505] When analyte is injected in a discrete pulse across a ligand surface, the resulting sensorgram can be divided into three essential phases: (i) Association of analyte with ligand during sample injection; (ii) Equilibrium or steady state during sample injection, where the rate of analyte binding is balanced by dissociation from the complex; (iii) Dissociation of analyte from the surface during buffer flow.
[0506] The association and dissociation phases provide information on the kinetics of analyte-ligand interaction (ka and k<], the rates of complex formation and dissociation, kd/ka = KD). The equilibrium phase provides information on the affinity of the analyte- ligand interaction (KD).
[0507] BIAevaluation software provides comprehensive facilities for curve fitting using both numerical integration and global fitting algorithms. With suitable analysis of the data, separate rate and affinity constants for interaction can be obtained from simple BIAcore investigations. The range of affinities measurable by this technique is very broad ranging from mM to pM.
[0508] Epitope specificity is an important characteristic of a monoclonal antibody.
Epitope mapping with BIAcore, in contrast to conventional techniques using radioimmunoassay, ELISA or other surface adsorption methods, does not require labeling or purified antibodies, and allows multi-site specificity tests using a sequence of several monoclonal antibodies. Additionally, large numbers of analyses can be processed automatically. [0509] Pair-wise binding experiments test the ability of two MAbs to bind simultaneously to the same antigen. MAbs directed against separate epitopes will bind independently, whereas MAbs directed against identical or closely related epitopes will interfere with each other's binding. These binding experiments with BIAcore are straightforward to carry out.
[0510] For example, one can use a capture molecule to bind the first Mab, followed by addition of antigen and second MAb sequentially. The sensorgrams will reveal: 1. how much of the antigen binds to first Mab, 2. to what extent the second MAb binds to the surface-attached antigen, 3. if the second MAb does not bind, whether reversing the order of the pair- wise test alters the results.
[0511] Peptide inhibition is another technique used for epitope mapping. This method can complement pair-wise antibody binding studies, and can relate functional epitopes to structural features when the primary sequence of the antigen is known. Peptides or antigen fragments are tested for inhibition of binding of different MAbs to immobilized antigen. Peptides which interfere with binding of a given MAb are assumed to be structurally related to the epitope defined by that MAb.
XI. PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION METHODS
[0512] Methods of preparing and administering α6β4 integrin-specific antibodies or immunospecific fragments thereof to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of the binding molecule, e.g., binding polypeptide, e.g., α6β4 integrin-specific antibody or immunospecific fragment thereof may be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. While all these forms of administration are clearly contemplated as being within the scope of the invention, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. Usually, a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. However, in other methods compatible with the teachings herein, binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
[0513] Preparations for parenteral administration includes sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
[0514] More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions, hi such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed. (1980).
[0515] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[0516] In any case, sterile injectable solutions can be prepared by incorporating an active compound (e.g., a binding molecule, e.g., a binding polypeptide, e.g., α6β4 integrin- specific antibody or immunospecific fragment thereof, by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S.S.N. 09/259,337 (US-2002-0102208 Al), which is incorporated herein by reference in its entirety. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to autoimmune or neoplastic disorders.
[0517] Effective doses of the compositions of the present invention, for treatment of hyperproliferative disorders as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
[0518] For treatment of hyperproliferative disorders with an antibody or fragment thereof, the dosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, lmg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention. Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis. An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within the ranges indicated.
[0519] α6β4 integrin-specific antibodies or immunospecifϊc fragments thereof disclosed herein can be administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of target polypeptide or target molecule in the patient. In some methods, dosage is adjusted to achieve a plasma polypeptide concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, binding molecules can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. The half-life of a binding molecule can also be prolonged via fusion to a stable polypeptide or moiety, e.g., albumin or PEG. In general, humanized antibodies show the longest half- life, followed by chimeric antibodies and nonhuman antibodies. In one embodiment, the binding molecules of the invention can be administered in unconjugated form, In another embodiment, the binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin- specific antibodies or immunospecific fragments thereof for use in the methods disclosed herein can be administered multiple times in conjugated form. In still another embodiment, the binding molecules of the invention can be administered in unconjugated form, then in conjugated form, or vise versa.
[0520] The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions comprising antibodies or a cocktail thereof are administered to a patient not already in the disease state or in a pre-disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose." In this use, the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose. A relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
[0521] In therapeutic applications, a relatively high dosage (e.g., from about 1 to 400 mg/kg of binding molecule, e.g., antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin- drug conjugated molecules) at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
[0522] In one embodiment, a subject can be treated with a nucleic acid molecule encoding an α6β4 integrin-specific antibody or immunospecific fragment thereof (e.g., in a vector). Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
[0523] Therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment, hi some methods, agents are injected directly into a particular tissue where α6β4 integrin-expressing cells have accumulated, for example intracranial injection. Intramuscular injection or intravenous infusion are preferred for administration of antibody, hi some methods, particular therapeutic antibodies are injected directly into the cranium, hi some methods, antibodies are administered as a sustained release composition or device, such as a Medipad™ device.
[0524] α6β4 integrin antibodies or fragments thereof of the invention can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).
[0525] Effective single treatment dosages (i.e., therapeutically effective amounts) Of 90Y- labeled binding polypeptides range from between about 5 and about 75 mCi, more preferably between about 10 and about 40 mCi. Effective single treatment non-marrow ablative dosages of 131I-labeled antibodies range from between about 5 and about 70 mCi, more preferably between about 5 and about 40 mCi. Effective single treatment ablative dosages (i.e., may require autologous bone marrow transplantation) of 131I-labeled antibodies range from between about 30 and about 600 mCi, more preferably between about 50 and less than about 500 mCi. In conjunction with a chimeric antibody, owing to the longer circulating half life vis-a-vis murine antibodies, an effective single treatment non-marrow ablative dosages of iodine-131 labeled chimeric antibodies range from between about 5 and about 40 mCi, more preferably less than about 30 mCi. Imaging criteria for, e.g., the 111In label, are typically less than about 5 mCi.
[0526] While a great deal of clinical experience has been gained with 131I and 90Y, other radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging. For example, additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 1231, 125I, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 1321, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac, 211At, and 213Bi. In this respect alpha, gamma and beta emitters are all compatible with in the instant invention. Further, in view of the instant disclosure it is submitted that one skilled in the art could readily determine which radionuclides are compatible with a selected course of treatment without undue experimentation. To this end, additional radionuclides which have already been used in clinical diagnosis include 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, as well as 111In. Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. Immunol. Cell Biol. 65: 111-125 (1987)). These radionuclides include 188Re and 186Re as well as 199Au and 67Cu to a lesser extent. U.S. Patent No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.
[0527] Whether or not α6β4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein are used in a conjugated or unconjugated form, it will be appreciated that a major advantage of the present invention is the ability to use these molecules in myelosuppressed patients, especially those who are undergoing, or have undergone, adjunct therapies such as radiotherapy or chemotherapy. That is, the beneficial delivery profile (i.e. relatively short serum dwell time, high binding affinity and enhanced localization) of the molecules makes them particularly useful for treating patients that have reduced red marrow reserves and are sensitive to myelotoxicity. In this regard, the unique delivery profile of the molecules make them very effective for the administration of radiolabeled conjugates to myelosuppressed cancer patients. As such, the α6β4 integrin-specific antibodies or immunospecific fragments thereof disclosed herein are useful in a conjugated or unconjugated form in patients that have previously undergone adjunct therapies such as external beam radiation or chemotherapy. In other preferred embodiments, binding molecules, e.g., binding polypeptides, e.g., α6β4 integrin-specific antibodies or immunospecific fragments thereof (again in a conjugated or unconjugated form) may be used in a combined therapeutic regimen with chemotherapeutic agents. Those skilled in the art will appreciate that such therapeutic regimens may comprise the sequential, simultaneous, concurrent or coextensive administration of the disclosed antibodies or other binding molecules and one or more chemotherapeutic agents. Particularly preferred embodiments of this aspect of the invention will comprise the administration of a radiolabeled binding polypeptide.
[0528] While α6β4 integrin-specific antibodies or immunospecific fragments thereof may be administered as described immediately above, it must be emphasized that in other embodiments conjugated and unconjugated binding molecules may be administered to otherwise healthy patients as a first line therapeutic agent. In such embodiments binding molecules may be administered to patients having normal or average red marrow reserves and/or to patients that have not, and are not, undergoing adjunct therapies such as external beam radiation or chemotherapy.
[0529] However, as discussed above, selected embodiments of the invention comprise the administration of α6β4 integrin-specific antibodies or immunospecific fragments thereof to myelosuppressed patients or in combination or conjunction with one or more adjunct therapies such as radiotherapy or chemotherapy (i.e. a combined therapeutic regimen). As used herein, the administration of α6β4 integrin-specific antibodies or immunospecific fragments thereof in conjunction or combination with an adjunct therapy means the sequential, simultaneous, coextensive, concurrent, concomitant or contemporaneous administration or application of the therapy and the disclosed binding molecules. Those skilled in the art will appreciate that the administration or application of the various components of the combined therapeutic regimen may be timed to enhance the overall effectiveness of the treatment. For example, chemotherapeutic agents could be administered in standard, well known courses of treatment followed within a few weeks by radioimmunoconjugates described herein. Conversely, cytotoxin-conjugated binding molecules could be administered intravenously followed by tumor localized external beam radiation. In yet other embodiments, binding molecules may be administered concurrently with one or more selected chemotherapeutic agents in a single office visit. A skilled artisan (e.g. an experienced oncologist) would be readily be able to discern effective combined therapeutic regimens without undue experimentation based on the selected adjunct therapy and the teachings of the instant specification.
[0530] In this regard it will be appreciated that the combination of a binding molecule
(with or without cytotoxin) and the chemotherapeutic agent may be administered in any order and within any time frame that provides a therapeutic benefit to the patient. That is, the chemotherapeutic agent and α6β4 integrin-specific antibody or immunospecific fragment thereof, may be administered in any order or concurrently. In selected embodiments α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention will be administered to patients that have previously undergone chemotherapy. hi yet other embodiments, α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention will be administered substantially simultaneously or concurrently with the chemotherapeutic treatment. For example, the patient may be given the binding molecule while undergoing a course of chemotherapy. In preferred embodiments the binding molecule will be administered within 1 year of any chemotherapeutic agent or treatment. hi other preferred embodiments the polypeptide will be administered within 10, 8, 6, 4, or 2 months of any chemotherapeutic agent or treatment. In still other preferred embodiments the binding molecule will be administered within 4, 3, 2 or 1 week of any chemotherapeutic agent or treatment, hi yet other embodiments the binding molecule will be administered within 5, 4, 3, 2 or 1 days of the selected chemotherapeutic agent or treatment. It will further be appreciated that the two agents or treatments may be administered to the patient within a matter of hours or minutes (i.e. substantially simultaneously).
