WO2020014306A1 - Anticorps anti-met, immunoconjugués et utilisations de ceux-ci - Google Patents

Anticorps anti-met, immunoconjugués et utilisations de ceux-ci Download PDF

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WO2020014306A1
WO2020014306A1 PCT/US2019/041128 US2019041128W WO2020014306A1 WO 2020014306 A1 WO2020014306 A1 WO 2020014306A1 US 2019041128 W US2019041128 W US 2019041128W WO 2020014306 A1 WO2020014306 A1 WO 2020014306A1
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seq
ala
antibody
immunoconjugate
met
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WO2020014306A8 (fr
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Stuart William HICKS
Katharine C. LAI
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Immunogen, Inc.
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • MET also known as c-Met, HGFR, RCCP2 or AUTS9, is a glycosylated receptor tyrosine kinase that plays a central role in epithelial morphogenesis and cancer development. It is also referred to as the hepatocyte growth factor or HGF receptor, the scatter factor or SF receptor, the met proto-oncogene tyrosine kinase or proto-oncogene c-Met.
  • MET is synthesized as a single chain precursor which undergoes co-translational proteolytic cleavage. This generates a mature MET that is a disulfide-linked dimer composed of a 50 kDa extracellular a chain and a 145 kDa transmembrane b chain (Birchmeier, C. et al. Nat. Rev. Mol. Cell Biol. 2003; 4:915; Corso, S. et al. Trends Mol. Med. 2005; 11:284).
  • the extracellular domain contains a seven bladed b-propeller sema domain, a cysteine-rich PS I/MRS domain, and four Ig-like E-set domains, while the cytoplasmic region includes the tyrosine kinase domain and an adaptor protein docking site (Gherardi, E. et al. Proc. Natl. Acad. Sci. 2003; 100:12039, Park, M. et al. Proc. Natl. Acad. Sci. 1987; 84:6379).
  • the sema domain which is formed by both the a and b chains of MET, mediates both ligand binding and receptor dimerization (Gherardi, E. et al. Proc. Natl. Acad. Sci. 2003; 100:12039, Kong- Beltran, M. et al. Cancer Cell 2004; 6:75).
  • Hepatocyte growth factor is the ligand for MET (Gheradi, E. et al. Proc. Natl. Acad. Sci. 2003; 100:12039). HGF is also known as scatter factor (SF) and hepatopoietin A and it belongs to the plasminogen subfamily of Sl peptidases. Human HGF is produced and secreted as an inactive 728 amino acid (AA) single chain propeptide. It is cleaved after the fourth Kringle domain by a serine protease to form the active form of HGF, which is a disulfide-linked heterodimer with a 60 kDa a and 30 kDa b chain.
  • AA 728 amino acid
  • HGF regulates epithelial morphogenesis by inducing cell scattering and branching tubulogenesis (Maeshima, A. et al. Kid. Int. 2000; 58:1511; Montesano, R. et al. Cell 1991; 67:901).
  • MET a cell scattering and branching tubulogenesis
  • Aberrant signaling by MET can be the result of multiple mechanisms including ligand-independent activation such as through MET overexpression or MET activating mutations and ligand-dependent activation in either paracrine or autocrine manner.
  • Paracrine induction of epithelial cell scattering and branching tubulogenesis results from the stimulation of MET on undifferentiated epithelium by HGF released from neighboring mesenchymal cells (Sonnenberg, E. et al. J. Cell Biol. 1993; 123:223).
  • Autocrine induction is a result of HGF production by MET positive cells.
  • MET may also form non-covalent complexes with a variety of membrane proteins including CD44v6, CD151, EGF R, Fas, Integrin a6/b4, Plexins Bl, 2, 3, and MSP R/Ron (Orian Rousseau, V. et al. Genes Dev. 2002; 16:3074; Follenzi, A. et al. Oncogene 2000; 19:3041). Ligation of one complex component triggers activation of the other, followed by cooperative signaling effects. Formation of some of these heteromeric complexes can lead to epithelial cell morphogenesis and tumor cell invasion (Trusolino, L. et al. Cell 2001;
  • MetMab Jin H, et al. Cancer Res 2008; 68, 4360-4368.
  • this one armed version cannot be considered a full antibody but rather represents an antibody fragment with undesirable properties including a diminished effector function and a reduced half-life.
  • ADC Antibody-drug conjugates
  • cytotoxic agent covalently linked to an antibody through specialized chemical linker.
  • the use of ADCs for the local delivery of cytotoxic or cytostatic agents, for example, drugs to kill or inhibit tumor cells in the treatment of cancer see Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151- 172; U.S. Pat. No.
  • Monoclonal Antibodies '84 Biological And Clinical Applications, A. Pinchera et al. (eds.), pp. 475-506). Maximal efficacy with minimal toxicity is sought. Both polyclonal antibodies and monoclonal antibodies have sometimes been reported as being useful in this regard. (See Rowland et ah, (1986) Cancer Immunol. Immunother., 21:183-87). Drugs that are known to be used in this fashion include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et ah, Cancer Immunol. Immunother. 21:183-87 (1986)).
  • Toxins used in antibody- toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins, such as ricin, small molecule toxins such as geldanamycin. Kerr et al (1997) Bioconjugate Chem. 8(6):78l- 784; Mandler et al (2000) Journal of the Nat. Cancer Inst. 92(19): 1573-1581; Mandler et al (2000) Bioorganic & Med. Chem. Letters 10: 1025-1028; Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928;
  • Toxins may exert cytotoxic and/or cytostatic effects through diverse mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Meyer, D. L. and Senter, P. D. "Recent Advances in Antibody Drug Conjugates for Cancer Therapy” in Annual Reports in Medicinal Chemistry, Vol 38 (2003) Chapter 23, 229-237. But many cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
  • the present invention provides anti-MET immunoconjugates exhibiting specific and potent cytotoxic activity in MET over-expressed, non-amplified and MET-amplifed settings.
  • the present invention provides an immunoconjugate represented by the following formula:
  • CB is an anti-cMET antibody or an antigen-binding fragment thereof;
  • L 2 is represented by one of the following formula:
  • R x , R y , R x and R y are independently H, -OH, halogen, -0-(Ci_ 4 alkyl), -S0 3 H, -NR 4 oR 4i R 42 + , or a C alkyl optionally substituted with -OH, halogen, SO 3 H or NR 4 oR 4i R 42 + , wherein R 4 o, R 4I and R 42 are each independently H or a Ci_ 4 alkyl;
  • I and k are each independently an integer from 1 to 10;
  • II is an integer from 2 to 5;
  • kl is an integer from 1 to 5;
  • sl indicates the site connected to the cell-binding agent CB and s3 indicates the site connected to the A group;
  • A is an amino acid residue or a peptide comprising 2 to 20 amino acid residues
  • R 1 and R 2 are each independently H or a Ci_ 3 alkyl
  • Li is represented by the following formula:
  • q is an integer from 1 to 20.
  • the anti-cMET antibody or an antigen-binding fragment is an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to an epitope in the extracellular region of human cMET, wherein said antibody or antigen binding fragment thereof comprises light chain complementary determining regions LC CDR1, LC CDR2, and LC CDR3 and heavy chain complementary determining regions HC CDR1, HC CDR2, and HC CDR3 having the sequences selected from the group consisting of:
  • the antibody is a murine, non-human mammal, chimeric, humanized, or human antibody.
  • the humanized antibody is a CDR- grafted antibody or resurfaced antibody.
  • the antibody is a full-length antibody.
  • the antigen-binding fragment is an Fab, Fab’, F(ab’) 2 , F d , single chain Fv or scFv, disulfide linked F v , V-NAR domain, IgNar, intrabody, IgGACH 2 , minibody, F(ab’) 3 , tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2 , (SCFV) 2 , or scFv-Fc.
  • the antibody or antigen-binding fragment thereof comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) having sequences that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to sequences selected from the group consisting of:
  • the antibody or antigen-binding fragment thereof comprises a light chain and a heavy chain having the sequences selected from the group consisting of:
  • the antibody comprises a light chain having the sequence of SEQ ID NO:49 and and a heavy chain having the sequence of SEQ ID NO:53.
  • the antibody comprises a light chain having the sequence of SEQ ID NO:49 and and a heavy chain having the sequence of SEQ ID NO:82.
  • the antibody comprises a light chain having the sequence of SEQ ID NO:49 and and a heavy chain having the sequence of SEQ ID NO:54.
  • the antibody comprises a light chain having the sequence of SEQ ID NO:49 and and a heavy chain having the sequence of SEQ ID NO:8l.
  • the isolated antibody, or antigen-binding fragment thereof is produced by any of hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, and 247.16.8.
  • the present invention provides a polypeptide comprising the VL and VH sequences described herein.
  • the immunoconjugate of the present invention is represented by the following formula:
  • CBA is an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to an epitope in the extracellular region of human cMET, wherein the antibody or antigen-binding fragment thereof comprises light chain complementary determining regions LC CDR1, LC CDR2, and LC CDR3 and heavy chain complementary determining regions HC CDR1, HC CDR2, and HC CDR3 having the sequences of SEQ ID NOs:4, 5, and 7 and SEQ ID NOs:l3, 14, and 15, respectively.
  • the isolated monoclonal antibody, or antigen-binding fragment thereof comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) having sequences of SEQ ID NO:32 and SEQ ID NO:36, respectively.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:54;
  • q 1 or 2;
  • the isolated monoclonal antibody, or antigen-binding fragment thereof comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) having sequences of SEQ ID NO:32 and SEQ ID NO:36, respectively.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:54.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:8l.
  • the immunoconjugate of the present invention is represented by the following formula:
  • CBA an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to an epitope in the extracellular region of human cMET, wherein said antibody or antigen-binding fragment thereof comprises light chain complementary determining regions LC CDR1, LC CDR2, and LC CDR3 and heavy chain complementary determining regions HC CDR1, HC CDR2, and HC CDR3 having the sequences of SEQ ID NOs:4, 5, and 7 and SEQ ID NOs:l3, 14, and 15, respectively;
  • q is an integer from 1 or 10;
  • the isolated monoclonal antibody, or antigen-binding fragment thereof comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) having sequences of SEQ ID NO:32 and SEQ ID NO:36, respectively.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:53.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:82.
  • the present invention also provides a pharmaceutical composition comprising an immunoconjugate described herein and a pharmaceutically acceptable carrier.
  • the present invention also provides a method for inhibiting aberrant cell proliferation comprising contacting a MET-expressing cell with an immunoconjugate described herein, wherein said contacting inhibits the aberrant proliferation of said cells.
  • the contacting induces apoptosis of the cells.
  • the MET-expressing cell is a cancer cell.
  • the cancer cell is cMet overexpressed, non- amplified.
  • the cancer cell is cMet amplified.
  • Also provided in the present invention is a method for treating a cell proliferation disorder in a patient, comprising administering to the patient a therapeutically effective amount of an immunoconjugate, or a pharmaceutical composition thereof described herein.