[0531] Moreover, in accordance with the present invention a myelosuppressed patient shall be held to mean any patient exhibiting lowered blood counts. Those skilled in the art will appreciate that there are several blood count parameters conventionally used as clinical indicators of myelosuppression and one can easily measure the extent to which myelosuppression is occurring in a patient. Examples of art accepted myelosuppression measurements are the Absolute Neutrophil Count (ANC) or platelet count. Such myelosuppression or partial myeloablation may be a result of various biochemical disorders or diseases or, more likely, as the result of prior chemotherapy or radiotherapy. In this respect, those skilled in the art will appreciate that patients who have undergone traditional chemotherapy typically exhibit reduced red marrow reserves. As discussed above, such subjects often cannot be treated using optimal levels of cytotoxin (i.e. radionuclides) due to unacceptable side effects such as anemia or immunosuppression that result in increased mortality or morbidity.
[0532] More specifically conjugated or unconjugated α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention may be used to effectively treat patients having ANCs lower than about 2000/mm3 or platelet counts lower than about 150,000/ mm3. More preferably α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention may be used to treat patients having ANCs of less than about 1500/ mm3, less than about 1000/mm3 or even more preferably less than about 500/ mm3. Similarly, α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention may be used to treat patients having a platelet count of less than about 75,000/mm3, less than about 50,000/mm3 or even less than about 10,000/mm3. In a more general sense, those skilled in the art will easily be able to determine when a patient is myelosuppressed using government implemented guidelines and procedures.
[0533] As indicated above, many myelosuppressed patients have undergone courses of treatment including chemotherapy, implant radiotherapy or external beam radiotherapy. In the case of the latter, an external radiation source is for local irradiation of a malignancy. For radiotherapy implantation methods, radioactive reagents are surgically located within the malignancy, thereby selectively irradiating the site of the disease. In any event, α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention may be used to treat disorders in patients exhibiting myelosuppression regardless of the cause.
[0534] In this regard it will further be appreciated that α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention may be used in conjunction or combination with any chemotherapeutic agent or agents (e.g. to provide a combined therapeutic regimen) that eliminates, reduces, inhibits or controls the growth of neoplastic cells in vivo. As discussed, such agents often result in the reduction of red marrow reserves. This reduction may be offset, in whole or in part, by the diminished myelotoxicity of the compounds of the present invention that advantageously allow for the aggressive treatment of neoplasias in such patients. In other embodiments, radiolabeled immunoconjugates disclosed herein may be effectively used with radiosensitizers that increase the susceptibility of the neoplastic cells to radionuclides. For example, radiosensitizing compounds may be administered after the radiolabeled binding molecule has been largely cleared from the bloodstream but still remains at therapeutically effective levels at the site of the tumor or tumors.
[0535] With respect to these aspects of the invention, exemplary chemotherapeutic agents that are compatible with the instant invention include alkylating agents, vinca alkaloids (e.g., vincristine and vinblastine), procarbazine, methotrexate and prednisone. The four- drug combination MOPP (mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazine and prednisone) is very effective in treating various types of lymphoma and comprises a preferred embodiment of the present invention, hi MOPP-resistant patients, ABVD (e.g., adriamycin, bleomycin, vinblastine and dacarbazine), ChIVPP (chlorambucil, vinblastine, procarbazine and prednisone), CABS (lomustine, doxorubicin, bleomycin and streptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone) combinations can be used. Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, in Harrison's Principles of Internal Medicine 1774-1788 (Kurt J. Isselbacher et al, eds., 13th ed. 1994) and V. T. DeVita et ah, (1997) and the references cited therein for standard dosing and scheduling. These therapies can be used unchanged, or altered as needed for a particular patient, in combination with one or more α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention.
[0536] Additional regimens that are useful in the context of the present invention include use of single alkylating agents such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide and leucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate and leucovorin) and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, fixed dose prednisone, bleomycin and leucovorin). Those skilled in the art will readily be able to determine standard dosages and scheduling for each of these regimens. CHOP has also been combined with bleomycin, methotrexate, procarbazine, nitrogen mustard, cytosine arabinoside and etoposide. Other compatible chemotherapeutic agents include, but are not limited to, 2-chlorodeoxyadenosine (2-CDA), 2'-deoxycoformycin and fludarabine.
[0537] For patients with intermediate- and high-grade malignancies, who fail to achieve remission or relapse, salvage therapy is used. Salvage therapies employ drugs such as cytosine arabinoside, cisplatin, carboplatin, etoposide and ifosfamide given alone or in combination, hi relapsed or aggressive forms of certain neoplastic disorders the following protocols are often used: IMVP- 16 (ifosfamide, methotrexate and etoposide), MIME (methyl-gag, ifosfamide, methotrexate and etoposide), DHAP (dexamethasone, high dose cytarabine and cisplatin), ESHAP (etoposide, methylpredisolone, HD cytarabine, cisplatin), CEPP(B) (cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin) and CAMP (lomustine, mitoxantrone, cytarabine and prednisone) each with well known dosing rates and schedules.
[0538] The amount of chemotherapeutic agent to be used in combination with the α6β4 integrin-specifϊc antibodies or immunospecific fragments thereof of the present invention may vary by subject or may be administered according to what is known in the art. See, for example, Bruce A Chabner et ai, Antineoplastic Agents, in Goodman & Gilman's The Pharmacological Basis of Therapeutics 1233-1287 (Joel G. Hardman et ah, eds., 9' ed. (1996)).
[0539] In another embodiment, an α6β4 integrin-specific antibody or immunospecific fragment thereof of the present invention is administered in conjunction with a biologic. Biologies useful in the treatment of cancers are known in the art and a binding molecule of the invention may be administered, for example, in conjunction with such known biologies. [0540] For example, the FDA has approved the following biologies for the treatment of breast cancer: Herceptin® (trastuzumab, Genentech Inc., South San Francisco, CA; a humanized monoclonal antibody that has anti-tumor activity in HER2 -positive breast cancer); Faslodex® (fulvestrant, AstraZeneca Pharmaceuticals, LP, Wilmington, DE; an estrogen-receptor antagonist used to treat breast cancer); Arimidex® (anastrozole, AstraZeneca Pharmaceuticals, LP; a nonsteroidal aromatase inhibitor which blocks aromatase, an enzyme needed to make estrogen); Aromasin® (exemestane, Pfizer Inc., New York, NY; an irreversible, steroidal aromatase inactivator used in the treatment of breast cancer); Femara® (letrozole, Novartis Pharmaceuticals, East Hanover, NJ; a nonsteroidal aromatase inhibitor approved by the FDA to treat breast cancer); and Nolvadex® (tamoxifen, AstraZeneca Pharmaceuticals, LP; a nonsteroidal antiestrogen approved by the FDA to treat breast cancer). Other biologies with which the binding molecules of the invention may be combined include: Avastin™ (bevacizumab, Genentech Inc.; the first FDA-approved therapy designed to inhibit angiogenesis); and Zevalin® (ibritumomab tiuxetan, Biogen Idee, Cambridge, MA; a radiolabeled monoclonal antibody currently approved for the treatment of B-cell lymphomas).
[0541] hi addition, the FDA has approved the following biologies for the treatment of colorectal cancer: Avastin™; Erbitux™ (cetuximab, ImClone Systems Inc., New York, NY, and Bristol-Myers Squibb, New York, NY; is a monoclonal antibody directed against the epidermal growth factor receptor (EGFR)); Gleevec® (imatinib mesylate; a protein kinase inhibitor); and Ergamisol® (levamisole hydrochloride, Janssen Pharmaceutica Products, LP, Titusville, NJ; an immunomodulator approved by the FDA in 1990 as an adjuvant treatment in combination with 5-fluorouracil after surgical resection in patients with Dukes' Stage C colon cancer).
[0542] For use in treatment of Non-Hodgkin's Lymphomas currently approved therapies include: Bexxar® (tositumomab and iodine 1-131 tositumomab, GlaxoSmithKline, Research Triangle Park, NC; a multi-step treatment involving a mouse monoclonal antibody (tositumomab) linked to a radioactive molecule (iodine 1-131)); Intron® A (interferon alfa-2b, Schering Corporation, Kenilworth, NJ; a type of interferon approved for the treatment of follicular non-Hodgkin's lymphoma in conjunction with anthracycline-containing combination chemotherapy (e.g., cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])); Rituxan® (rituximab, Genentech Inc., South San Francisco, CA, and Biogen Idee, Cambridge, MA; a monoclonal antibody approved for the treatment of non-Hodgkin's lymphoma; Ontak® (denileukin diftitox, Ligand Pharmaceuticals Inc., San Diego, CA; a fusion protein consisting of a fragment of diphtheria toxin genetically fused to interleukin-2); and Zevalin® (ibritumomab tiuxetan, Biogen Idee; a radiolabeled monoclonal antibody approved by the FDA for the treatment of B-cell non-Hodgkin's lymphomas).
[0543] For treatment of Leukemia, exemplary biologies which may be used in combination with the binding molecules of the invention include Gleevec®; Campath®- IH (alemtuzumab, Berlex Laboratories, Richmond, CA; a type of monoclonal antibody used in the treatment of chronic Lymphocytic leukemia). In addition, Genasense (oblimersen, Genta Corporation, Berkley Heights, NJ; a BCL-2 antisense therapy under development to treat leukemia may be used (e.g., alone or in combination with one or more chemotherapy drugs, such as fludarabine and cyclophosphamide) may be administered with the claimed binding molecules.
[0544] For the treatment of lung cancer, exemplary biologies include Tarceva™ (erlotinib
HCL, OSI Pharmaceuticals Inc., Melville, NY; a small molecule designed to target the human epidermal growth factor receptor 1 (HERl) pathway).
[0545] For the treatment of multiple myeloma, exemplary biologies include Velcade®
Velcade (bortezomib, Millennium Pharmaceuticals, Cambridge MA; a proteasome inhibitor). Additional biologies include Thalidomid® (thalidomide, Clegene Corporation, Warren, NJ; an immunomodulatory agent and appears to have multiple actions, including the ability to inhibit the growth and survival of myeloma cells and anti-angiogenesis).
[0546] Other exemplary biologies include the MOAB IMC-C225, developed by ImClone
Systems, Inc., New York, NY.
[0547] As previously discussed, α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention, or recombinants thereof may be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian hyperproliferative disorders, hi this regard, it will be appreciated that the disclosed antibodies will be formulated so as to facilitate administration and promote stability of the active agent. Preferably, pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. For the purposes of the instant application, a pharmaceutically effective amount of α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention, or recombinant thereof, conjugated or unconjugated to a therapeutic agent, shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell. In the case of tumor cells, the binding molecule will be preferably be capable of interacting with selected immunoreactive antigens on neoplastic or immunoreactive cells, or on non neoplastic cells, e.g., vascular cells associated with neoplastic cells, and provide for an increase in the death of those cells. Of course, the pharmaceutical compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the binding molecule.
[0548] In keeping with the scope of the present disclosure, α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect. The α6β4 integrin-specific antibodies or immunospecific fragments thereof of the present invention can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody of the invention with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of binding molecules according to the present invention may prove to be particularly effective.
[0549] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al, ed., Cold Spring Harbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual, Sambrook et al, ed., Cold Springs Harbor Laboratory, New York (1992), DNA Cloning, D. N. Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds. (1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, TRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology, Academic Press, Inc., N. Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., (1986); and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).