  • the present invention also provides an immunoconjugate, or a pharmaceutical composition thereof described herein for use in treating a cell proliferation disorder in a patient. Also provided in the present invention is an use of an immunoconjugate, or a pharmaceutical composition thereof for the manufacture of a medicament for treating a cell proliferation disorder in a patient.
  • the patient has been identified having cMet overexpressed, non-amplified. In certain embodiments, the patients has been identified having cMet amplified.
  • the cell proliferation disorder is cancer.
  • the cancer is a cancer selected from the group consisting of glioblastoma, pancreatic cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer,
  • HCC hepatocellular carcinoma
  • SCLC small-cell lung cancer
  • NSCLC non-small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • kidney cancer renal cancer
  • esophageal cancer esophageal cancer
  • thyroid cancer esophageal cancer
  • the cancer is Met-amplified NSCLC.
  • FIG. 1 depicts the results of HGF-binding assay using MKN45 (solid bars) or BxPC3 cells (open bars) incubated with hybridoma supernatant obtained from fusion 247 containing various anti-MET antibodies.
  • FIG. 2 depicts the results of HGF-binding assay using MKN45 (solid bars) or BxPC3 cells (open bars) incubated with hybridoma supernatant obtained from fusion 248 containing various anti-MET antibodies.
  • FIGs. 3A and 3B show sequence alignment of CDR-grafted hucMET-27 constructs.
  • FIGs. 4A and 4B show positions of hackmutations in CDR-grafted hucMET-27 constructs.
  • FIG. 5 shows binding of hucMET-27 antibodies to NCI-H441 cells expressing human cMET antigen as determined by FACS.
  • FIG. 6 shows FACS binding data of hucMET-27 antibodies and conjugates to EBC-l cells.
  • FIG. 7 shows pErk stimulation of hucMET-27 antibodies in NCI-H441 cells.
  • FIG. 8 shows pAkt stimulation of hucMET-27 antibodies in NCI-H441 cells.
  • FIG. 9 shows cell proliferation of different cMET reference antibodies and hucMET- 27 antibodies with and without hinge modifications.
  • FIG. 10 shows pERK stimulation of different cMET reference antibodies and hucMET-27 antibodies with and without hinge modifications.
  • FIG. 11 shows pAKT stimulation of different cMET reference antibodies and hucMET-27 antibodies with and without hinge modifications.
  • FIGs. 12A, 12B and 12C show synthetic schemes for preparing exemplary maytansinoid compounds and immunoconjugates of the present invention.
  • FIG. 13 shows in vitro cytotoxicity of anti-cMET LDL-DM conjugates in cMET- expressing tumor cell lines.
  • FIG. 14 shows the anti-tumor activity of hucMET27Gvl.3-sSPDB-DM4 (2.5 mg/kg and 5 mg/kg) and hucMet27Gvl.3 -GMBS -LDL-DM (1.25 mg/kg, 2.5 mg/kg and 5 mg/kg) in the Hs746T gastric MET-amplified xenograft model.
  • FIG. 15 shows the anti-tumor activity of hucMET27Gvl.3-sSPDB-DM4 (2.5 mg/kg and 5 mg/kg) and hucMet27Gv 1.3 -GMBS -LDL-DM (1.25 mg/kg, 2.5 mg/kg and 5 mg/kg) in EBC-l, a human non-small cell lung cancer MET-amplified xenograft model.
  • FIG. 15 shows the anti-tumor activity of hucMET27Gvl.3-sSPDB-DM4 (2.5 mg/kg and 5 mg/kg) and hucMet27Gv 1.3 -GMBS -LDL-DM (1.25 mg/kg, 2.5 mg/kg and 5 mg/kg) in EBC-l, a human non-small cell lung cancer MET-amplified xenograft model.
  • 16 shows the anti-tumor activity of hucMET27Gvl.3Hinge28-sSPDB-DM4 (100 mg/kg, based on payload) and hucMet27Gvl.3Hinge28-GMBS-LDL-DM (100 mg/kg, based on payload) in NCI-H1975, a human non-small cell adeno-carcinoma cMet over-expressed xenograft model.
  • FIG. 17 shows the antitumor activity of hucMet27Gvl.3Hinge28-sSPDB-DM4 (100 mg/kg, based on payload), hucMet27Gvl.3Hinge28-GMBS-LDL-DM (50 mg/kg and 100 mg/kg, based on payload) and hucMet27Gvl.3Hinge28-VC-MMAE (100 mg/kg, based on payload) in Detroit 562, a human head and neck squamous cell carcinoma cMet over expressed xenograft model.
  • FIG. 18 shows the antitumor activity of hucMet27Gvl.3Hinge28-sSPDB-DM4 (100 mg/kg, based on payload), hucMet27Gvl.3Hinge28-GMBS-LDL-DM (50 mg/kg and 100 mg/kg, based on payload) and hucMet27Gvl.3Hinge28-VC-MMAE (100 mg/kg, based on payload) in NCI-H441, a human non-small cell lung cancer cMet over-expressed xenograft model.
  • the term“MET” or“c-MET” or“cMET” or“MET antigen” or HGFR or HGF receptor refers to polypeptides and any variants, isoforms and species homologs of MET that are naturally expressed or are expressed on cells transfected with the HGFR gene, or the like.
  • Human MET is also known as the hepatocyte growth factor or HGF receptor, the scatter factor or SF receptor, and is a member of the receptor tyrosine kinase family.
  • Transcript Variant 1 represents the longer transcript corresponding to GenBank ID (GI) 42741654. It encodes the longer isoform (a) and comprises a 1408 amino acid protein described by GenBank Protein ID 42741655.
  • Transcript Variant 2 uses an alternate in-frame splice junction at the end of an exon compared to variant 1 and corresponds to GenBank ID (GI) 188595715.
  • the resulting isoform (b) comprises a 1390 amino acid protein described by GenBank Protein ID 188595716 and has the same bl and C-termini but is shorter compared to isoform (a).
  • “aberrant MET receptor activation” refers to the dysregulation of MET expression and/or MET signaling including, but not limited to, overexpression of c-Met and/or HGF (e.g., in the presence or absence of gene amplification, e.g., cMET
  • “aberrant MET receptor activation” may mean and include any heightened or altered expression or overexpression of MET protein in a tissue, e.g. an increase in the amount of a protein, caused by any means including enhanced expression or translation, modulation of the promoter or a regulator of the protein, amplification of a gene for a protein, or enhanced half-life or stability, such that more of the protein exists or can be detected at any one time, in contrast to a non-overexpressed state.
  • Aberrant MET expression includes and contemplates any scenario or alteration wherein the MET protein expression or post-translational modification is overexpressed, including wherein an altered MET protein, as in mutated MET protein or variant due to sequence alteration, deletion or insertion, or altered folding is expressed.
  • “aberrant MET receptor activation” may refer to enhanced MET receptor signaling activity that leads to the activation of key oncogenic signaling pathways including, but not limited to, RAS, PI3 kinase, STAT, b-catenin, Notch, Src, MAPK and Akt signaling pathways.“Aberrant MET receptor activation” may be associated with enhanced angiogenesis and cell metastasis.
  • “aberrant MET receptor activation” refers to MET receptor activation, receptor dimerization and associated activation of tyrosine kinase and/or serine/threonine kinase activity.
  • “aberrant MET receptor activation” is present when MET receptor associated tyrosine kinase activity is activated.
  • a MET receptor associated tyrosine kinase activity is activated when the MET associated tyrosine kinase activity is detectable.
  • an“antibody” or fragment and the like includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain variable region or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of an antigen or antigen receptor or binding protein, which can be incorporated into an antibody to MET of the present invention.
  • CDR complementarity determining region
  • Such antibody optionally further affects a specific ligand, such as, but not limited to, where such antibody modulates, decreases, increases, antagonizes, agonizes, partially agonizes, partially antagonizes, mitigates, alleviates, blocks, inhibits, abrogates and/or interferes with at least one antigen activity or binding, or with antigen receptor activity or binding, in vitro, in situ, in vivo and ex vivo.
  • a specific ligand such as, but not limited to, where such antibody modulates, decreases, increases, antagonizes, agonizes, partially agonizes, partially antagonizes, mitigates, alleviates, blocks, inhibits, abrogates and/or interferes with at least one antigen activity or binding, or with antigen receptor activity or binding, in vitro, in situ, in vivo and ex vivo.
  • MET specific antibodies are disclosed, wherein a specified portion or variant can bind at least one antigen molecule, or specified portions, variants
  • a suitable antigen specific antibody, specified portion, or variant can also optionally affect at least one activity or function, such as, but not limited to, ligand binding, receptor dimerization, receptor phosphorylation, receptor signaling, membrane association, cell migration, cell proliferation, receptor binding activity, RNA, DNA or protein production and/or synthesis.
  • activity or function such as, but not limited to, ligand binding, receptor dimerization, receptor phosphorylation, receptor signaling, membrane association, cell migration, cell proliferation, receptor binding activity, RNA, DNA or protein production and/or synthesis.
  • Antibodies are heterotetrameric glycoproteins, composed of two identical light chains (LC) and two identical heavy chains (HC). Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • VH variable domain
  • VL variable domain at one end
  • Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequences of their constant domains.
  • Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence.
  • IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4.
  • antibody also includes fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof.
  • Functional fragments include antigen-binding fragments that bind to a mammalian antigens, such as MET, alone or in combination with other antigens.
  • antibody fragments capable of binding to antigen or portions thereof include, but are not limited to, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the present invention (see, e.g., Colligan, Immunology).
  • Fab e.g., by papain digestion
  • Fab' e.g., by pepsin digestion and partial reduction
  • F(ab')2 e.g., by pepsin digestion
  • facb e.g., by plasmin digestion
  • Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a combination gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and/or hinge region of the heavy chain.
  • the various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • antibody fragment refers to a portion of an intact antibody, generally the antigen binding or variable region of an intact antibody.
  • antibody fragments include, but are not limited to Fab, Fab', F(ab')2, single chain (scFv) and Fv fragments, diabodies; linear antibodies; single-chain antibody molecules; single Fab arm“one arm” antibodies and multispecific antibodies formed from antibody fragments, among others.
  • Antibody fragments include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, or at least one portion of an antigen or antigen receptor or binding protein, which can be incorporated into an antibody to MET of the present invention.
  • CDR complementarity determining region
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR).
  • CDRs complementarity-determining regions
  • FR framework
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen -binding site of antibodies.
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the Rabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Rabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the amino acid position numbering as in Rabat refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain can include a single amino acid insert (residue 52a according to Rabat) after residue 52 of H2 and inserted residues (e.g.
  • Rabat residues 82a, 82b, and 82c, etc. according to Rabat after heavy chain FR residue 82.
  • the Rabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Rabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
  • the end of the Chothia CDR-H1 loop when numbered using the Rabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Rabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).
  • the AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
  • epitope refers to a protein determinant capable of specific binding to an antibody.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • the antigen is a polypeptide
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • blocking antibody is one which inhibits or reduces the biological activity of the antigen it binds such as MET.
  • Preferred blocking antibodies substantially or completely inhibit the biological activity of the antigen. Desirably, the biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.
  • the blocking antibody reduces the MET associated tyrosine kinase activity 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.
  • An “isolated” antibody is one separated and/or recovered from its natural
  • the antibody will be purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the MET antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • a "human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.
  • chimeric antibodies refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g. mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.
  • humanized antibody refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences.
  • humanized antibodies are human immunoglobulins in which residues from the
  • CDR complementary determining region
  • non human species e.g. mouse, rat, rabbit, hamster
  • Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability.
  • the humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability.
  • the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539.
  • engineered antibody or“altered antibody” includes an antibody with significant human frameworks and constant regions (CL, CH domains (e.g., CH1, CH2, CH3), and hinge), and CDRs derived from antigen binding antibodies such as anti-MET antibodies or fragments thereof.
  • Fully human frameworks comprise frameworks that correspond to human germline sequences as well as sequences with somatic mutations.
  • CDRs may be derived from one or more CDRs that associate with or bind to antigen in or outside of the context of any antibody framework.
  • the CDRs of the human engineered antibody of the present invention directed to MET may be derived from CDRs that bind antigen in the context of a mouse antibody framework and then are engineered to bind antigen in the context of a human framework.
  • the human engineered antibody is substantially non-immunogenic in humans.
  • antibodies designated primate monkey, baboon, chimpanzee, etc.
  • rodent mouse, rat, rabbit, guinea pig, hamster, and the like
  • other mammals designate such species, sub-genus, genus, sub-family, and family specific antibodies.
  • chimeric antibodies can include any combination of the above. Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans or other species relative to non-modified antibodies.
  • a human engineered antibody is distinct from a chimeric or humanized antibody.
  • An engineered antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human or human engineered immunoglobulin (e.g., heavy chain and/or light chain) genes.
  • an engineered antibody when it is a single chain antibody, it can comprise a linker peptide that is not found in native human or non-human antibodies.
  • an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin.
  • Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used that are monoclonal, preferably, human, human engineered, resurfaced or humanized, antibodies that have binding specificities for at least two different antigens such as MET and a non-MET antigen. In the present case, one of the binding specificities is for at least one antigenic protein, the other one is for another antigenic protein.
  • Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)).
  • Antibody effector functions refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); and antibody-dependent cell- mediated phagocytosis (ADCP).
  • Human effector cells are leukocytes which express one or more FcRs and perform effector functions. In certain aspects, the cells express at least FcyRIII and perform ADCC or ADCP effector function(s). Examples of human leukocytes which mediate ADCC or ADCP include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells
  • monocytes cytotoxic T cells and neutrophils.
  • the effector cells may be isolated from a native source, e.g., from blood.
  • CBA cell binding agent
  • a linker is any chemical moiety that is capable of linking a maytansinoid compound described herein to a cell-binding agent such as an anti-MET antibody or a fragment thereof in a stable, covalent manner.
  • Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active.
  • Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.
  • Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.
  • ABSORFERRED cell growth or“aberrant cell proliferation”, as used herein, unless otherwise indicated, refers to cell growth that is independent of normal regulatory
  • tumor cells tumor cells
  • mutated tyrosine kinase a mutated tyrosine kinase or over expression of a receptor tyrosine kinase
  • benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs any tumors that proliferate by receptor tyrosine kinases; (4) any tumors that proliferate by aberrant serine/threonine kinase activation
  • benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation occurs and (6) benign and malignant cells of other proliferative diseases.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • a “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, myeloma, leukemia or lymphoid malignancies.
  • carcinoma a malignant neoplasm originating from a malignant neoplasm originating from a malignant fibroblasts, and lymphoid malignancies.
  • cancer or “cancerous.” as defined herein, includes“pre-cancerous” conditions that, if not treated, can evolve into a cancerous condition.
  • cancer cell refers to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non- tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).
  • cytotoxic agent refers to a substance that inhibits or prevents one or more cellular functions and/or causes cell death.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, methods and compositions of the invention are useful in attempts to delay development of a disease or disorder.
  • A“ therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • A“therapeutically effective amount” of a therapeutic agent e.g., a conjugate or immunoconjugate
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.
  • HGF hepatocyte growth factor
  • A“therapeutic agent” encompasses both a biological agent such as an antibody, a peptide, a protein, an enzyme, a chemotherapeutic agent, or a conjugate or immunoconjugate.
  • polynucleotide or“nucleic acid”, as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure can be imparted before or after assembly of the polymer.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.
  • any of the hydroxyl groups ordinarily present in the sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to solid supports.
  • the 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms.
  • Other hydroxyls can also be derivatized to standard protecting groups.
  • Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-0-methyl-, 2'-0-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.
  • One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(0)S
  • each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—0—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.
  • vector means a construct, which is capable of delivering, and optionally expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • polypeptide “peptide”, and“protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.
  • identity or percent“identity” is a measure of the relationship between two polynucleotides or two polypeptides, as determined by comparing their sequences. Identity or similarity with respect to a sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) or similar (i.e., amino acid residue from the same group based on common side- chain properties, see below) to anti-MET antibody residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • N-terminal, C-terminal, or internal extensions, deletions, or insertions into the antibody sequence outside of the variable domain shall be construed as affecting sequence identity or similarity.
  • the two sequences to be compared are aligned to give a maximum correlation between the sequences.
  • the alignment of the two sequences is examined and the number of positions giving an exact amino acid or nucleotide correspondence between the two sequences determined, divided by the total length of the alignment and multiplied by 100 to give a % identity figure.
  • This % identity figure may be determined over the whole length of the sequences to be compared, which is particularly suitable for sequences of the same or very similar length and which are highly homologous, or over shorter defined lengths, which is more suitable for sequences of unequal length or which have a lower level of homology. Likewise percent similarity can be determined in an analogous manner based on the presence of both identical and similar residues.
  • the percent identity can be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.
  • One such non limiting example of a sequence alignment algorithm is the algorithm described in
  • BLAST-2 Altschul et ah, 1996, Methods in Enzymology, 266:460-480
  • ALIGN ALIGN-2
  • ALIGN-2 Genentech, South San Lrancisco, California
  • Megalign Megalign
  • the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6).
  • the GAP program in the GCG software package which
  • the algorithm of Needleman and Wunsch J. Mol. Biol. (48):444-453 (1970) can be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).
  • the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)).
  • the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM 120 with residue table, a gap length penalty of 12 and a gap penalty of 4.
  • Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art.
  • the default parameters of the alignment software are used.
  • the percentage identity“X” of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.
  • whether any particular polynucleotide has a certain percentage sequence identity can, in certain embodiments, be determined using 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 such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • two nucleic acids or polypeptides of the invention are provided.
  • substantially identical meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • Identity can exist over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length or any integral value therebetween, and can be over a longer region than 60-80 residues, for example, at least about 90-100 residues, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.
  • A“conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including, for example, 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).
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), to which the polypeptide or antibody binds.
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummell et al., Biochem. 32: 1180- 1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc.
  • BxPC3 tumor cells refer to a human pancreatic tumor cell line (ATCC No: CRL-1687; Tan MH, et al. Characterization of a new primary human pancreatic tumor line. Cancer Invest. 4: 15-23, 1986).
  • MKN45 tumor cells refer to a human gastric adenocarcinoma cell line (DSMZ no.: ACC 409; Naito et al., Virchows Arch B Cell Pathol Incl Mol Pathol 46: 145-154 (1984); Motoyama et al., Acta Pathol Jpn 36: 65-83 (1986); Rege-Cambrin et al., Cancer Genet Cytogenet 64: 170-173 (1992); DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures)).
  • Alkyl refers to a saturated linear or branched-chain monovalent hydrocarbon radical of one to twenty carbon atoms.
  • alkyl include, but are not limited to, methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-methyl- 1 -propyl, -CH 2 CH(CH 3 ) 2 ),
  • the alkyl has one to ten carbon atoms. More preferably, the alkyl has one to four carbon atoms.
  • the number of carbon atoms in a group can be specified herein by the prefix“C x-xx ”, wherein x and xx are integers.
  • “Ci_ 4 alkyl” is an alkyl group having from 1 to 4 carbon atoms.
  • the term“compound” or“cytotoxic compound,” or“cytotoxic agent” are used interchangeably. They are intended to include compounds for which a structure or formula or any derivative thereof has been disclosed in the present invention or a structure or formula or any derivative thereof that has been incorporated by reference.
  • the term also includes, stereoisomers, geometric isomers, tautomers, solvates, metabolites, and salts (e.g., pharmaceutically acceptable salts) of a compound of all the formulae disclosed in the present invention.
  • the term also includes any solvates, hydrates, and polymorphs of any of the foregoing.
  • stereoisomer refers to compounds that have identical chemical constitution and connectivity, but different orientations of their atoms in space that cannot be interconverted by rotation about single bonds.
  • Diastereomer refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can separate under high resolution analytical procedures such as crystallization, electrophoresis and chromatography.
  • Enantiomers refer to two stereoisomers of a compound that are non- superimposable mirror images of one another.
  • optically active compounds i.e., they have the ability to rotate the plane of plane-polarized light.
  • the prefixes D and L, or R and S are used to denote the absolute configuration of the molecule about its chiral center(s).
  • the prefixes d and I or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory.
  • a compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another.
  • a specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur where there has been no stereoselection or stereospecificity in a chemical reaction or process.
  • the terms“racemic mixture” and“racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
  • tautomer or“tautomeric form” refers to structural isomers of different energies that are interconvertible via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • Valence tautomers include
  • the term“cation” refers to an ion with positive charge.
  • the cation can be monovalent (e.g., Na + , K + , NH 4 + etc.), bi-valent (e.g., Ca 2+ , Mg 2+ , etc.) or multi-valent (e.g., Al 3+ etc.).
  • the cation is monovalent.
  • pharmaceutically acceptable salt refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention.
  • Exemplary salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate“mesylate,” ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate ( i.e ., l,l’-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts,
  • a pharmaceutically acceptable salt can involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counter ion.
  • the counter ion can be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt can have more than one charged atom in its structure.
  • a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counter ion.
  • the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesul
  • an inorganic acid such as hydrochloric
  • the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.
  • an inorganic or organic base such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or alkaline earth metal hydroxide, or the like.
  • suitable salts include, but are not limited to, organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
  • amino acids such as glycine and arginine
  • ammonia such as glycine and arginine
  • primary, secondary, and tertiary amines such as piperidine, morpholine and piperazine
  • inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.
  • solvate means a compound that further includes a stoichiometric or non- stoichiometric amount of solvent such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2-propanol, or the like, bound by non-covalent intermolecular forces.
  • Solvates or hydrates of the compounds are readily prepared by addition of at least one molar equivalent of a hydroxylic solvent such as methanol, ethanol, 1 -propanol, 2-propanol or water to the compound to result in solvation or hydration of the imine moiety.