[0550] General principles of antibody engineering are set forth in Antibody Engineering,
2nd edition, C.A.K. Borrebaeck, Ed., Oxford Univ. Press (1995). General principles of protein engineering are set forth in Protein Engineering, A Practical Approach, Rickwood, D., et al, Eds., IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principles of antibodies and antibody-hapten binding are set forth in: Nisonoff, A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, MA (1984); and Steward, M.W., Antibodies, Their Structure and Function, Chapman and Hall, New York, NY (1984). Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al. (eds) , Basic and Clinical -Immunology (8th ed.), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York (1980).
[0551] Standard reference works setting forth general principles of immunology include
Current Protocols in Immunology, John Wiley & Sons, New York; Klein, J., Immunology: The Science of Self-Nonself Discrimination, John Wiley & Sons, New York (1982); Kennett, R., et al., eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, New York (1980); Campbell, A., "Monoclonal Antibody Technology" in Burden, R., et al., eds., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunology 4th ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A. Osborne, H. Freemand & Co. (2000); Roitt, L, Brostoff, J. and Male D., Immunology 6th ed. London: Mosby (2001); Abbas A., Abul, A. and Lichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier Health Sciences Division (2005); Kontermann and Dubel, Antibody Engineering, Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII, Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer Cold Spring Harbor Press (2003). [0552] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.
EXAMPLES Example 1
Generation of Soluble Protein Reagents and Cell Lines
[0553] cDNAs encoding cDNAs encoding the α6 & β4 ectodomains were generated by
RT-PCR from commercially available human skin cDNA and inserted into mammalian expression vectors pV90/pV100 using standard molecular techniques. The expression vectors pV90/pV100 are vectors in which heterologous gene expression is driven by a strong constitutive CMV-IE promoter (with intron) and a human growth hormone transcription termination and polyA signal. pV90 carries an SV40 early-driven dhfr selectable marker, while pV100, which is a pV90 derivative, carries a phosphoglycerate kinase-driven neo selectable marker. They share pUC -derived plasmid backbones.
[0554] Immunoadhesin versions of each ectodomain were generated by inserting α6 or β4 cDNA as an in-frame fusion with a human IgGl Fc cDNA in pV90, thus generating α6Fc and β4Fc constructs. Stable Chinese hamster ovary (CHO) cell lines co-expressing α6Fc with β4 ectodomain or α6 ectodomain with β4Fc were generated using standard techniques. The resulting cell lines secreted α6Fcβ4 and α6β4Fc homodimers. Proteins were purified from culture supernatant by affinity chromatography, then characterized by SDS gel electrophoresis and size exclusion chromatography. Figure 1 shows the purification of α6Fcβ4 as an example.
[0555] Stable CHO cell lines expressing full-length versions of human α6β4, α6βl and murine α6β4 were generated for antibody screening purposes, as discussed in the following sections. Full length cDNAs encoding the various integrin subunits were constructed by standard PCR and cloning methods using commercially available ESTs in addition to the above-described ectodomain constructs as templates. Full-length cDNAs of the appropriate integrin subunits were co- inserted into proprietary mammalian expression vector DOUBLE ONE, a derivative of the NEOSPLA vector fully described in US patent 5,733,779, for expression in CHO cells.
Example 2
Generation and Conversion of Phage-Display-Derived Fab Antibodies
[0556] Purified α6Fcβ4 and α6β4Fc proteins were used to screen a human naive phagemid Fab library containing 3.5 x 1010 unique clones (Nat Biotechnol. 23(3):344-8 (2005)). Proteins were captured on streptavidin-coated magnetic beads prior to incubation with the phage library. A biotinylated anti-Fc antibody was captured on the magnetic beads, followed by captured of the Fc fusion protein. Selections were performed as described previously (Nat Biotechnol. 23(3):344-8 (2005)). After 3 rounds of panning, the 479 bp gene III stump was removed by MM digestion, and the vector was religated for soluble Fab expression in TGl cells. ELISA analysis of 1920 clones from both arms yielded 305 positive clones, containing 39 unique sequences. Unique clones were purified and binding was reconfirmed by ELISA against immobilized a6Fcb4Fc and FACS against α6β4 expressing CHO cells. Based on binding data, 5 of the 39 unique clones isolated were selected for further analysis.
[0557] The DNA sequences encoding the selected anti-α6β4 Fab antibodies - transferred into vectors for expression of full-length human IgG4.P. AU five M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 - were antibodies use the VH3-23 human heavy chain germline fragment. The variable heavy chain was removed from the soluble Fab expression vector and inserted into a derivative of the pV90 expression vector described above such that the resulting plasmid contained the heavy chain signal peptide (MGWSCIILFLVATATGAHS (SEQ ID NO: 96)) followed by the anti-α6β4 VH and constant regions for human IgG4.P.
[0558] All five antibodies contain kappa light chains. The variable light chain was amplified by PCR and ultimately inserted into a derivative of the pVlOO expression vector described above between the immunoglobulin light chain signal peptide (MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO:97)) and the constant kappa domain. Light and heavy chain expression vectors were co-expressed in CHO cells, and antibodies purified from CHO cell supernatant by protein A affinity chromatography.
Example 3
Generation of Murine Monoclonal Antibodies
[0559] 6-8 week old female Rbf mice were immunized with 50μg of purified α6Fcβ4 protein in Freund's complete adjuvant and boosted with protein in Freund's incomplete adjuvant four times over a six week period. Serum titer and specificity was determined by ELISA against immobilized α6Fcβ4 protein compared to human IgGlFc and confirmed by FACS against α6β4 expressing cell lines. Mice exhibiting the highest titer of α6β4 antibodies were sacrificed and splenic B-lymphocytes aseptically harvested. Splenic B-lymphocytes were washed and prepared for use in PEG mediated lymphocyte somatic cell fusions to either FL653 or SP2/0-Agl4 myeloma cells using standard immunological techniques (Kennett, R.H., McKearn, TJ., and Bechtol, K.B., Monoclonal Antibodies: A New Dimension in Biological Analyses, 1982, Plenum Press, New York). Fused cells were plated into 24-well sterile tissue culture plates and fed with adenine, aminopterin and thymidine (AAT) or hypoxanthine, aminopterin and thymidine (HAT) containing culture media, for FL653 or SP2/0-Agl4 myeloma based fusions respectively. After ten days of culture, hybridoma supernatants were screened by ELISA and FACS as described above. Colonies displaying the desired binding characteristics were subcloned by limiting dilution (~1 cell/well) and microscopically scored upon growth to assure integrity of the selected clones. Selected clones were expanded into IL spinner flasks and subclass characterized using IsoStrip (Roche). Antibodies were purified from 7-10 day culture supernatant by protein A affinity chromatography.
Example 4
Cloning of murine hybridoma 1P2D10.1 and 1P5B10.2 immunoglobulin variable regions
[0560] Total cellular RNA from murine hybridoma cells was prepared using a Qiagen
RNeasy mini kit following the manufacturer's recommended protocol. cDNAs encoding the variable regions of the heavy and light chains were cloned by RT-PCR from total cellular RNA using random hexamers for priming. For PCR amplification of the murine immunoglobulin variable domains with intact signal sequences, a cocktail of degenerate forward primers hybridizing to multiple murine immunoglobulin gene family signal sequences and a single back primer specific for 5' end of the murine constant domain. The PCR products were gel-purified and subcloned into Invitrogen's pCR2. ITOPO vector using their TOPO cloning kit following the manufacturer's recommended protocol. Inserts from multiple independent subclones were sequenced to establish a consensus sequence. Deduced mature immunoglobulin N-termini were consistent with those determined by Edman degradation from the hybridomas.
[0561] P2D10's IgGl, kappa isotype was confirmed by RT-PCR (i.e., sufficient constant domain sequences were cloned along with the variable domains to unambiguously assign isotype). Shown below as SEQ ID NO:77 is the P2D10.1 mature heavy chain variable domain protein sequence, with CDRs (Kabat definitions) underlined:
1 QVQLQQPGAE LVKPGASVKL SCEASGYTFT SYWMHWVKOR
PGQGLEWIGE
51 INPYNGRPNY NEKFKNKATL TADKTSNTAY MQLSSLTSED SAVYYCTRPS
101 YYDYDGAWFA YWGQGTLVTV SA
[0562] This is a murine subgroup II(B) heavy chain. Shown below as SEQ ID NO: 76 is the DNA sequence of the P2D10.1 mature heavy chain variable domain (from pYL340):
1 CAGGTCCAAC TGCAGCAGCC TGGGGCTGAA CTGGTGAAGC CTGGGGCTTC
51 AGTGAAACTG TCCTGCGAGG CTTCTGGCTA CACCTTCACC AGCTACTGGA
101 TGCACTGGGT GAAGCAGAGG CCTGGACAAG GCCTTGAGTG GATTGGAGAA
151 ATTAATCCTT ACAACGGTCG TCCTAACTAC AATGAGAAGT TCAAGAATAA
201 GGCCACACTG ACTGCAGACA AAACCTCCAA CACAGCCTAC ATGCAACTCA 251 GCAGCCTGAC ATCTGAGGAC TCTGCGGTCT ATTACTGTAC AAGACCCTCC
301 TATTATGATT ACGACGGGGC CTGGTTTGCC TACTGGGGCC AAGGGACTCT
351 GGTCACTGTC TCTGCA
[0563] Shown below as SEQ ID NO:82 is the P2D10.1 mature light chain variable domain protein sequence, with CDRs underlined:
1 DIVMSQSPSS LAVSAGEKVT LSCKSSOSLL NSRSRKNYLA WYQQKPGQSP
51 KLLISWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKOSYNL
101 YTFGGGTKLE IK
[0564] This is a murine subgroup kappa I light chain. Shown below as SEQ ID NO: 81 is the DNA sequence of the P2D10.1 mature light chain variable domain (from pYL351):
1 GACATTGTGA TGTCACAGTC TCCATCCTCC CTGGCTGTGT CAGCAGGAGA
51 GAAGGTCACT TTGAGCTGCA AATCCAGTCA GAGTCTGCTC AACAGTAGAT
101 CCCGAAAGAA CTACTTGGCT TGGTACCAGC AGAAACCAGG ACAGTCTCCT
151 AAACTGCTGA TCTCCTGGGC ATCCACTAGG GAATCTGGGG TCCCTGATCG
201 CTTCACAGGC AGTGGATCTG GGACAGATTT CACTCTCACC ATCAGCAGTG
251 TGCAGGCTGA AGACCTGGCA GTTTATTACT GCAAGCAATC TTATAATCTC
301 TACACGTTCG GAGGGGGGAC CAAGCTGGAG ATAAAA
[0565] P5B10's IgGl, kappa isotype was confirmed by RT-PCR. Shown below as SEQ
ID NO:87 is the P5B10.2 mature heavy chain variable domain protein sequence, with CDRs underlined:
1 QVQLKQSGPS LVQPSQSLSI TCTVSGFSLT SYGVHWFROS PGKGLEWLGV 51 IWRGGNTDYN AAFMSRLSIT KDNSKSQVFF KMNSLQPDDT AIYYCVKNYR
101 FGGSAYWGQG TLVTVSA
[0566] This is a murine subgroup 1(B) heavy chain. Shown below as SEQ ID NO: 86 is the DNA sequence of the P5B10.2 mature heavy chain variable domain (from pYL342):
1 CAGGTGCAGC TGAAGCAGTC AGGACCCAGC CTAGTGCAGC CCTCACAGAG
51 CCTGTCCATA ACCTGCACAG TCTCTGGCTT CTCATTAACT AGCTATGGTG
101 TACACTGGTT TCGCCAGTCT CCAGGAAAGG GTCTGGAGTG GCTGGGAGTG
151 ATATGGAGAG GTGGAAACAC AGACTACAAT GCAGCTTTCA TGTCCAGACT
201 GAGCATCACC AAGGACAACT CCAAGAGCCA AGTTTTCTTT AAAATGAACA
251 GTCTGCAACC TGATGACACT GCCATATACT ACTGTGTCAA AAACTATAGG
301 TTCGGAGGGT CTGCTTACTG GGGCCAAGGG ACTCTGGTCA CTGTCTCTGC
[0567] Shown below as SEQ ID NO:92 is the P5B10.2 mature light chain variable domain protein sequence, with CDRs underlined:
1 DIQMTQTTSS LSASLGDRVT ISCRASODIS NHLNWYOOKP DGTVKLLIYY
5i TSTLHSGVPS RFSGSGSGTD YSLTISNLEP EDIATYΎCQQ DLNLPWTFGG
101 GTKLEIK
[0568] This is a murine subgroup kappa V light chain. Shown below as SEQ ED NO:91 is the DNA sequence of the P5B10.2 mature light chain variable domain (from pYL344):
1 GATATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCT CTCTGGGAGA 51 CAGAGTCACC ATTAGTTGCA GGGCAAGTCA GGACATTAGC AACCATTTAA
101 ACTGGTATCA GCAGAAACCA GATGGTACTG TTAAACTCCT GATCTACTAC
151 ACATCAACAT TACACTCAGG CGTCCCATCA AGGTTCAGTG GCAGTGGGTC
201 TGGGACAGAT TATTCTCTCA CCATTAGCAA CCTGGAACCG GAAGATATTG
251 CCACTTACTA TTGCCAACAG GATCTTAATC TTCCGTGGAC GTTCGGTGGA 301 GGCACCAAGC TGGAAATCAA A
Example 5
Characterization of murine antibody specificities by flow cytometry
[0569] Binding affinities of purified murine antibodies were determined by flow cytometry analysis on integrin expressing CHO cells. In all experiments, CHO cells were incubated with test antibodies at a range of concentrations for twenty minutes then washed and incubated with PE-conjugated secondary antibody. The control antibody was a Biogen Idee proprietary isotype matched murine antibody to CD20. Cells were analyzed using a BD FACSarray, gating for live cells. Figure 2 shows FACS titrations of 2 murine monoclonal antibodies on CHO-α6β4 cells. These data show that the murine α6β4 monoclonal antibodies bind specifically to α6β4 integrin with good binding affinities. No binding to CHO-α6βl was detected, indicating the antibodies specifically recognize the α6β4 integrin.
[0570] Figure 3 shows an example of a competition experiment whereby antibody binding to CHO-α6β4 cells is inhibited by addition of increasing concentrations of soluble α6Fcβ4 protein. Addition of an irrelevant Fc protein (sZIP4) has no effect on antibody binding. This experiment further confirms the specificity of the antibodies for α6β4.
[0571] The antibodies were also tested for their ability to recognize endogenously expressed α6β4 on human tumor cell lines. The control antibody was a Biogen Idee proprietary isotype matched murine antibody to CD20. Representative data are shown in Figure 4, where strong binding to colorectal (SW620) (top panel) and breast (MD A-MB- 231 and BT474) (bottom panel) tumor cell lines was observed. These data confirm that murine α6β4 antibodies raised against recombinant α6β4 protein recognize the endogenous antigen on tumor cells, and therefore will be able to detect and bind to the antigen in human tumors.
Example 6
Characterization of Human Antibody Specificities by flow cytometry
[0572] Binding affinities of purified human antibodies was determined by flow cytometry on integrin expressing CHO cells. For all experiments, CHO cells (expressing either human α6β4, murine α6β4 or untransfected control CHO cells) were harvested and washed twice with wash buffer (PBS/10% normal goat serum/0.2% BSA/0.1% sodium azide) then resuspended in wash buffer at a concentration of 1,000,000 cells/mL. Cells were aliquoted into 96-well plates at 500,000 cells/well and incubated with test antibodies at concentrations ranging from 80ug/ml to 0.0003ug/ml for twenty minutes then washed and incubated with PE-conjugated secondary antibody.
[0573] Cells were analyzed using a BD FACSarray, with propidium iodide staining to gate for live cells. GraphPad Prism version 4.0 software was used for data analysis. Representative FACS titrations of 3 of these human antibodies on CHO-α6β4 are shown in Figure 5. A FACS titration of these human antibodies on CHO cells expressing murine α6β4 is shown in Figure 6. These data indicate that these human antibodies bind to both human and murine α6β4. Figure 7 demonstrates the specificity of 59B05 for α6β4, with no binding to α6βl (top panel) as well as the ability of 59B05 to recognize murine as well as human α6β4 (bottom panel).
[0574] To determine antibody specificity, flow cytometry analysis on CHO-α6β4 was carried out at a fixed antibody concentration in the presence of increasing concentrations of purified oc6Fcβ4 protein or an irrelevant Fc protein (sZip4) as a control. An example of a specificity experiment is shown in Figure 8, where the irrelevant control protein (sZip4) has no effect on antibody binding to CHO-α6β4 cells, but the α6Fcβ4 protein competes for binding. This experiment further confirms the specificity of the antibodies for cc6β4. Figure 9 demonstrates the ability of 59B05 to recognize endogenously expressed α6β4 on colorectal (SW620) and breast (MDA-MB-231 and BT474) tumor cell lines. Example 7
Human and Murine α6β4 Antibodies Inhibit Anchorage Independent Growth of Tumor
Cell Lines
[0575] The human and murine antibodies were tested for their ability to inhibit anchorage independent growth of a breast (MDA-MB-231) and colorectal (SW620) cell line as follows. A bottom layer of ImI IX Media (MDA-MB-231: RPMI, SW620: Leibovitz L- 15) containing 0.6% Nobel Agar (Difco), 10% FBS and lOμg/ml of the appropriate antibody was prepared in 12 well-ultra low cluster cell culture plates (Costar). After the bottom layer solidified, cells (MDA-MB-231: 15,000cells/well, SW620: 10,000 cells/well) are added in a ImI top layer containing 0.3% agar, 10% FBS and 10 μg/ml antibody. The samples are prepared in duplicate and incubated in 5% CO2 (MDA-MB- 231) or 0% CO2 (SW620) incubator at 37°C. Colonies measuring >100μm were counted on Nikon Eclipse TE2000-U system after 21-27 days. α6β4 antibodies were tested alone or in combination with anti-EGFR antibody C225 (Calbiochem). Control antibodies used in the experiment were mouse isotype control (BD Pharmingen) for the EGFR antibody and isotype matched control human antibody to CD4 (Biogen Idee proprietary) for the human antibodies. The antibody concentrations were fixed at lOμg/ml, which represents at least a 30-fold excess over the EC50 values for binding to α6β4 on the tumor cell lines as determined by flow cytometry.
[0576] Figure 10 shows the effects of antibody treatment on anchorage independent growth in colorectal cell line SW620, while Figure 11 shows the effects in breast cancer cell line MDA-MB-231. Table 7 below shows the percent inhibition in the breast (MDA- MB-231) and colorectal (SW620) cancer cell lines, respectively. A subset of the α6β4 antibodies inhibited colony formation compared to isotype control antibodies in both cell types. An antibody to epidermal growth factor receptor (EGFR) (anti-EGFR antibody C225) had a similar inhibitory effect. Furthermore, an enhanced effect, e.g., additive or synergistic, on colony growth was observed when four of the α6β4 antibodies (59B05, 67F05, 1P2D10.1 and 1P5B10.2) were combined with the anti-EGFR antibody 225. These data suggest that α6β4 antibodies may be effective in inhibiting tumor growth, and their efficacy may be enhanced when used in combination with other targeted therapies. Table 7. Effects of Antibody Treatment on Anchorage Independent Growth in Colorectal and Breast Cancer Cell Lines
Figure imgf000174_0001
Figure imgf000174_0002
Example 8
Human and Murine α6β4 Antibodies Inhibit α6β4 Mediated Adhesion Interactions
[0577] For cellular adhesion assays, 96 well plates coated with laminin rich matrix secreted by squamous carcinoma cell line SCC25 were prepared as described previously (Rouselle P, Aumailley M. (1994) J. Cell Biol. 125(1), 205-14.). Colorectal tumor cell lines SW480 and SW620 were starved for 24 hours in serum free media, harvested using cell dissociation buffer (Invitrogen), and resuspended at 1x106 cells/ml in Leibovitz L- 15 media. For antibody treatments, 10 μg/ml of the indicated antibodies were added to the resuspended cells. Cells were incubated on ice for 1 hour prior to plating on the SCC25 matrix coated plates at 100,000 cells per well. After a 1 hour incubation at 4°C, plates were washed with Leibovitz L- 15 media and adherent cells quantified using Cell Titer GIo cell viability assay (Promega, Madison, WL). Luminescence was detected using a SpectraMax M5 (Molecular Devices) plate reader. The data in Figure 12 show that murine antibody 1P5B10.2 along with human antibodies 59B05 and 67F05 inhibit cellular adhesion to the laminin matrix in both cell lines tested.
Example 9
Human and Murine α6β4 Antibodies Increase Apoptosis in Human Tumor Cell Lines
[0578] Tumor cell lines were plated at 2 x 106cells/dish in culture medium containing
10% FBS and incubated for 24 hours, after which media was replaced with low serum (0.5%FBS) medium supplemented with 20μg/ml of α6β4 antibodies. Twenty- four hours after antibody addition, taxol was added at concentrations ranging from 0.0125-lμM and cells were incubated for an additional 48 hours in the presence of both agents. Cells were subsequently harvested, washed and stained with PE conjugated annexin-V and 7AAD (BD Biosciences) to assess apoptosis. Staurosporine treatment at lμM for 24 hours was used as a positive control to induce apoptosis. Fluorescence was measured using a FACSarray. Figure 13 shows that murine antibodies 1P5B10.2 and 1.P2D10.1 along with human antibody 61C03 induced apoptosis in MDA-MB-231 breast cancer cells and human antibody 61C03 induced apoptosis in SW620 colorectal tumor cells.