  • A“metabolite” or“catabolite” is a product produced through metabolism or catabolism in the body of a specified compound, a derivative thereof, or a conjugate thereof, or salt thereof. Metabolites of a compound, a derivative thereof, or a conjugate thereof, can be identified using routine techniques known in the art and their activities determined using tests such as those described herein. Such products can result for example from the oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound.
  • the invention includes metabolites of compounds, a derivative thereof, or a conjugate thereof, of the invention, including compounds, a derivative thereof, or a conjugate thereof, produced by a process comprising contacting a compound, a derivative thereof, or a conjugate thereof, of this invention with a mammal for a period of time sufficient to yield a metabolic product thereof.
  • phrase“pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.
  • the term“protecting group” or“protecting moiety” refers to a substituent that is commonly employed to block or protect a particular functionality while reacting other functional groups on the compound, a derivative thereof, or a conjugate thereof.
  • an“amine-protecting group” or an“amino-protecting moiety” is a substituent attached to an amino group that blocks or protects the amino functionality in the compound.
  • Such groups are well known in the art (see for example P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis , Chapter 7, J.
  • carbamates such as methyl and ethyl carbamate, FMOC, substituted ethyl carbamates, carbamates cleaved by l,6-P-elimination (also termed“self immolative”), ureas, amides, peptides, alkyl and aryl derivatives.
  • Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9- fluorenylmethylenoxycarbonyl (Fmoc).
  • amino acid refers to naturally occurring amino acids or non- naturally occurring amino acid.
  • amino acid or an amino acid residue is referenced without indicating the specific stererochemistry of the alpha carbon, it is meant to include both the L- and R- isomers.
  • “Ala” includes both L-alanine and R-alanine.
  • peptide refers to short chains of amino acid monomers linked by peptide (amide) bonds. In some embodiments, the peptides contain 2 to 20 amino acid residues. In other embodiments, the peptides contain 2 to 10 amino acid residus. In yet other
  • the peptides contain 2 to 5 amino acid residues.
  • a peptide when a peptide is a portion of a cytotoxic agent or a linker described herein represented by a specific sequence of amino acids, the peptide can be connected to the rest of the cytotoxic agent or the linker in both directions.
  • a dipeptide XI -X2 includes XI -X2 and X2-X1.
  • a tripeptide X1-X2-X3 includes X1-X2-X3 and X3-X2-X1 and a tetrapeptide XI- X2-X3-X4 includes X1-X2-X3-X4 and X4-X2-X3-X1.
  • XI, X2, X3 and X4 represents an amino acid residue.
  • stereochemistry of one or more amino acid or amino acid residue in the peptide or peptide residue is specified as D-isomer
  • the other amino acid or amino acid residue in the peptide or peptide residue without specified stereochemistry is meant to include only the natural L-isomer.
  • “Ala- Ala- Ala” meant to include peptides or peptide residues, in which each of the Ala can be either L- or R-isomer
  • Al-D-Ala-Ala meant to include L-Ala-D-Ala-L-Ala.
  • reactive ester group refers to a group an ester group that can readily react with an amine group to form amide bond.
  • exemplary reactive ester groups include, but are not limited to, N-hydroxysuccinimide esters, N-hydroxyphthalimide esters, N-hydroxy sulfo-succinimide esters, para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters and their derivatives, wherein said derivatives facilitate amide bond formation.
  • the reactive ester group is a N-hydroxysuccinimide ester or a
  • amine reactive group refers to a group that can react with an amine group to form a covalent bond.
  • exemplary amine reactive groups include, but are not limited to, reactive ester groups, acyl halides, sulfonyl halide, imidoester, or a reactive thioester groups.
  • the amine reactive group is a reactive ester group.
  • the amine reactive group is a N-hydroxysuccinimide ester or a N-hydroxy sulfo-succinimide ester.
  • thiol-reactive group refers to a group that can react with a thiol (-SH) group to form a covalent bond.
  • exemplary thiol-reactive groups include, but are not limited to, maleimide, haloacetyl, aloacetamide, vinyl sulfone, vinyl sulfonamide or vinyal pyridine.
  • the thiol-reactive group is maleimide.
  • the present invention provides agents that specifically bind MET. These agents are referred to herein as "MET binding agents.” Full-length amino acid sequences for human MET are known in the art.
  • the MET binding agents are antibodies, antibody fragments, or immunoconjugates. In some embodiments, the MET binding agents are humanized antibodies.
  • the MET -binding agents have one or more of the following effects: inhibit proliferation of tumor cells, reduce the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, inhibit tumor growth, trigger cell death of tumor cells, differentiate tumorigenic cells to a non-tumorigenic state, or prevent metastasis of tumor cells.
  • the MET-binding agents are bivalent anti-MET antibodies.
  • the MET-binding agents are bivalent anti-MET antibodies, antibody fragments, or immunoconjugates that inhibit HGF binding to MET expressing cells.
  • the MET-binding agents are bivalent anti-MET antibodies, antibody fragments, or immunoconjugates that inhibit proliferation.
  • the MET-binding agents are bivalent anti-MET antibodies, antibody fragments, or immunoconjugates that are capable of inhibiting HGF-induced proliferation, while not inducing proliferation of MET-expressing cells in the absence of HGF.
  • the MET-binding agents are bivalent anti-MET antibodies, antibody fragments, or immunoconjugates that are capable of inhibiting HGF binding to MET expressing cells and inhibiting HGF-induced proliferation, while not inducing proliferation in the absence of HGF.
  • a“c-MET binding agent” may be a c-MET binding polypeptide identified using recombinant procedures, for example, phage display or two hybrid screening and the like.
  • Preferred antigen- specific MET antibodies of the invention are described below.
  • Preferred antibodies are polypeptides comprised of one of the individual variable light chains or variable heavy chains described herein.
  • Antibodies and polypeptides can also comprise both a variable light chain and a variable heavy chain.
  • the variable light chain and variable heavy chain sequences of murine anti-MET antibodies are, for example, produced by hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, and 247.16.8.
  • polypeptides that comprise: (a) a polypeptide having at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% contour 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any of the heavy chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, and 247.16.8 and/or (b) a polypeptide having at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any of the light chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.
  • the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of any of the heavy chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, and 247.16.8.
  • the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to the amino acid sequence of any of the heavy chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, and 247.16.8, and/or (b) a polypeptide having at least about 95% sequence identity to the amino acid sequence of any of the light chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26,
  • the polypeptide comprises (a) a polypeptide having the amino acid sequence of any of the heavy chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26,
  • the polypeptide is an antibody and/or the polypeptide specifically binds MET.
  • the polypeptide is a murine, chimeric, or humanized or re surfaced antibody that specifically binds MET.
  • the polypeptide having a certain percentage of sequence identity to the amino acid sequence of any of the heavy chain variable regions or the light chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, and 247.16.8 differs from the amino acid sequence of any of the heavy chain variable regions or the light chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26,
  • Preferred antibodies are polypeptides containing one of the CDR sequences described herein.
  • an antigen specific antibody of the invention includes one of the light chain CDR sequences (i.e., LC CDR1, LC CDR2, and LC CDR3) and/or one of the heavy chain CDR sequences (i.e., HC CDR1, HC CDR2, and HC CDR3) shown below in Table 1.
  • Table 1 CDR sequences for exemplary c-MET-22 and c-MET-27 antibodies
  • the anti-MET antibodies or anti-MET antibody fragment thereof comprises a LC CDR1, a LC CDR2, and a LC CDR3 and a HC CDR1, a HC CDR2, and a HC CDR3 having the sequences selected from the group consisting of:
  • the anti-MET antibody or anti-MET antibody fragment thereof comprises a LC CDR1, a LC CDR2, and a LC CDR3 and a HC CDR1, a HC CDR2, and a HC CDR3 having the sequence of SEQ ID NOs:4, 5, and 7 and SEQ ID NOs: 13, 14, and 15, respectively.
  • humanized antibodies that comprise one of the individual variable light chains or variable heavy chains described herein.
  • the humanized antibodies can also comprise both a variable light chain and a variable heavy chain.
  • the variable light chain and variable heavy chain sequences of chimeric and humanized cMET-22 and cMET-27 antibodies are found in Table 2 below.
  • the anti-MET antibodies or fragment thereof comprises a variable light chain (VL) and a variable heavy chain (VH) having sequences that are at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the sequences as follows:
  • the anti-MET antibody or fragment thereof comprises a VL and VH having the sequences of SEQ ID NO:32 and SEQ ID NO:36, respectively.
  • the polypeptide is an antibody and/or the polypeptide specifically binds MET.
  • the polypeptide is a murine, chimeric, or humanized (by resurfacing methods or by CDR-grafting methods) antibody that specifically binds MET.
  • the polypeptide having a certain percentage of sequence identity to SEQ ID NOs: 18-36 by conservative amino acid substitutions.
  • polypeptides that comprise one of the individual light chains or heavy chains described herein. These can also comprise both a light chain and a heavy chain.
  • the light chain and heavy chain sequences of humanized cMET-22 and cMET-27 antibodies are below in Table 4.
  • the anti-MET antibodies or fragment thereof comprises a light chain and heavy chain sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the sequences as follows:
  • the anti-MET antibody or fragment thereof comprises a light chain and heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:54, respectively. In some embodiments, the anti-MET antibody or fragment thereof comprises a light chain and heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:53, respectively. In some embodiments, the anti-MET antibody or fragment thereof comprises a light chain and heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:82, respectively. In some embodiments, the anti-MET antibody or fragment thereof comprises a light chain and heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:8l, respectively.
  • the anti-MET antibody or fragment thereof comprises a light chain encoded by the plasmid DNA deposited with American Type Culture Collection (ATCC®, Manassas, Virginia, USA) on June 29, 2018 and having ATCC deposit no. PTA- 125143.
  • ATCC® American Type Culture Collection
  • the anti-MET antibody or fragment thereof comprises a heavy chain encoded by the plasmid DNA deposited with ATCC on June 29, 2018 and having ATCC deposit no. PTA-125144.
  • the anti-MET antibody or fragment thereof comprises a heavy chain encoded by the plasmid DNA deposited with ATCC on June 29, 2018 and having ATCC deposit no. PTA-125145.
  • the anti-MET antibody comprises a light chain encoded by the plasmid DNA deposited with ATCC on June 29, 2018 and having ATCC deposit no. PTA-125143, and a heavy chain encoded by the plasmid DNA deposited with ATCC on June 29, 2018 and having ATCC deposit no. PTA-125144.
  • the anti-MET antibody comprises a light chain encoded by the plasmid DNA deposited with ATCC on June 29, 2018 and having ATCC deposit no. PTA-125143, and a heavy chain encoded by the plasmid DNA deposited with the ATCC on June 29, 2018 and having ATCC deposit no. PTA-125145.
  • the polypeptide is an antibody and/or the polypeptide specifically binds MET.