Example 10
Human and Murine α6β4 Antibodies Inhibit PI3 Kinase Mediated Signaling in Human
Tumor Cell Lines
[0579] Phosphorylation of AKT at Thr308 and Ser473 occurs downstream of PI3 kinase activation in tumor cell lines. Alterations of phospho-AKT levels are therefore used as a readout of the PI3 kinase pathway. To evaluate the effects of α6β4 antibodies on phospho AKT, BT-474 cells were starved for 24 hours in serum free media before each experiment. Cells were then harvested using cell dissociation buffer, resuspended at 5 X 106 cells/ml in IX DMEM media and incubated in suspension at 370C in a 5% CO2 incubator. After 30 minutes of incubation lOμg/ml of α6β4 or control (human and rat isotype) antibodies were added to the suspension, and incubation continued for an additional 30 minutes, after which time cells were plated at 5 x 106 cells/well into 6 well culture dishes precoated with 50μg/ml of human laminin. Mab 13 (BD Pharmingen), a rat antibody to human βl integrin was used as a positive control. Plates were incubated 24 hours at 370C in 5% CO2 incubator, then washed 3X with PBS and lysed using 200μl /well of RIPA buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using Bio-Rad DC Protein Assay kit and 20μg of each lysate was run on NuPAGE 4-12% Bis-Tris gel, then transferred to a PVDF membrane. Membranes were probed first for phospho-AKT308 (Cell Signal antibody #2965L), then stripped and reprobed for total- AKT (Cell Signal antibody #469 IL) following standard Western blotting procedures. Invitrogen See-Blue molecular weight markers were used as standards. The 6OkDa AKT protein runs between the 49kDa (glutamic dehydrogenase) and 62kDa (bovine serum albumin) standards on the gel, as indicated by the arrows in Figure 14. Figure 14 shows that human antibodies 59B05 and 61C03 inhibited the phosphorylation of AKT in breast tumor cells. These data indicate that the human α6β4 antibodies inhibit PI3 kinase mediated signaling in human tumor cell lines.
Example 11
Comparison of Full Length IgG4 versus Fab Versions of Human Antibodies 59B05 and
61C03
[0580] Fab fragments ("Fabs") of full length 59B05 and 61C03 were generated by overnight papain digestion, followed by cation exchange chromatography using a sodium chloride gradient for Fab elution. Free cysteines were capped by addition of 1.2 fold molar excess of N-ethylmaleimide, then pooled column fractions were dialyzed into 20 mM Hepes pH 7.5, 150 mM sodium chloride.
[0581] Full length and Fab versions of 59B05 and 61C03 were compared in a cellular adhesion assay using colorectal tumor cell line SW480 (Figure 15) and an anchorage independent growth assay using breast carcinoma tumor cell line BT474 (Figure 16) as shown. Assays were performed as described in the application at Examples 7 and 8, except that in the case of the adhesion assay, 96 well plates were coated overnight with lμg/ml purified rat laminin-5 (Chemicon) instead of SCC-25 deposited matrix as in Example 8. All antibodies were used at a concentration of lOμg/ml. These data show that Fab versions of the antibodies behave in a similar manner as the full length IgG4 from which they were derived. Example 12
Evaluation of Apoptotic Activity of 59B05
[0582] Breast cancer cells (MCF7) were plated at a density of 0.5x106 cells/dish in culture medium containing 10% FBS and incubated for 24 hours, after which dishes were either left untreated or treated with human antibody 59B05 or isotype control antibody at 20 μg/ml. Twenty-four hours after treatment, the culture medium was changed to contain either complete FBS or hormone free FBS, and cells were incubated for an additional 3 days. Cells were then collected by trypsinization and apoptosis was measured by Annexin V staining on viable cells (7AAD negative) by flow cytometry using a FACS Array. The results of this experiment are depicted in Figure 17, which shows that treatment with human antibody 59B05 causes an increase in apoptosis when cells are in a hormone-deprived environment.
Example 13
Epitope Mapping of Human Antibody 61C03
[0583] Gel fragments containing β4 and β4-Fc polypeptides were isolated from SDS- polyacrylamide gels run under non-reducing conditions. The polypeptides were subjected to CNBr cleavage for lhr. Peptides from the digestion were extracted using 1%SDS, 15OmM NaCl, subjected to reducing SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes for western blot analysis with either 61C03 or anti-Fc antibody. Peptides recognized by these antibodies were submitted for N-terminal sequencing. Sequencing was carried out on Applied Biosystems Procise 494 high throughput sequencers (HT, SAM), (SAM and HT), run in the pulsed liquid PVDF and pulsed-liquid modes respectively. The resulting phenylthionhydantoin (PTH) amino acids were separated using an ABI 140C Microgradient System with a PTH C18 Column (2.1 mm X 220 mm) and analyzed online using an ABI 785 A programmable absorbance detector. Ten cycles of data were collected and analyzed using the ABI 610A data analysis software.
[0584] Figure 18 depicts a western blot of the CNBr digested material reacted with human antibody 61C03 and anti-Fc as indicated, hi each panel, material in lane 1 was derived from a β4 digest, while that in lane 2 from a β4-Fc digest. The box overlay indicates the fragments excised for N-terminal sequencing. Table 8 below lists the N- terminal peptides identified by sequence analysis, indicating whether the β4 fragments containing this sequence were recognized by human antibody 61C03.
Table 8. CNBr peptides identified by N-terminal sequencing
N-terminal peptide Recognized by Antibody 61C03 Anti-Fc
MRPEKLKEPWP (SEQ ID NO: 98) Yes Yes MDAGπCDVXT (SEQ ID NO: 99) Yes No MSRNDERCHLD (SEQ ID NOr IOO) Yes No
[0585] Shown below as SEQ ED NO:101 is the sequence of the human β4 ectodomain, where potential CNBr cleavage methionines are bolded, the actual peptides identified by sequencing are italics, and the β4 sequence predicted to contain the human antibody 61C03 epitope based on these analyses is underlined.
MAGPRPSPWA RLLLAALISV SLSGTLANRC KKAPVKSCTE CVRVDKDCAY CTDEMFRDRR CNTQAELLAA GCQRESIWM ESSFQITEET QIDTTLRRSQ MSPQGLRVRL RPGEERHFEL EVFEPLESPV DLYILMDFSN SMSDDLDNLK KMGQNLARVL SQLTSDYTIG FGKFVDKVSV PQTDMKPEKL XEPIVPWSDPP FSFKNVISLT EDVDEFRNKL QGERISGNLD APEGGFDAIL QTAVCTRDIG WRPDSTHLLV FSTESAFHYE ADGANVLAGI KSRNDERCHL DTTGTYTQYR TQDYPSVPTL VRLLAKHNII PIFAVTNYSY SYYEKLHTYF PVSSLGVLQE DSSNIVELLE EAFNRIRSNL DIRALDSPRG LRTEVTSKMF QKTRTGSFHI RRGEVGIYQV QLRALEHVDG THVCQLPEDQ KGNIHLKPSF SDGLKMDAGI ICDVCTCELQ KEVRSARCSF NGDFVCGQCV CSEGWSGQTC NCSTGSLSDI
QPCLREGEDK PCSGRGECQC GHCVCYGEGR YEGQFCEYDN FQCPRTSGFL
CNDRGRCSMG QCVCEPGWTG PSCDCPLSNA TCIDSNGGIC NGRGHCECGR
CHCHQQSLYT DTICEINYSA IHPGLCEDLR SCVQCQAWGT GEKKGRTCEE
CNFKVKMVDE LKRAEEWVR CSFRDEDDDC TYSYTMEGDG APGPNSTVLV HKKKDCPPGS F
[0586] These analyses indicate the human antibody 61C03 epitope is contained between amino acids 446-686 in the β4 sequence.

Claims

WHAT IS CLAIMED IS:
1. An isolated antibody or antigen-binding fragment thereof which specifically binds to the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
2. An isolated antibody or antigen-binding fragment thereof which specifically binds to α6β4 integrin, wherein said antibody or fragment thereof competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59- B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, and M12-G04, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2 from binding to α6β4 integrin.
3. An isolated antibody or antigen-binding fragment thereof which specifically binds to α6β4 integrin, wherein said antibody or fragment thereof is comprises an antigen binding domain identical to that of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05, or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
4. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the heavy chain variable region (VH) of said antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
5. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the light chain variable region (VL) of said antibody or fragment thereof comprises an amino acid sequence at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ BD NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92.
6. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH of said antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
7. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VL of said antibody or fragment thereof comprises an amino acid sequence identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO:92.
8. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH of said antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ E) NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87.
9. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VL of said antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ED NO: 92.
10. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH and VL of said antibody or fragment thereof comprise, respectively, amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ID NO: 62; SEQ ID NO: 67 and SEQ ID NO: 72; SEQ ID NO: 77 and SEQ ID NO: 82; and SEQ ID NO: 87 and SEQ ID NO: 92.
11. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH and VL of said antibody or fragment thereof comprise, respectively, amino acid sequences identical, except for 20 or fewer conservative amino acid substitutions each, to reference amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ID NO: 62; SEQ ID NO: 67 and SEQ ID NO:72; SEQ ID NO: 77 and SEQ ID NO: 82; and SEQ ID NO: 87 and SEQ ID NO: 92.
12. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH and VL of said antibody or fragment thereof comprise, respectively, amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ED NO: 62; SEQ ED NO: 67 and SEQ ED NO: 72; SEQ ED NO:77 and SEQ ID NO:82; and SEQ ED NO: 87 and SEQ ED NO: 92.
13. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH of said antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region- 1 (VH-CDRl) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ED NO: 18, SEQ ED NO: 28, SEQ ED NO: 38, SEQ ED NO: 48, SEQ ED NO: 58, SEQ ED NO: 68, SEQ ID NO: 78, and SEQ ED NO: 88.
14. The antibody or fragment thereof of claim 13, wherein said VH-CDRl amino acid sequence is selected from the group consisting of: SEQ ED NO: 18, SEQ ED NO: 28, SEQ ED NO: 38, SEQ ED NO: 48, SEQ ED NO: 58, SEQ ED NO: 68 SEQ ED NO: 78, and SEQ ED NO: 88.
15. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH of said antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-2 (VH-CDR2) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ E) NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 79 and SEQ ID NO: 89.
16. The antibody or fragment thereof of claim 15, wherein said VH-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 49, SEQ ID NO: 59, SEQ ID NO: 69 SEQ ID NO: 79 and SEQ ID NO: 89.
17. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH of said antibody or fragment thereof comprises a Kabat heavy chain complementarity determining region-3 (VH-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ BD NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90.
18. The antibody or fragment thereof of claim 17, wherein said VH-CDR3 amino acid sequence is selected from the group consisting of: SEQ ED NO: 20, SEQ ED NO: 30, SEQ ED NO: 40, SEQ EO NO: 50, SEQ EO NO: 60, SEQ ID NO: 70 SEQ ED NO: 80, and SEQ ED NO: 90.
19. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VL of said antibody or fragment thereof comprises a Kabat light chain complementarity determining region- 1 (VL-CDRl) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ EO NO: 53, SEQ EO NO: 63, SEQ ID NO: 73, SEQ E) NO: 83, and SEQ E) NO: 93.
20. The antibody or fragment thereof of claim 19, wherein said VL-CDRl amino acid sequence is selected from the group consisting of: SEQ E) NO: 23, SEQ E) NO: 33, SEQ E) NO: 43, SEQ E) NO: 53, SEQ E) NO: 63, SEQ E) NO: 73 SEQ E) NO: 83, and SEQ E) NO: 93.
21. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VL of said antibody or fragment thereof comprises a Kabat light chain complementarity determining region-2 (VL-CDR2) amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO:
44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74, SEQ ID NO: 84, and SEQ ID NO: 94.
22. The antibody or fragment thereof of claim 21, wherein said VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, SEQ ID NO: 74 SEQ ID NO: 84, and SEQ ID NO: 94.
23. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VL of said antibody or fragment thereof comprises a Kabat light chain complementarity determining region-3 (VL-CDR3) amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 25, SEQ TD NO: 35, SEQ ID NO:
45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, and SEQ ID NO: 95.
24. The antibody or fragment thereof of claim 23, wherein said VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75 SEQ ID NO: 85, and SEQ ID NO: 95.
25. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH of said antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90, except for one, two, three, or four amino acid substitutions in at least one of said VH-CDRs.
26. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VH of said antibody or fragment thereof comprises VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ED NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70 SEQ ID NOs: 78, 79 and 80; and SEQ ID NOs: 88, 89 and 90.
27. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VL of said antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95, except for one, two, three, or four amino acid substitutions in at least one of said VL-CDRs.
28. An isolated antibody or fragment thereof which specifically binds to α6β4 integrin, wherein the VL of said antibody or fragment thereof comprises VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ED NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ED NOs: 83, 84 and 85; and SEQ ID NOs 93, 94 and 95.
29. The antibody or fragment thereof of any one of claims 1 to 28, wherein the VH framework regions are human, except for five or fewer amino acid substitutions.
30. The antibody or fragment thereof of any one of claims 1 to 29, wherein the VL framework regions are human, except for five or fewer amino acid substitutions.
31. The antibody or fragment thereof of any one of claims 1 to 30, which binds to a linear epitope.
32. The antibody or fragment thereof of any one of claims 1 to 30, which binds to a nonlinear conformational epitope.
33. The antibody or fragment thereof of any one of claims 1 to 32, which is a multivalent, and comprises at least two heavy chains and at least two light chains.
34. The antibody or fragment thereof of any one of claims 1 to 33, which is multispecifϊc.
35. The antibody or fragment thereof of claim 34, which is bispecific.
36. The antibody or fragment thereof of any one of claims 1 to 35, which is bispecific.
37. The antibody or fragment thereof of any one of claims 1 to 36, wherein the heavy and light chain variable domains are fully human.
38. The antibody or fragment thereof of claim 37, wherein said heavy and light chain variable domains are from a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05.
39. 39. The antibody or fragment thereof of any one of claims 1 to 36, wherein the heavy and light chain variable domains are murine.
40. The antibody or fragment thereof of claim 39, wherein said heavy and light chain variable domains are from a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
41. The antibody or fragment thereof of any one of claims 1 to 36, which is humanized.
42. The antibody or fragment thereof of any one of claims 1 to 36, which is chimeric.
43. The antibody or fragment thereof of any one of claims 1 to 36, which is primatized.
44. The antibody or fragment thereof of any one of claims 1 to 38, which is fully human.
45. The antibody or fragment thereof of any one of claims 1 to 44, which is an Fab fragment.
46. The antibody or fragment thereof of any one of claims 1 to 44, which is an Fab' fragment.
47. The antibody or fragment thereof of any one of claims 1 to 44, which is an F(ab)2 fragment.
48. The antibody or fragment thereof of any one of claims 1 to 44, which is an Fv fragment.
49. The antibody or fragment thereof of any one of claims 1 to 44, which is a single chain antibody.
50. The antibody or fragment thereof of any one of claims 1 to 47 and 49, which comprises a light chain constant regions selected from the group consisting of a human kappa constant region and a human lambda constant region.
51. The antibody or fragment thereof of any one of claims 1 to 47 and 49, which comprises at a heavy chain constant region or fragment thereof.
52. The antibody or fragment thereof of claim 51, wherein said heavy chain constant region or fragment thereof is human IgG4.
53. The antibody or fragment thereof of any one of claims 1 to 52, which specifically binds to an α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) which is less than the KD for said reference monoclonal antibody.
54. The antibody or fragment thereof of any one of claims 1 to 53, which specifically binds to an α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M, 10-4 M, 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 10-6 M, 5 x 10-7 M, 10-7 M, 5 x 10-8 M, 10-8 M, 5 x 10-9 M, 10-9 M, 5 x 10-10 M, 10- 10 M, 5 x 10-11 M, 10-11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M.
55. The antibody or fragment thereof of any one of claims 1 to 54, which preferentially binds to a human α6β4 integrin polypeptide or fragment thereof, relative to a murine α6β4 integrin polypeptide or fragment thereof.
56. The antibody or fragment thereof of any one of claims 1 to 55, which binds to α6β4 integrin expressed on the surface of a cell.
57. The antibody or fragment thereof of claim 56, wherein said cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
58. The antibody or fragment thereof of any one of claims 1 to 57, which blocks laminin from binding to α6β4 integrin.
59. The antibody or fragment thereof of any one of claims 1 to 58, which inhibits α6β4 integrin association with growth factor receptors.
60. The antibody or fragment thereof of claim 59, wherein said growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
61. The antibody or fragment thereof of any one of claims 1 to 60, which inhibits α6β4 integrin-mediated activation of PD-K.
62. The antibody or fragment thereof of any one of claims 1 to 61, which inhibits α6β4 integrin-mediated activation of the Ras/MAPK signaling pathway.
63. The antibody or fragment thereof of any one of claims 1 to 62, which inhibits α6β4 integrin-mediated cell proliferation.
64. The antibody or fragment thereof of any one of claims 1 to 63, which inhibits tumor cell growth.
65. The antibody or fragment thereof of any one of claims 1 to 64, which induces apoptosis.
66. The antibody or fragment thereof of any one of claims 1 to 65, further comprising a heterologous polypeptide fused thereto.
67. The antibody or fragment thereof of any one of claims 1 to 66, wherein said antibody is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
68. The antibody or fragment thereof of claim 67, wherein said cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents.
69. The antibody or fragment thereof of claim 67, wherein said detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
70. A composition comprising the antibody or fragment thereof of any one of claims 1 to 69, and a carrier.
71. An isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, wherein the amino acid sequence of said VH polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and wherein an antibody or antigen binding fragment thereof comprising said VH polypeptide specifically binds to α6β4 integrin.
72. The polynucleotide of claim 71, wherein the amino acid sequence of said VH polypeptide is selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO:
87.
73. The polynucleotide of claim 71 or 72, wherein the nucleotide sequence encoding said VH polypeptide is optimized for increased expression without changing the amino acid sequence of said VH polypeptide.
74. The polynucleotide of claim 73, wherein said optimization comprises identification and removal of splice donor and splice acceptor sites.
75. The polynucleotide of claim 73 or 74, wherein said optimization comprises optimization of codon usage for the cells expressing said polynucleotide.
76. The polynucleotide of any one of claims 71 to 75, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 66, SEQ ID NO: 76, and SEQ ID NO: 86.
77. An isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein the amino acid sequence of said VL polypeptide is at least 90% identical to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ED NO: 32, SEQ ID NO: 42, SEQ ED NO: 52, SEQ ED NO: 62, SEQ ED NO: 72, SEQ ED NO: 82, and SEQ ED NO: 92; and wherein an antibody or antigen binding fragment thereof comprising said VL polypeptide specifically binds to α6β4 integrin.
78. The polynucleotide of claim 77, wherein the amino acid sequence of said VL polypeptide is selected from the group consisting of: SEQ ED NO: 22, SEQ ED NO: 32, SEQ ED NO: 42, SEQ ED NO: 52, SEQ ED NO: 62, SEQ ED NO: 72, SEQ ED NO: 82, and SEQ ED NO: 92.
79. The polynucleotide of claim 77 or 78, wherein the nucleotide sequence encoding said VL polypeptide is optimized for increased expression without changing the amino acid sequence of said VL polypeptide.
80. The polynucleotide of claim 79, wherein said optimization comprises identification and removal of splice donor and splice acceptor sites.
81. The polynucleotide of claim 79 or 80, wherein said optimization comprises optimization of codon usage for the cells expressing said polynucleotide.
82. The polynucleotide of any one of claims 77 to 81, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of: SEQ ED NO: 21, SEQ ED NO: 31, SEQ ED NO: 41, SEQ ED NO: 51, SEQ ED NO: 61, SEQ ED NO: 71, SEQ ED NO: 81, and SEQ ED NO: 91.
83. An isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, wherein the amino acid sequence of said VH polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ED NO: 17, SEQ ED NO: 27, SEQ ED NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, SEQ ID NO: 77, and SEQ ID NO: 87; and wherein an antibody or antigen binding fragment thereof comprising said VH polypeptide specifically binds to α6β4 integrin.
84. An isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein the amino acid sequence of said VL polypeptide is identical, except for 20 or fewer conservative amino acid substitutions, to a reference amino acid sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO: 92; and wherein an antibody or antigen binding fragment thereof comprising said VL polypeptide specifically binds to α6β4 integrin.
85. An isolated polynucleotide comprising a nucleic acid which encodes a VH-CDRl amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VH-CDRl amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ED NO: 48, SEQ ID NO: 58, SEQ ED NO: 68, SEQ ED NO: 78, and SEQ ED NO: 88; and wherein an antibody or antigen binding fragment thereof comprising said VH-CDRl specifically binds to α6β4 integrin.
86. The polynucleotide or fragment thereof of claim 85, wherein said VH-CDRl amino acid sequence is selected from the group consisting of: SEQ ED NO: 18, SEQ ED NO: 28, SEQ ED NO: 38, SEQ ED NO: 48, SEQ ED NO: 58, SEQ ED NO: 68, SEQ ID NO: 78, and SEQ ED NO: 88.
87. An isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR2 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR2 amino acid sequence selected from the group consisting of: SEQ ED NO: 19, SEQ ED NO: 29, SEQ ED NO: 39, SEQ ED NO: 49, SEQ ED NO: 59, SEQ ED NO: 69, SEQ ED NO: 79, and SEQ ED NO: 89; and wherein an antibody or antigen binding fragment thereof comprising said VH-CDR2 specifically binds to α6β4 integrin.
88. The polynucleotide or fragment thereof of claim 87, wherein said VH-CDR2 amino acid sequence is selected from the group consisting of: SEQ ED NO: 19, SEQ ED NO: 29, SEQ ED NO: 39, SEQ ED NO: 49, SEQ ED NO: 59, SEQ ED NO: 69, SEQ ED NO: 79, and SEQ ED NO: 89.
89. An isolated polynucleotide comprising a nucleic acid which encodes a VH-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VH-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90; and wherein an antibody or antigen binding fragment thereof comprising said VH-CDR3 specifically binds to α6β4 integrin.
90. The polynucleotide or fragment thereof of claim 89, wherein said VH-CDR3 amino acid sequence is selected from the group consisting of: SEQ ED NO: 20, SEQ ID NO: 30, SEQ ID NO: 40, SEQ ID NO: 50, SEQ ID NO: 60, SEQ ID NO: 70, SEQ ID NO: 80, and SEQ ID NO: 90.
91. An isolated polynucleotide comprising a nucleic acid which encodes a VL-CDRl amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDRl amino acid sequence selected from the group consisting of: SEQ DD NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDRl specifically binds to α6β4 integrin.