  • the polypeptide is a murine, chimeric, or humanized (by resurfacing methods or CDR-grafting methods) antibody that specifically binds MET.
  • the polypeptide having a certain percentage of sequence identity to SEQ ID NOs:39-54 by conservative amino acid substitutions.
  • the anti-MET antibodies of the invention include a hinge region modification to reduce agonistic activity of the antibody, where the modification includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-108.
  • the anti-MET antibodies or anti-MET antibody fragment thereof comprises a LC CDR1, a LC CDR2, and a LC CDR3 and a HC CDR1, a HC CDR2, and a HC CDR3 having the sequences of SEQ ID NOs:4, 5, and 7 and SEQ ID NOs: 13, 14, and 15, respectively, where the antibody or anti-MET antibody fragment thereof are further characterized in that the antibody or fragment thereof also comprise a hinge region modification including an amino acid sequence as disclosed in Table 5.
  • the anti-MET antibodies and fragments thereof, conjugates, compositions and methods of the invention can be mutant antibodies and the like.
  • the anti-MET antibody can be an“engineered antibody” or an altered antibody such as an amino acid sequence variant of the anti-MET antibody wherein one or more of the amino acid residues of the anti-MET antibody have been modified.
  • the modifications to the amino acid sequence of the anti-MET antibody include, for example, modifications to the polypeptide and/or polynucleotide sequence to improve affinity or avidity of the antibody or fragment for its antigen, improve stability, and/or modifications to the polypeptide and/or polynucleotide sequence to improve production of the antibody, and/or modifications to the Fc portion of the antibody to improve effector function unless otherwise indicated herein or known.
  • the modifications may be made to any known anti-MET antibodies or anti-MET antibodies identified as described herein. Such altered antibodies necessarily have less than 100% sequence identity or similarity with a reference anti-MET antibody.
  • the altered antibody will have an amino acid sequence having at least 20%, 25%, 35%, 45%, 55%, 65%, or 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the anti-MET antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
  • the altered antibody will have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, or 75% amino acid sequence identity or similarity with the amino acid sequence of the heavy chain CDR1, CDR2, or CDR3 of the anti-MET antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
  • the altered antibody will have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, or 75% amino acid sequence identity or similarity with the amino acid sequence of light chain CDR1, CDR2, or CDR3 of the anti- MET antibody, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, 99%.
  • the altered antibody will maintain human MET binding capability.
  • the anti- MET antibody of the invention comprises a heavy chain that is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences of the amino acid sequences of the heavy chain variable regions of the antibodies produced by hybridoma 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, or 247.16.8.
  • the anti-MET antibody of the invention comprises a light chain that is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequences of the amino acid sequences of the light chain variable regions of the antibodies produced by hybridoma 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, or 247.16.8.
  • the anti-MET antibody can be an“engineered antibody” or an altered antibody such as an amino acid sequence variant of the anti-MET antibody wherein one or more of the amino acid residues of the anti-MET antibody have been modified.
  • the altered antibody will have an amino acid sequence having at least 1-5, 1-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 conservative amino acid substitutions when compared with the amino acid sequence of either the heavy or light chain variable domain of the anti-MET antibody.
  • the altered antibody will have an amino acid sequence having at least 1-20, 1-15, 1-10, 1-5, 1-3, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions when compared with the amino acid sequence of the heavy chain CDR1, CDR2, or CDR3 of the anti-MET antibody.
  • the altered antibody will have an amino acid sequence having at least 1-20, 1-15, 1-10, 1-5, 1-3, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions when compared with the amino acid sequence of the light chain CDR1, CDR2, or CDR3 of the anti-MET antibody produced by hybridoma 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, or 247.16.8.
  • the altered antibody will maintain human MET binding capability.
  • the anti-MET antibody of the invention comprises a heavy chain having an amino acid sequence that has about 1-20, 1-15, 1-10, 1-5, 1-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 conservative amino acid substitutions when compared with the amino acid sequence of the amino acid sequences of the heavy chain variable regions of the antibodies produced by hybridoma 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, or 247.16.8.
  • the anti-MET antibody of the invention comprises a light chain having an amino acid sequence that has about 1-20, 1-15, 1-10, 1-5, 1-3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 conservative amino acid substitutions when compared with the amino acid sequence of the light chain variable regions of the antibodies produced by hybridoma 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, or 247.16.8.
  • the altered antibody will have an amino acid sequence having at least 1-20, 1-15, 1-10, 1-5, 1-3, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions when compared with the amino acid sequence of the heavy chain CDR1, CDR2, or CDR3 of the anti-MET antibody produced by hybridoma 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, or 247.16.8.
  • amino acid alterations e.g., amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino
  • substitutions are introduced in one or more of the hypervariable regions of an antibody.
  • one or more alterations (e.g., substitutions) of framework region residues may be introduced in an anti-MET antibody where these result in an improvement in the binding affinity of the antibody mutant for the antigen.
  • framework region residues to modify include those which non-covalently bind antigen directly (Amit et ah, Science, 233:747-753 (1986)); interact with/effect the conformation of a CDR (Chothia et ah, J. Mol. Biol., 196:901-917 (1987)); and/or participate in the VL VH interface.
  • modification of one or more of such framework region residues results in an enhancement of the binding affinity of the antibody for the antigen.
  • framework residues e.g. 1, 2, 3, 4 or 5
  • this may be sufficient to yield an antibody with an enhancement of the binding affinity, even where none of the hypervariable region residues have been altered.
  • an altered antibody will comprise additional hypervariable region alteration(s).
  • the hypervariable region residues which are altered may be changed randomly, especially where the starting binding affinity of an anti-MET antibody for the antigen is such that such randomly produced altered antibody can be readily screened.
  • alanine scanning mutagenesis (Cunningham and Wells, Science, 244:1081-1085 (1989)).
  • One or more of the hypervariable region residue(s) are replaced by alanine or polyalanine residue(s) to affect the interaction of the amino acids with the antigen.
  • Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing additional or other mutations at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • the Ala-mutants produced this way are screened for their biological activity as described herein and/or as known in the art.
  • Another procedure for generating such an altered antibody involves affinity maturation using phage display (Hawkins et ah, J. Mol. Biol., 254:889-896 (1992) and Lowman et al., Biochemistry, 30(45): 10832-10837 (1991)).
  • Mutations in antibody sequences may include substitutions, deletions, including internal deletions, additions, including additions yielding fusion proteins, or conservative substitutions of amino acid residues within and/or adjacent to the amino acid sequence, but that result in a "silent" change, in that the change produces a functionally equivalent anti- MET antibody or fragment.
  • Conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • glycine and proline are residues can influence chain orientation. Non conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
  • non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody sequence.
  • Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, oc-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, C oc-methyl amino acids,
  • N oc-methyl amino acids and amino acid analogs generally.
  • Any technique for mutagenesis known in the art can be used to modify individual nucleotides in a DNA sequence, for purposes of making amino acid substitution(s) in the antibody sequence, or for creating/deleting restriction sites to facilitate further manipulations.
  • Such techniques include, but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Hutchinson, C. et ah,
  • a humanized, resurfaced or similarly engineered antibody may have one or more amino acid residues from a source that is non human, e.g., but not limited to, mouse, rat, rabbit, non-human primate or other mammal. These non-human amino acid residues are replaced by residues that are often referred to as "import" residues, which are typically taken from an "import" variable, constant or other domain of a known human sequence.
  • IG immunoglobulins
  • TR T cell receptors
  • MHC major histocompatibility complex
  • RPI major histocompatibility complex
  • Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.
  • the CDR residues are directly and most substantially involved in influencing MET binding. Accordingly, part or all of the non-human or human CDR sequences are maintained while the non-human sequences of the variable and constant regions may be replaced with human or other amino acids.
  • Antibodies can also optionally be humanized, resurfaced, engineered or human antibodies engineered with retention of high affinity for the antigen MET and other favorable biological properties.
  • humanized (or human) or engineered anti-MET antibodies and resurfaced antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered, and humanized sequences. Three- dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • the present invention provides formulation of proteins comprising a variant Fc region. That is, a non-naturally occurring Fc region, for example an Fc region comprising one or more non-naturally occurring amino acid residues. Also encompassed by the variant Fc regions of the present invention are Fc regions which comprise amino acid deletions, additions and/or modifications.
  • the antibody comprises an altered (e.g., mutated) Fc region.
  • the Fc region has been altered to reduce or enhance the effector functions of the antibody, alter serum half life or other functional properties of the antibody.
  • the Fc region is an isotype selected from IgM, IgA, IgG, IgE, or other isotype.
  • Fc region as used herein includes the polypeptides comprising the constant region of an antibody excluding the first constant region
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains.
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3 ) and the hinge between Cyl (Cyl) and Cy2 (Cy2).
  • the human IgG heavy chain Fc region is usually defined to comprise residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.).
  • the "EU index as set forth in Kabat” refers to the residue numbering of the human IgGl EU antibody as described in Kabat et al., supra.
  • Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.
  • An Fc variant protein may be an antibody, Fc fusion, or any protein or protein domain that comprises an Fc region.
  • proteins comprising variant Fc regions, which are non-naturally occurring variants of an Fc.
  • Polymorphisms have been observed at a number of Fc positions, including, but not limited to, Kabat 270, 272, 312, 315, 356, and 358, and thus slight differences between the presented sequence and sequences in the prior art may exist and would be known to one of skill in the art based on the present teachings.
  • Fc mutations can be introduced into engineered antibodies to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties.
  • FcRn neonatal Fc receptor
  • a collection of human Fc variants with improved binding to the FcRn has been described and include, for example, those disclosed in Shields et al., 2001. High resolution mapping of the binding site on human IgGl for FcyRI, FcyRII, FcyRIII, and FcRn and design of IgGl variants with improved binding to the FcyR, J. Biol. Chem. 276:6591-6604), which is hereby entirely incorporated by reference.
  • the serum half-life of proteins comprising Fc regions may be increased by increasing the binding affinity of the Fc region for FcRn.
  • the Fc variant protein has enhanced serum half life relative to comparable molecule.
  • the Fc hinge region can also be engineered to alter Fab arm flexibility as a means to manipulate antibody binding, effector potency or other functional properties of the antibody. The flexibility of the antibody’s Fc hinge is largely a function of its length and the number and placement of cysteine residues.
  • Amino acid changes to the Fc hinge cysteine residues or length have been described which can elicit altered ADCC or CDC activity (Dall’Acqua WF et al., 2006; J Immunol; 177:1129-38), or other antibody binding mediated functional activities (WO 2010064090). It may therefore be desirable to make such amino acid modifications, including amino acid deletions and substitutions, in the Fc hinge region.
  • anti-MET antibody of the invention may also be desirable to modify the anti-MET antibody of the invention with respect to effector function, so as to enhance the effectiveness of the antibody in treating a cancer, for example.
  • In vitro assays known in the art can be used to determine whether the anti-MET antibodies, compositions, conjugates and methods of the invention, for example, are capable of mediating effector functions such as ADCC or CDC, such as those described herein.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs Fc receptors
  • cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • NK Natural Killer
  • macrophages e.g., neutrophils, and macrophages
  • High-affinity IgG antibodies directed to the surface of target cells "arm" the cytotoxic cells and afford such killing. Lysis of the target cell is extracellular, requires direct cell-to-cell contact, and does not involve complement.
  • ADCC activity the cell-mediated cytotoxicity resulting from the activity of an Fc fusion protein is also referred to herein as ADCC activity.
  • any particular Fc variant protein to mediate lysis of the target cell by ADCC can be assayed.
  • an Fc variant protein of interest is added to target cells in combination with immune effector cells, which may be activated by the antigen antibody complexes resulting in cytolysis of the target cell. Cytolysis is generally detected by the release of label (e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins) from the lysed cells.
  • label e.g., radioactive substrates, fluorescent dyes or natural intracellular proteins
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • ADCC assays are described in Wisecarver et al., 1985, 79:277-282; Bruggemann et al., 1987, J Exp Med, 166:1351-1361; Wilkinson et al., 2001, J Immunol Methods, 258:183-191; and Patel et al., 1995, J Immunol Methods, 184:29-38.
  • ADCC activity of the Fc variant protein of interest may be assessed in vivo , e.g., in an animal model such as that disclosed in Clynes et al., 1998, PNAS USA, 95:652-656.
  • Complement dependent cytotoxicity and “CDC” refer to the lysing of a target cell in the presence of complement.
  • the complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule, an antibody for example, complexed with a cognate antigen.
  • a CDC assay e.g. as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods, 202:163, may be performed.
  • the Fc region of an antibody of the present invention can be designed with altered effector functions including, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc.
  • effector functions may require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.)
  • a variant Fc region of the engineered anti-MET antibody with improved Clq binding and improved FcyRIII binding e.g., having both improved ADCC activity and improved CDC activity.
  • a variant Fc region can be engineered with reduced CDC activity and/or reduced ADCC activity.
  • only one of these activities may be increased, and, optionally, also the other activity reduced (e.g., to generate an Fc region variant with improved ADCC activity, but reduced CDC activity, and vice versa).
  • An exemplary Fc mutant is the triple residue change, S239D, A330F, and I332E (EU numbering system) in which ADCC is enhanced and CDC activity is diminished.
  • Non-limiting methods for designing such mutants can be found, for example, in Fazar et al. (2006, Proc. Natl. Acad. Sci. U.S.A. 103(11): 4005-4010) and Okazaki et al. (2004, J. Mol. Biol. 336(5): 1239-49). See also WO 03/074679, WO 2004/029207, WO 2004/099249, W02006/047350,
  • cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and/or antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • Homodimeric antibodies with enhanced activity may also be prepared using hetero-bifunctional cross-linkers as described in Wolff et al., Cancer Research, 53:2560-2565 (1993).
  • an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson et al., Anti- Cancer Drug Design, 3:219-230 (1989).
  • the present invention encompasses Fc variant proteins which have altered binding properties for an Fc ligand (e.g., an Fc receptor, Clq) relative to a comparable molecule (e.g., a protein having the same amino acid sequence except having a wild type Fc region).
  • Fc ligand e.g., an Fc receptor, Clq
  • comparable molecule e.g., a protein having the same amino acid sequence except having a wild type Fc region.
  • binding properties include, but are not limited to, binding specificity, equilibrium dissociation constant (KD), dissociation and association rates (K 0ff and K on ), binding affinity and/or avidity. It is generally understood that a binding molecule (e.g., a
  • Fc variant protein such as an antibody
  • a low K D is preferable to a binding molecule with a high K D .
  • the value of the K on or K 0ff may be more relevant than the value of the K D -
  • One skilled in the art can determine which kinetic parameter is most important for a given antibody application.
  • the affinities and binding properties of an Fc domain for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-FcyR interactions, i.e., specific binding of an Fc region to an FcyR including but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE. TM analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and
  • chromatography e.g., gel filtration
  • detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • the ratio of binding affinities can indicate if the ADCC activity of an Fc variant is enhanced or decreased.
  • a decrease in the ratio of FcyRIIIA/FcyRIIB equilibrium dissociation constants (KD) will correlate with improved ADCC activity, while an increase in the ratio will correlate with a decrease in ADCC activity.
  • modifications that enhanced binding to Clq would be preferable for enhancing CDC activity while modification that reduced binding to Clq would be preferable for reducing or eliminating CDC activity.
  • the Fc variants of the invention bind FcyRIIIA with increased affinity relative to a comparable molecule. In another aspect, the Fc variants of the invention bind FcyRIIIA with increased affinity and bind FcyRIIB with a binding affinity that is unchanged relative to a comparable molecule. In still another aspect, the Fc variants of the invention bind FcyRIIIA with increased affinity and bind FcyRIIB with a decreased affinity relative to a comparable molecule. In yet another aspect, the Fc variants of the invention have a ratio of FcyRIIIA/FcyRIIB equilibrium dissociation constants (K D ) that is decreased relative to a comparable molecule.
  • K D FcyRIIIA/FcyRIIB equilibrium dissociation constants
  • the Fc variant protein has enhanced binding to one or more Fc ligand relative to a comparable molecule.
  • the Fc variant protein has an affinity for an Fc ligand that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or at least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold greater than that of a comparable molecule.
  • the Fc variant protein has an affinity for an Fc ligand that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or at least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold greater than that of
  • Fc variant protein has enhanced binding to an Fc receptor.
  • the Fc variant protein has enhanced binding to the Fc receptor FcyRIIIA.
  • the Fc variant protein has enhanced binding to the Fc receptor FcRn.
  • the Fc variant protein has enhanced binding to Clq relative to a comparable molecule.
  • an Fc variant of the invention has an equilibrium dissociation constant (KD) that is decreased between about 2 fold and about 10 fold, or between about 5 fold and about 50 fold, or between about 25 fold and about 250 fold, or between about 100 fold and about 500 fold, or between about 250 fold and about 1000 fold relative to a comparable molecule.
  • an Fc variant of the invention has an equilibrium dissociation constant (KD) that is decreased between 2 fold and 10 fold, or between 5 fold and 50 fold, or between 25 fold and 250 fold, or between 100 fold and 500 fold, or between 250 fold and 1000 fold relative to a comparable molecule.
  • the Fc variants have an equilibrium dissociation constants (K D ) for FcyRIIIA that is reduced by at least
  • an Fc variant protein has enhanced ADCC activity relative to a comparable molecule.
  • an Fc variant protein has ADCC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 10 fold, or at least 50 fold, or at least 100 fold greater than that of a comparable molecule.
  • an Fc variant protein has ADCC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 10 fold, or at least 50 fold, or at least 100 fold greater than that of a comparable molecule.
  • Fc variant protein has enhanced binding to the Fc receptor FcyRIIIA and has enhanced ADCC activity relative to a comparable molecule. In other aspects, the Fc variant protein has both enhanced ADCC activity and enhanced serum half life relative to a comparable molecule.
  • an Fc variant protein has enhanced CDC activity relative to a comparable molecule.
  • an Fc variant protein has CDC activity that is at least 2 fold, or at least 3 fold, or at least 5 fold, or at least 10 fold, or at least 50 fold, or at least 100 fold greater than that of a comparable molecule.
  • the Fc variant protein has both enhanced CDC activity and enhanced serum half life relative to a comparable molecule.
  • the present invention provides formulations, wherein the Fc region comprises a non-naturally occurring amino acid residue at one or more positions selected from the group consisting of 234, 235, 236, 239, 240, 241, 243, 244, 245, 247, 252, 254, 256, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 326, 327, 328, 329, 330, 332, 333, and 334 as numbered by the EU index as set forth in Kabat.
  • the EU index as set forth in Kabat.
  • Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO 02/06919;
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non-naturally occurring amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241 R.
  • the Fc region comprises at least one non-naturally occurring amino acid residue selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y,
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least a non-naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330 and 332, as numbered by the
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat.
  • the Fc region may further comprise an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat.
  • the present invention provides an Fc variant protein formulation, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat, and at least one non-naturally occurring amino acid at one or more positions are selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.
  • the Fc variants of the present invention may be combined with other known Fc variants such as those disclosed in Ghetie et ah, 1997, Nat. Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et ah, 1991, J. Immunol., 147:2657-2662; Lund et al, 1992, Mol. Immunol., 29:53-59; Alegre et al, 1994, Transplantation 57:1537- 1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA, 92:11980-11984; Jefferis et al, 1995, Immunol.
  • Fc regions which comprise deletions, additions and/or modifications. Still other modifications/substitutions/additions/deletions of the Fc domain will be readily apparent to one skilled in the art.
  • amino acid modifications with one or more further amino acid modifications that alter Clq binding and/or the complement dependent cytotoxicity (CDC) function of the Fc region of an antigen binding molecule.
  • the starting polypeptide of particular interest may be one that binds to Clq and displays complement dependent cytotoxicity.
  • Polypeptides with pre-existing Clq binding activity, optionally further having the ability to mediate CDC, may be modified such that one or both of these activities are enhanced.
  • Amino acid modifications that alter Clq and/or modify its complement dependent cytotoxicity function are described, for example, in W00042072, which is hereby entirely incorporated by reference.
  • amino acid substitutions and/or deletions can be generated by mutagenesis methods, including, but not limited to, site-directed mutagenesis (e.g., Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492 (1985)), PCR mutagenesis (e.g., Higuchi, in "PCR Protocols: A Guide to Methods and Applications", Academic Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis (e.g., Wells et ah, Gene, 34:315-323 (1985)).
  • site-directed mutagenesis e.g., Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492 (1985)
  • PCR mutagenesis e.g., Higuchi, in "PCR Protocols: A Guide to Methods and Applications", Academic Press, San Diego, pp. 177-183 (1990)
  • cassette mutagenesis e.g., Wells e
  • mutagenesis is performed by the overlap-extension PCR method (e.g., Higuchi, in "PCR Technology: Principles and Applications for DNA Amplification", Stockton Press, New York, pp. 61-70 (1989)).
  • Other exemplary methods useful for the generation of variant Fc regions are known in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Publication Nos. 2004/0002587 and PCT Publications WO 94/29351; WO 99/58572; WO 00/42072; WO 02/060919; WO 04/029207;
  • an Fc variant protein comprises one or more engineered glycoforms, i.e., a carbohydrate composition that is covalently attached to the molecule comprising an Fc region.
  • Engineered glycoforms may be useful for a variety of purposes, including but not limited to enhancing or reducing effector function.
  • Engineered glycoforms may be generated by methods disclosed herein and any method known to one skilled in the art, for example by using engineered or variant expression strains, by using growth conditions or media affecting glycosylation, by co-expression with one or more enzymes, for example DI N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an enzymes, for example DI N- acetylglucosaminyltransferase III (GnTIII), by expressing a molecule comprising an enzymes, for example DI N- acetylglucosaminyltransferase III (GnTIII), by
  • Fc region in various organisms or cell lines from various organisms or by modifying carbohydrate(s) after the molecule comprising Fc region has been expressed.