92. The polynucleotide or fragment thereof of claim 91, wherein said VL-CDRl amino acid sequence is selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 53, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID NO: 83, and SEQ ID NO: 93.
93. An isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR2 amino acid sequence identical, except for two or fewer amino acid substitutions, to a reference VL-CDR2 amino acid sequence selected from the group consisting of: SEQ DD NO: 24, SEQ DD NO: 34, SEQ ID NO: 44, SEQ DD NO: 54, SEQ DD NO: 64, SEQ DD NO: 74, SEQ DD NO: 84 and SEQ DD NO: 94; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR2 specifically binds to α6β4 integrin.
94. The polynucleotide or fragment thereof of claim 93, wherein said VL-CDR2 amino acid sequence is selected from the group consisting of: SEQ DD NO: 24, SEQ DD NO: 34, SEQ DD NO: 44, SEQ DD NO: 54, SEQ DD NO: 64, SEQ DD NO: 74, SEQ DD NO: 84 and SEQ DD NO: 94.
95. An isolated polynucleotide comprising a nucleic acid which encodes a VL-CDR3 amino acid sequence identical, except for four or fewer amino acid substitutions, to a reference VL-CDR3 amino acid sequence selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, and SEQ ID NO: 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to α6β4 integrin.
96. The polynucleotide or fragment thereof of claim 95, wherein said VL-CDR3 amino acid sequence is selected from the group consisting of: SEQ ID NO: 25, SEQ ID NO: 35, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 75, SEQ ID NO: 85, and SEQ ID NO: 95.
97. An isolated polynucleotide comprising a nucleic acid which encodes an antibody VH polypeptide, wherein said VH polypeptide comprises VH-CDRl, VH-CDR2, and VH- CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ID NOs: 78, 79, and 80; and SEQ ID NOs: 88, 89 and 90; and wherein an antibody or antigen binding fragment thereof comprising said VH-CDR3 specifically binds to α6β4 integrin.
98. An isolated polynucleotide comprising a nucleic acid which encodes an antibody VL polypeptide, wherein said VL polypeptide comprises VL-CDRl, VL-CDR2, and VL- CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ID NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ ID NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84, and 85; and SEQ ID NO: 93, 94, and 95; and wherein an antibody or antigen binding fragment thereof comprising said VL-CDR3 specifically binds to α6β4 integrin.
99. The polynucleotide of any one of claims 71 to 98, further comprising a nucleic acid encoding a signal peptide fused to said antibody VH polypeptide.
100. The polynucleotide of any one of claims 71 to 98, further comprising a nucleic acid encoding a signal peptide fused to said antibody VL polypeptide.
101. The polynucleotide of any one of claims 71 to 100, further comprising a nucleic acid encoding a heavy chain constant region CHl domain fused to said VH polypeptide.
102. The polynucleotide of any one of claims 71 to 101, further comprising a nucleic acid encoding a heavy chain constant region CH2 domain fused to said VH polypeptide.
103. The polynucleotide of any one of claims 71 to 102, further comprising a nucleic acid encoding a heavy chain constant region CH3 domain fused to said VH polypeptide.
104. The polynucleotide of any one of claims 71 to 103, further comprising a nucleic acid encoding a heavy chain hinge region fused to said VH polypeptide.
105. The polynucleotide of any one of claims 101 to 104, wherein said heavy chain constant region is human IgG4.
106. The polynucleotide of any one of claims 71 to 105, further comprising a nucleic acid encoding a light chain constant region domain fused to said VL polypeptide.
107. The polynucleotide of claim 106, wherein said light chain constant region is human kappa.
108. The polynucleotide of any one of claims 71 to 107, wherein an antibody or antigen- binding fragment thereof comprising a polypeptide encoded by said nucleic acid specifically binds the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65- AI l, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
109. The polynucleotide of any one of claims 71 to 108, wherein an antibody or antigen- binding fragment thereof comprising a polypeptide encoded by said nucleic acid competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
110. The polynucleotide of any one of claims 71 to 109, wherein the framework regions of said VH polypeptide are human, except for five or fewer amino acid substitutions.
111. The polynucleotide of any one of claims 71 to 110, wherein the framework regions of said VL polypeptide are human, except for five or fewer amino acid substitutions.
112. The polynucleotide of any one of claims 71 to 111, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid binds to a linear epitope.
113. The polynucleotide of any one of claims 71 to 111, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid binds to a non-linear conformational epitope.
114. The polynucleotide of any one of claims 71 to 113, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is a multivalent, and comprises at least two heavy chains and at least two light chains.
115. The polynucleotide of any one of claims 71 to 114, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is multispecific.
116. The polynucleotide of any one of claims 71 to 115, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is bispecific.
117. The polynucleotide of any one of claims 71 to 116, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid comprises heavy and light chain variable domains which are fully human.
118. The polynucleotide of claim 117, wherein said heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05.
119. The polynucleotide of any one of claims 71 to 116, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid comprises heavy and light chain variable domains which are murine.
120. The polynucleotide of claim 119, wherein said heavy and light chain variable domains are identical to those of a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
121. The polynucleotide of any one of claims 71 to 116, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is humanized.
122. The polynucleotide of any one of claims 71 to 116, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is chimeric.
123. The polynucleotide of any one of claims 71 to 116, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is primatized.
124. The polynucleotide of any one of claims 71 to 118, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is fully human.
125. The polynucleotide of any one of claims 71 to 124, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is an Fab fragment.
126. The polynucleotide of any one of claims 71 to 124, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is an Fab' fragment.
127. The polynucleotide of any one of claims 71 to 124, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is an F(ab)2 fragment.
128. The polynucleotide of any one of claims 71 to 124, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is an Fv fragment.
129. The polynucleotide of any one of claims 71 to 124, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is a single chain antibody.
130. The polynucleotide of any one of claims 71 to 129, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid specifically binds to an α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M, 10-4 M, 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 10-6 M, 5 x 10-7 M, 10-7 M, 5 x 10-8 M, 10-8 M, 5 x 10-9 M, 10-9 M, 5 x 10-10 M, 10-10 M, 5 x 10-11 M, 10-11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M.
131. The polynucleotide of any one of claims 71 to 130, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid preferentially binds to a human α6β4 integrin polypeptide or fragment thereof, relative to a murine α6β4 integrin polypeptide or fragment thereof.
132. The polynucleotide of any one of claims 71 to 131, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid binds to α6β4 integrin expressed on the surface of a cell.
133. The polynucleotide of claim 132, wherein said cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
134. The polynucleotide of any one of claims 71 to 133, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid blocks laminin from binding to α6β4 integrin.
135. The polynucleotide of any one of claims 71 to 134, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid inhibits α6β4 integrin association with growth factor receptors.
136. The polynucleotide of claim 135, wherein said growth factor receptor was selected from the group consisting of erbB2, Met, and Ron.
137. The polynucleotide of any one of claims 71 to 136, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid inhibits α6β4 integrin-mediated activation of PI3-K.
138. The polynucleotide of any one of claims 71 to 137, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid inhibits α6β4 integrin-mediated activation of the Ras/MAPK signaling pathway.
139. The polynucleotide of any one of claims 71 to 138, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid inhibits α6β4 integrin-mediated cell proliferation.
140. The polynucleotide of any one of claims 71 to 139, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid inhibits tumor cell growth.
141. The polynucleotide of any one of claims 71 to 140, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid induces apoptosis.
142. The polynucleotide of any one of claims 71 to 141, further comprising a nucleic acid encoding a heterologous polypeptide.
143. The polynucleotide of any one of claims 71 to 142, wherein an antibody or antigen- binding fragment thereof comprising the polypeptide encoded by said nucleic acid is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
144. The polynucleotide of claim 143, wherein said cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents.
145. The polynucleotide of claim 143, wherein said detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
146. A composition comprising the polynucleotide any one of claims 71 to 145, and a carrier.
147. A vector comprising the polynucleotide of any one of claims 71 to 145.
148. The vector of claim 143, wherein said polynucleotide is operably associated with a promoter.
149. A host cell comprising the vector of claim 147 or claim 148.
150. A method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising culturing the host cell of claim 149, and recovering said antibody, or fragment thereof.
151. An isolated polypeptide produced by the method of claim 150.
152. An isolated polypeptide encoded by the polynucleotide of any one of claims 71 to 145.
153. The isolated polypeptide of claim 152, wherein an antibody or fragment thereof comprising said polypeptide specifically binds to α6β4 integrin.
154. An isolated antibody or fragment thereof comprising the polypeptide of claim 152 or 153.
155. A composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, wherein said VH encoding polynucleotide and said VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences at least 90% identical to reference amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ID NO: 62; SEQ ID NO: 67 and 72; SEQ ID NO: 77 and 82; and SEQ ID NO: 87 and 92; and wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides specifically binds α6β4 integrin.
156. The composition of claim 155, wherein said VH encoding polynucleotide and said VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ID NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ID NO: 62; SEQ ID NO: 67 and 72; SEQ ID NO: 77 and 82; and SEQ ID NO: 87 and 92.
157. A composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, wherein said VH encoding polynucleotide and said VL encoding polynucleotide, respectively, comprise nucleic acids encoding amino acid sequences identical, except for less than 20 conservative amino acid substitutions, to reference amino acid sequences selected from the group consisting of: SEQ ID NO: 17 and SEQ ED NO: 22; SEQ ID NO: 27 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 42; SEQ ID NO: 47 and SEQ ID NO: 52; SEQ ID NO: 57 and SEQ ID NO: 62; SEQ ID NO: 67 and 72; SEQ ID NO: 77 and 82; and SEQ ID NO: 87 and 92; and wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides specifically binds α6β4 integrin.
158. A composition comprising an isolated VH encoding polynucleotide and an isolated VL encoding polynucleotide, wherein said VH encoding polynucleotide encodes a VH polypeptide comprising VH-CDRl, VH-CDR2, and VH-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 18, 19, and 20; SEQ ID NOs: 28, 29, and 30; SEQ ID NOs: 38, 39, and 40; SEQ ID NOs: 48, 49, and 50; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 68, 69, and 70; SEQ ED NOs: 78, 79 and 80; and SEQ ED NOs: 88, 89, and 90; and wherein said VL encoding polynucleotide encodes a VL polypeptide comprising VL-CDRl, VL-CDR2, and VL-CDR3 amino acid sequences selected from the group consisting of: SEQ ID NOs: 23, 24, and 25; SEQ ED NOs: 33, 34, and 35; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 53, 54, and 55; SEQ DD NOs: 63, 64, and 65; SEQ ID NOs: 73, 74, and 75; SEQ ID NOs: 83, 84, and 85; and SEQ ID NOs: 93, 94 and 95; and wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides specifically binds α6β4 integrin.
159. The composition of any one of claims 155-158, wherein said VH encoding polynucleotide further comprises a nucleic acid encoding a signal peptide fused to said antibody VH polypeptide.
160. The composition of any one of claims 155-158, wherein said VL encoding polynucleotide further comprises a nucleic acid encoding a signal peptide fused to said antibody VL polypeptide.