  • Methods for generating engineered glycoforms are known in the art, and include but are not limited to those described in Umana et ah, 1999, Nat. Biotechnok, 17:176-180; Davies et ah, 20017 Biotechnol Bioeng., 74:288-294; Shields et ah, 2002, J Biol. Chem., 277:26733-26740;
  • an Fc variant protein with engineered glycoforms contains carbohydrate structures attached to the Fc region that lack fucose. Such variants have improved ADCC function.
  • Examples of publications related to "defucosylated” or “fucose- deficient” antibodies include: US Pat. Appl. No. US 2003/0157108 (Presta, F.) and
  • Antibodies with a bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc region of the antibody are referenced in, for example, WO 2003/011878, Jean-Mairet et al. and US Pat. No. 6,602,684, Umana et al.
  • Antibodies with at least one galactose residue in the oligosaccharide attached to an Fc region of the antibody are reported in, for example, WO 1997/30087, Patel et al. See also, WO 1998/58964 and WO 1999/22764 (Raju, S.) concerning antibodies with altered carbohydrate attached to the Fc region thereof.
  • US 2005/0123546 Umana et al.
  • Non-limiting examples of cell lines producing defucosylated antibodies include Fee 13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533- 545 (1986); US Pat. Appl. No. US 2003/0157108 Al, Presta, F; and WO 2004/056312 Al, Adams et al., especially at Example 11), knockout cell lines, such as alpha-l,6- fucosyltransferase gene, FUT8, knockout CHO cells (Yamane- Ohnuki et al., Biotech.
  • the antibody of the present invention is expressed in cells that express beta (l,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnT III adds GlcNAc to the human engineered antigen specific antibody.
  • GnT III beta (l,4)-N-acetylglucosaminyltransferase III
  • Methods for producing antibodies in such a fashion are provided in WO/9954342, W 0/03011878, patent publication 20030003097A1, and Umana et al., Nature Biotechnology, 17:176-180, February 1999.
  • Another method to alter the glycosylation pattern of the Fc region of an antibody is through amino acid substitution(s). Glycosylation of an Fc region is, for example, either N-linked or O-linked.
  • N-linked generally refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain peptide sequences are asparagine-X- serine and asparagine-X-threonine, where X is any amino acid except proline.
  • X is any amino acid except proline.
  • O-linked glycosylation generally refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
  • glycosylation pattern of an antibody or fragment thereof may be altered, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and/or adding one or more glycosylation site(s) that are not present in the polypeptide. Removal of glycosylation sites in the Fc region of an antibody or antibody fragment is conveniently accomplished by altering the amino acid sequence such that it eliminates one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • An exemplary glycosylation variant has an amino acid substitution of residue N297 to A297 (EU numbering system) of the heavy chain.
  • the removal of an O-linked glycosylation site may also be achieved by the substitution of one or more glycosylated serine or threonine residues with any amino acid besides serine or threonine.
  • Functional equivalents further include fragments of antibodies that have the same, or comparable binding characteristics to those of the whole or intact antibody. Such fragments may contain one or both Fab fragments or the F(ab') 2 fragment. Preferably the antibody fragments contain all six complementarity determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as one, two, three, four or five CDRs, are also functional. Further, the functional equivalents may be or may combine members of any one of the following immunoglobulin classes: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.
  • the anti-MET antibodies can be modified to produce fusion proteins; i.e., the antibody, or a fragment fused to a heterologous protein, polypeptide or peptide.
  • the protein fused to the portion of an anti-MET antibody is an enzyme component of ADEPT.
  • proteins or polypeptides that can be engineered as a fusion protein with an anti-MET antibody include, but are not limited to, toxins such as ricin, abrin, ribonuclease, DNase I, Staphylococcal enterotoxin-A, pokeweed anti-viral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and
  • Enzymatically active toxins and fragments thereof which can be used include, but are not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa ), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.
  • Non-limiting examples Non-limiting examples
  • DNA shuffling may be employed to alter the activities of the antibodies or fragments thereof (e.g., an antibody or a fragment thereof with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721;
  • the antibody can further be a binding- domain immunoglobulin fusion protein as described in U.S. Publication 20030118592,
  • the anti-MET antibodies of the compositions and methods of the invention can be domain antibodies, e.g., antibodies containing the small functional binding units of antibodies, corresponding to the variable regions of the heavy (VH) or light (VL) chains of human antibodies.
  • domain antibodies include, but are not limited to, those available from Domantis Limited (Cambridge, UK) and Domantis Inc. (Cambridge, Mass., USA), that are specific to therapeutic targets (see, for example, W004/058821;
  • the anti-MET antibodies of the invention comprise a MET functional binding unit and a Fc gamma receptor functional binding unit.
  • Diabodies refers to small antibody fragments with two antigen binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. Diabodies are described more fully in, for example, EP 404,097;
  • the anti-MET antibodies are vaccibodies.
  • Vaccibodies are dimeric polypeptides. Each monomer of a vaccibody consists of a scFv with specificity for a surface molecule on an APC connected through a hinge region and a Cg3 domain to a second scFv.
  • vaccibodies containing as one of the scFv's an anti-MET antibody fragment may be used to juxtapose B cells to be destroyed and an effector cell that mediates ADCC. For example, see, Bogen et al.,
  • the anti-MET antibodies are linear antibodies.
  • Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH- CH1) which form a pair of antigen-binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • Non-limiting examples of linear antibodies are disclosed in, for example, Zapata et al., Protein Eng., 8(10): 1057-1062 (1995).
  • the anti-MET antibody is a parent antibody.
  • a "parent antibody” is an antibody comprising an amino acid sequence which lacks, or is deficient in, one or more amino acid residues in or adjacent to one or more hypervariable regions thereof compared to an altered/mutant antibody as herein disclosed. Thus, the parent antibody has a shorter hypervariable region than the corresponding hypervariable region of an antibody mutant as herein disclosed.
  • the parent polypeptide may comprise a native sequence (i.e., a naturally occurring) antibody (including a naturally occurring allelic variant) or an antibody with pre-existing amino acid sequence modifications (such as other insertions, deletions and/or substitutions) of a naturally occurring sequence.
  • the parent antibody is a humanized antibody or a human antibody.
  • Antibody fragments comprise a portion of a full-length antibody, generally the antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single- chain antibody molecules; single Fab arm“one arm” antibodies and multispecific antibodies formed from antibody fragments, among others.
  • fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et ah, Journal of Biochemical and Biophysical Methods, 24: 107-117 (1992) and Brennan et ah, Science, 229:81 (1985)).
  • fragments can now be produced directly by recombinant host cells.
  • the antibody fragments can be isolated from the antibody phage libraries as discussed herein.
  • Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio Technology, 10: 163-167 (1992)).
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture.
  • Single Fab arm“one arm” antibodies can be made by generating Fc“knob and hole” mutations such that the resulting molecule can be expressed in bacterial or mammalian hosts containing a single Fab arm with a full dimeric Fc region (Merchant et al., Nat. Biotechnol., 1998 Jul., 16(7):677-81, WO 2005/063816 A2).
  • Other techniques for the production of antibody fragments are apparent to the skilled practitioner given the detailed teachings in the present specification.
  • the antibody of choice is a single-chain Fv fragment (scFv). See, for example, WO 93/16185.
  • the antibody is not a single-chain Fv fragment (scFv).
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of MET. Other such antibodies may bind MET and further bind a second antigen. Alternatively, a MET binding arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (FcyR), so as to focus cellular defense mechanisms to the target. Bispecific antibodies may also be used to localize cytotoxic agents to the target.
  • a triggering molecule such as a T cell receptor molecule (e.g., CD2 or CD3), or Fc receptors for IgG (FcyR)
  • bispecific antibodies possess a cell marker-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferonoc, vinca alkaloid, ricin A chain, methola-exate or radioactive isotope hapten).
  • cytotoxic agent e.g., saporin, anti-interferonoc, vinca alkaloid, ricin A chain, methola-exate or radioactive isotope hapten.
  • Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab'): bispecific antibodies).
  • the compositions and methods comprise a bispecific murine antibody or fragment thereof and/or conjugates thereof with specificity for human MET and the CD3 epsilon chain of the T cell receptor, such as the bispecific antibody described by Daniel et al., Blood, 92:4750-4757 (1998).
  • the anti-MET antibody or fragments thereof and/or conjugates thereof of the compositions and methods of the invention is bispecific
  • the anti-MET antibody is human or humanized and has specificity for human MET and an epitope on a T cell or is capable of binding to a human effector-cell such as, for example, a monocyte/macrophage and/or a natural killer cell to effect cell death.
  • the antibodies of the invention bind human MET, with a wide range of affinities (KD).
  • at least one mAb of the present invention can optionally bind human antigen with high affinity.
  • a human or human engineered or humanized or resurfaced mAb can bind human antigen with a K D equal to or less than about 10 M, such as but not limited to, 0.1-9.9 (or any range or value therein)xlO 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 or any range or value therein, as determined by flow cytometry base assays, enzyme-linked immunoabsorbent assay (ELISA), surface plasmon resonance (SPR) or the KinExA® method, as practiced by those of skill in the art.
  • the anti-MET antibodies bind with a Kd of about l0 9 M or less, more specifically about l0 9 to 10 10 M.
  • the affinity or avidity of an antibody for an antigen is determined experimentally using any suitable method well known in the art, e.g. flow cytometry, enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., BIACORE TM analysis).
  • ELISA enzyme-linked immunoabsorbent assay
  • RIA radioimmunoassay
  • kinetics e.g., BIACORE TM analysis.
  • Direct binding assays as well as competitive binding assay formats can be readily employed. (See, for example, Berzofsky, et al., "Antibody- Antigen Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); and methods described herein.
  • the measured affinity of a particular antibody- antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH, temperature).
  • affinity and other antigen-binding parameters e.g., K D or K d , K on , K 0ff
  • K D or K d , K on , K 0ff are preferably made with standardized solutions of antibody and antigen, and a standardized buffer, as known in the art and such as the buffer described herein.
  • binding assays can be performed using flow cytometry on cells expressing the MET antigen on the surface.
  • MET -positive cells are incubated with varying concentrations of anti-MET antibodies using 1 xlO 5 cells per sample in 100 pL FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum). Then, the cells are pelleted, washed, and incubated for 1 h with 100 pL of FITC-conjugated goat anti-mouse IgG-antibody (such as obtainable from Jackson ImmunoRe search) in FACS buffer.
  • FACS buffer RPMI-1640 medium supplemented with 2% normal goat serum
  • the cells are pelleted again, washed with FACS buffer and resuspended in 200 pF of PBS containing 1% formaldehyde.
  • Samples are acquired, for example, using a FACSCalibur flow cytometer with the HTS multiwell sampler and analyzed using CellQuest Pro (all from BD Biosciences, San Diego, US).