161. The composition of any one of claims 155 to 160, wherein said VH encoding polynucleotide further comprises a nucleic acid encoding a heavy chain constant region CHl domain fused to said VH polypeptide.
162. The composition of any one of claims 155 to 161, wherein said VH encoding polynucleotide further comprises a nucleic acid encoding a heavy chain constant region CH2 domain fused to said VH polypeptide.
163. The composition of any one of claims 155 to 162, wherein said VH encoding polynucleotide further comprises a nucleic acid encoding a heavy chain constant region CH3 domain fused to said VH polypeptide.
164. The composition of any one of claims 155 to 163, wherein said VH encoding polynucleotide further comprises a nucleic acid encoding a heavy chain hinge region fused to said VH polypeptide.
165. The composition of any one of claims 161 to 164, wherein said heavy chain constant region is human IgG4.
166. The composition of any one of claims 155 to 165, wherein said VL encoding polynucleotide further comprises a nucleic acid encoding a light chain constant region domain fused to said VL polypeptide.
167. The composition of claim 166, wherein said light chain constant region is human kappa.
168. The composition of any one of claims 155 to 167, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides specifically binds the same α6β4 integrin epitope as a reference monoclonal Fab antibody fragment selected from the group consisting of M59-B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
169. The composition of any one of claims 155 to 167, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides competitively inhibits a reference monoclonal Fab antibody fragment selected from the group consisting of M59- B05, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05 or a reference monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D10.1 and 1.P5B10.2.
170. The composition of any one of claims 155 to 169, wherein the framework regions of said VH and VL polypeptides are human, except for five or fewer amino acid substitutions.
171. The composition of any one of claims 155 to 170, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides binds to a linear epitope.
172. The composition of any one of claims 155 to 170, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides binds to a non-linear conformational epitope.
173. The composition of any one of claims 155 to 170, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is multivalent, and comprises at least two heavy chains and at least two light chains.
174. The composition of any one of claims 155 to 173, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is multispecific.
175. The composition of any one of claims 155 to 174, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is bispecific.
176. The composition of any one of claims 155 io 175, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are fully human.
177. The composition of claim 176, wherein said heavy and light chain variable domains are identical to those of a monoclonal Fab antibody fragment selected from the group consisting of M59-BO5, M61-C02, M61-C03, M65-A11, M66-H09 and M67-F05.
178. The composition of any one of claims 155 to 175, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides comprises heavy and light chain variable domains which are murine.
179. The composition of claim 178, wherein said heavy and light chain variable domains are identical to those of a monoclonal antibody produced by a hybridoma selected from the group consisting of 1.P2D 10.1 and 1.P5B10.2.
180. The composition of any one of claims 155 to 175, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is humanized.
181. The composition of any one of claims 155 to 175, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is chimeric.
182. The composition of any one of claims 155 to 175, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is primatized.
183. The composition of any one of claims 155 to 177, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is fully human.
184. The composition of any one of claims 155 to 183, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is an Fab fragment.
185. The composition of any one of claims 155 to 183, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is an Fab' fragment.
186. The composition of any one of claims 155 to 183, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is an F(ab)2 fragment.
187. The composition of any one of claims 155 to 183, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is an Fv fragment.
188. The composition of any one of claims 155 to 183, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is a single chain antibody.
189. The composition of any one of claims 155 to 188, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides specifically binds to an α6β4 integrin polypeptide or fragment thereof, or an α6β4 integrin variant polypeptide, with an affinity characterized by a dissociation constant (KD) no greater than 5 x 10-2 M, 10-2 M, 5 x 10-3 M, 10-3 M, 5 x 10-4 M, 10-4 M, 5 x 10-5 M, 10-5 M, 5 x 10-6 M, 10-6 M, 5 x 10-7 M, 10-7 M, 5 x 10-8 M, 10-8 M, 5 x 10-9 M, 10-9 M, 5 x 10-10 M, 10- 10 M, 5 x 10-11 M, 10-11 M, 5 x 10-12 M, 10-12 M, 5 x 10-13 M, 10-13 M, 5 x 10-14 M, 10-14 M, 5 x 10-15 M, or 10-15 M.
190. The composition of any one of claims 155 to 189, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides preferentially binds to a human α6β4 integrin polypeptide or fragment thereof, relative to a murine α6β4 integrin polypeptide or fragment thereof.
191. The composition of any one of claims 155 to 190, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides binds to α6β4 integrin expressed on the surface of a cell.
192. The composition of claim 191, wherein said cell is a malignant cell, a neoplastic cell, a tumor cell, or a metastatic cell.
193. The composition of any one of claims 155 to 192, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides blocks laminin from binding to α6β4 integrin.
194. The composition of any one of claims 155 to 193, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides inhibits α6β4 integrin association with growth factor receptors.
195. The composition of claim 194, wherein said growth factor receptor is selected from the group consisting of erbB2, Met, and Ron.
196. The composition of any one of claims 155 to 195, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides inhibits α6β4 integrin- mediated activation of PI3-K.
197. The composition of any one of claims 155 to 196, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides inhibits α6β4 integrin- mediated activation of the Ras/MAPK signaling pathway.
198. The composition of any one of claims 155 to 197, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides inhibits α6β4 integrin- mediated cell proliferation.
199. The composition of any one of claims 155 to 198, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides inhibits tumor cell growth.
200. The composition of any one of claims 155 to 199, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides induces apoptosis.
201. The composition of any one of claims 155 to 200, wherein said VH encoding polynucleotide, said VL encoding polynucleotide, or both said VH and said VL encoding polynucleotides further comprise a nucleic acid encoding a heterologous polypeptide.
202. The composition of any one of claims 155 to 201, wherein an antibody or fragment thereof encoded by said VH and VL encoding polynucleotides is conjugated to an agent selected from the group consisting of cytotoxic agent, a therapeutic agent, cytostatic agent, a biological toxin, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, polyethylene glycol (PEG), and a combination of two or more of any said agents.
203. The composition of claim 202, wherein said cytotoxic agent is selected from the group consisting of a radionuclide, a biotoxin, an enzymatically active toxin, a cytostatic or cytotoxic therapeutic agent, a prodrugs, an immunologically active ligand, a biological response modifier, or a combination of two or more of any said cytotoxic agents.
204. The composition of claim 202, wherein said detectable label is selected from the group consisting of an enzyme, a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, or a combination of two or more of any said detectable labels.
205. The composition of any one of claims 155 to 204, wherein said VH encoding polynucleotide is contained on a first vector and said VL encoding polynucleotide is contained on a second vector.
206. The composition of claim 205, wherein said VH encoding polynucleotide is operably associated with a first promoter and said VL encoding polynucleotide is operably associated with a second promoter.
207. The composition of claim 206, wherein said first and second promoters are copies of the same promoter.
208. The composition of claim 206, wherein said first and second promoters non-identical.
209. The composition of any one of claims 205 to 208, wherein said first vector and said second vector are contained in a single host cell.
210. The composition of any one of claims 205 to 208, wherein said first vector and said second vector are contained in a separate host cells.
211. A method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising culturing the host cell of claim 209, and recovering said antibody, or fragment thereof.
212. A method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising co-culturing the separate host cells of claim 210, and recovering said antibody, or fragment thereof.
213. A method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising separately culturing the separate host cells of claim 210, combining said VH and VL encoding polypeptides, and recovering said antibody, or fragment thereof.
214. An antibody or fragment thereof which specifically binds α6β4 integrin, produced by the method of any one of claims 211 to 213.
215. The composition of any one of claims 155 to 204, wherein said VH encoding polynucleotide and said VL encoding polynucleotide are on the same vector.
216. The vector of claim 215.
217. The vector of claim 216, wherein said VH encoding polynucleotide and said VL encoding polynucleotide are each operably associated with a promoter.
218. The vector of claim 216, wherein said VH encoding polynucleotide and said VL encoding polynucleotide are fused in frame, are co-transcribed from a single promoter operably associated therewith, and are cotranslated into a single chain antibody or antigen-binding fragment thereof.
219. The vector of claim 216, wherein said VH encoding polynucleotide and said VL encoding polynucleotide are co-transcribed from a single promoter operably associated therewith, but are separately translated.
220. The vector of claim 219, further comprising an IRES sequence disposed between said VH encoding polynucleotide and said VL encoding polynucleotide.
221. The vector of claim 216, wherein said polynucleotide encoding a VH and said polynucleotide encoding a VL are separately transcribed, each being operably associated with a separate promoter.
222. The vector of claim 221 wherein said separate promoters are copies of the same promoter.
223. The vector of claim 221, wherein said separate promoters are non-identical.
224. A host cell comprising the vector of any one of claims 216 to 223.
225. A method of producing an antibody or fragment thereof which specifically binds α6β4 integrin, comprising culturing the host cell of claim 224, and recovering said antibody, or fragment thereof.
226. An antibody or fragment thereof which specifically binds α6β4 integrin, produced by the method of claim 225.
227. A method for treating a hyperproliferative disorder in an animal, comprising administering to an animal in need of treatment a composition comprising:
a) the isolated antibody or fragment thereof of any one of claims 1 to 69, 154, 214 and 226; and b) a pharmaceutically acceptable carrier.
228. The method of claim 227, wherein said hyperproliferative disease or disorder is selected from the group consisting of cancer, a neoplasm, a tumor, a malignancy, or a metastasis thereof.
229. The method of claim 228, wherein said antibody or fragment thereof specifically binds to α6β4 integrin expressed on the surface of a malignant cell.
230. The method of claim 229, wherein binding of said antibody or fragment thereof to said malignant cell results in growth inhibition of said malignant cell.
231. The method of any one of claims 227 to 230, wherein said antibody or fragment thereof inhibits tumor cell proliferation.
232. The method of claim 231, wherein tumor cell proliferation is inhibited through the prevention or retardation of metastatic growth.
233. The method of any one of claims 227 to 230, wherein said antibody or fragment thereof inhibits tumor cell migration.
234. The method of claim 231, wherein tumor cell proliferation is inhibited through the prevention or retardation of tumor spread to adjacent tissues.
235. The method of claim 228, wherein said hyperproliferative disease or disorder is a neoplasm located in the: prostate, colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, adrenal gland, parathyroid gland, pituitary gland, testicles, ovary, thymus, thyroid, eye, head, neck, central nervous system, peripheral nervous system, lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, or urogenital tract.
236. The method of claim 228, wherein said hyperproliferative disease is cancer, said cancer selected from the group consisting of: epithelial squamous cell cancer, melanoma, leukemia, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, renal cancer, prostate cancer, testicular cancer, thyroid cancer, and head and neck cancer.
237. The method of claim 236, wherein said cancer is selected from the group consisting of stomach cancer, renal cancer, brain cancer, bladder cancer, colon cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, and prostate cancer.
238. The method of any one of claims 227 to 237, wherein said animal is a mammal.
239. The method of claim 238, wherein said mammal is a human.
240. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein said reference monoclonal Fab antibody fragment is M59-B05.
241. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein said reference monoclonal Fab antibody fragment is M61-C03.
242. The isolated antibody or antigen-binding fragment thereof of claim 241, wherein said α6β4 integrin epitope is contained between amino acids 446-686 of SEQ ID NO: 101.
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