  • MFI mean fluorescence intensity for FF1
  • a sigmoidal dose-response curve is fitted for binding curves and EC50 values are calculated using programs such as GraphPad Prism v4 with default parameters (GraphPad software, San Diego, CA). EC50 values can be used as a measure for the apparent dissociation constant“Kd” or“KD” for each antibody.
  • the anti-MET antibodies can be modified to alter their binding affinity for the MET and antigenic fragments thereof. Binding properties may be determined by a variety of in vitro assay methods known in the art, e.g. enzyme-linked immunoabsorbent assay (EFISA), or radioimmunoassay (RIA)), or kinetics (e.g.,
  • antibodies or antibody fragments specifically bind MET and antigenic fragments thereof with a dissociation constant or KD or Kd (k 0ff /k 0n ) of less than 10 5 M, or of less than 10 6 M, or of less than 10 7 M, or of less than 10 8 M, or of less than 10 9 M, or of less than 10 10 M, or of less than 10 11 M, or of less than 10 12 M, or of less than 10 13 M.
  • the antibody or fragment of the invention binds to MET and/or antigenic fragments thereof with a K 0ff of less than 1x10 3 s-1 , or less than 3x10 3 s-1.
  • the antibody binds to HGFR and antigenic fragments thereof with a K 0ff less than 10 3 s 1 less than 5xl0 3 s 1 , less than 10 4 s 1 , less than 5xl0 4 s 1 , less than 10 5 s 1 , less than 5x10 5 s 1 , less than 10 6 s 1 , less than 5xl0 6 s 1 , less than 10 7 s 1 , less than 5xl0 7 s 1 , less than 10-8 s 1 , less than 5xl0 8 s- 1 , less than 10 9 s 1 , less than 5xl0 9 s 1 , or less than 10 10 s 1 .
  • the antibody or fragment of the invention binds to MET and/or antigenic fragments thereof with an association rate constant or k on rate of at least 10 5 M 1 s 1 , at least 5x10 5 M 1 s 1 , at least 10 6 M 1 s 1 , at least 5xl0 6 M 1 s 1 , at least 10 7 M 1 s 1 , at least 5xl0 7 M 1 s _1 , or at least 10 8 M 1 s 1 , or at least 10 9 M _1 s 1 .
  • conjugates of the invention may have the same properties as those described herein.
  • the anti-MET antibodies can be modified to alter their isoelectric point (pi).
  • Antibodies like all polypeptides, have a pi, which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pi) of the protein.
  • the pi value is defined as the pi of the predominant charge form.
  • the pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see, e.g., Bjellqvist et ah, 1993, Electrophoresis, 14: 1023).
  • the thermal melting temperatures (Tm) of the Fab domain of an antibody can be a good indicator of the thermal stability of an antibody and may further provide an indication of the shelf-life.
  • Tm indicates more aggregation/less stability, whereas a higher Tm indicates less aggregation/more stability.
  • antibodies having higher Tm are preferable.
  • Tm of a protein domain e.g., a Fab domain
  • an additional non-exclusive aspect of the present invention includes modified antibodies that have certain preferred biochemical characteristics, such as a particular isoelectric point (pi) or melting temperature (Tm).
  • certain preferred biochemical characteristics such as a particular isoelectric point (pi) or melting temperature (Tm).
  • the present invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention or epitope-binding fragments thereof.
  • polynucleotides encoding such anti-MET antibodies as described above.
  • polynucleotide having least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% rule 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide that encodes for or transcribes the amino acid sequence of any of the heavy chain variable regions of the antibodies produced by hybridomas 247.27.16, 247.2.26, 247.48.38, 247.3.14, 247.22.2, 248.69.4, and 247.16.8 and/or (b) a polynucleotide having at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% consult 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to
  • the invention provides a polynucleotide encoding a polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs:55-72.
  • the invention further provides a polynucleotide comprising a humanized variable region DNA sequence selected from those shown in Tables 6 and 7 below.
  • polynucleotide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequences in Table 6 (SEQ ID NOs:55-67).
  • a polynucleotide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequences in Table 6 (SEQ ID NOs:55-67).
  • a polynucleotide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequences in Table 6 (SEQ ID NOs:55-67).
  • polynucleotide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the following sequences:
  • polynucleotide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequences in Table 7 (SEQ ID NOs:68-72 and 109-116).
  • polynucleotide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the following sequences:
  • the polynucleotide has the sequence of SEQ ID NO:68 and SEQ ID NO:68
  • the present invention further provides variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and derivatives.
  • the polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments the polynucleotide variants contain alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. In some embodiments, nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).
  • the present invention also encompasses polynucleotides encoding a polypeptide that can bind MET and that hybridizes under stringent hybridization conditions to polynucleotides that encode an antibody of the present invention, wherein said stringent hybridization conditions include: pre-hybridization for 2 hours at 60°C in 6x SSC, 0.5% SDS, 5x
  • polynucleotides may be obtained, and the nucleotide sequence of the
  • polynucleotides determined, using methods 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 ah, 1994, BioTechniques 17:242) which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • chemically synthesized oligonucleotides e.g., as described in Kutmeier et ah, 1994, BioTechniques 17:242
  • oligonucleotides e.g., as described in Kutmeier et ah, 1994, BioTechniques 17:242
  • 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., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • U.S. Patent No. 7,538,195 has been referred to in the present disclosure, the teachings of which are hereby incorporated in its entirety by reference.
  • diverse antibodies and antibody fragments, as well as antibody mimics may be readily produced by mutation, deletion and/or insertion within the variable and constant region sequences that flank a particular set of CDRs.
  • different classes of antibody are possible for a given set of CDRs by substitution of different heavy chains, whereby, for example, IgGl-4, IgM, IgA 1-2, IgD, IgE antibody types and isotypes may be produced.
  • artificial antibodies within the scope of the invention may be produced by embedding a given set of CDRs within an entirely synthetic framework.
  • variable is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its antigen.
  • variability is not usually evenly distributed through the variable domains of the antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of heavy and light chains each comprise four framework regions, largely adopting a beta- sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see, for example, E. A. Rabat et al. Sequences of Proteins of Immunological Interest, fifth edition, 1991, NIH).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • Humanized antibodies or antibodies adapted for non-rejection by other mammals, may be produced using several technologies such as resurfacing and CDR grafting.
  • resurfacing technology molecular modeling, statistical analysis and mutagenesis are combined to adjust the non-CDR surfaces of variable regions to resemble the surfaces of known antibodies of the target host.
  • Strategies and methods for the resurfacing of antibodies, and other methods for reducing immunogenicity of antibodies within a different host, are disclosed in, for example, US Patent 5,639,641, which is hereby incorporated in its entirety by reference.
  • the murine heavy and light chain CDRs are grafted into a fully human framework sequence.
  • the invention also includes functional equivalents of the antibodies described in this specification.
  • Functional equivalents have binding characteristics that are comparable to those of the antibodies, and include, for example, chimerized, humanized and single chain antibodies as well as fragments thereof.
  • Exemplary methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319, European Patent Application No. 239,400; PCT Application WO 89/09622; European Patent Application 338,745; and European Patent Application EP 332,424, which are incorporated in their respective entireties by reference.
  • Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the invention. "Substantially the same" as applied to an amino acid sequence is defined herein as a sequence with at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, and 99% sequence identity to another amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448 (1988).
  • Chimeric antibodies preferably can have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region from a mammal other than a human.
  • Humanized forms of the antibodies can be made by substituting the complementarity determining regions of, for example, a mouse antibody, into a human framework domain, e.g., PCT Pub. No. W092/22653.
  • Humanized antibodies preferably can have constant regions and variable regions other than the complementarity determining regions (CDRs) derived substantially or exclusively from the corresponding human antibody regions and CDRs derived substantially or exclusively from a mammal other than a human.
  • CDRs complementarity determining regions
  • Functional equivalents also include single-chain antibody fragments, also known as single-chain antibodies (scFvs). These fragments contain at least one fragment of an antibody variable heavy-chain amino acid sequence (V H ) tethered to at least one fragment of an antibody variable light-chain sequence (V L ) with or without one or more interconnecting linkers.
  • V H antibody variable heavy-chain amino acid sequence
  • V L antibody variable light-chain sequence
  • Such a linker may be a short, flexible peptide selected to assure that the proper three-dimensional folding of the (VL) and (V H ) domains occurs once they are linked so as to maintain the target molecule binding- specificity of the whole antibody from which the single chain antibody fragment is derived.
  • the carboxyl terminus of the (V L ) or (V H ) sequence may be covalently linked by such a peptide linker to the amino acid terminus of a complementary (VL) and (VH) sequence.
  • Single-chain antibody fragments may be generated by molecular cloning, antibody phage display library or similar techniques. These proteins may be produced either in eukaryotic cells or prokaryotic cells, including bacteria.
  • Single-chain antibody fragments may contain amino acid sequences having at least one of the variable or complementarity determining regions (CDRs) of the intact antibodies described in this specification, but are lacking some or all of the constant domains of those antibodies. These constant domains are not necessary for antigen binding, but constitute a major portion of the structure of intact antibodies. Single-chain antibody fragments may therefore overcome some of the problems associated with the use of antibodies containing a part or all of a constant domain. For example, single-chain antibody fragments tend to be free of undesired interactions between biological molecules and the heavy-chain constant region, or other unwanted biological activity.
  • CDRs complementarity determining regions
  • single-chain antibody fragments are considerably smaller than intact or whole antibodies and may therefore have greater capillary permeability than intact antibodies, allowing single-chain antibody fragments to localize and bind to target antigen-binding sites more efficiently.
  • antibody fragments can be produced on a relatively large scale in prokaryotic cells, thus facilitating their production.
  • the relatively small size of single-chain antibody fragments makes them less likely to provoke an immune response in a recipient than intact antibodies.
  • variants of the primary antibody have been generated by changing the sequences of the heavy and light chain genes in the CDR1, CDR2, CDR3, or framework regions, using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E. coli (Vaughan, T. J., et al., 1998, Nature Biotechnology, 16, 535-539; Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in "Phage Display of Peptides and Proteins", Eds. Kay, B. K. et al., Academic Press).
  • the antibody sequences described in this invention can be used to develop anti- MET antibodies with improved functions, such as those methods described in patent application publication 20090246195, the contents of which is incorporated in its entirety herein by reference.
  • the present invention relates to immunoconjugates comprising a MET- binding agent (e.g., an anti-MET antibody or an antigen-binding fragment thereof) described herein conjugated or covalently linked to a maytansinoid compound described herein.
  • a MET- binding agent e.g., an anti-MET antibody or an antigen-binding fragment thereof
  • the cytotoxic agent may be coupled or conjugated either directly to the MET-binding agent or indirectly, through a linker using techniques known in the art to produce an “immunoconjugate,”“conjugate,” or“ADC.”
  • the immunoconjugate of the present invention comprises a MET-binding agent (e.g., an anti-MET antibody or an antigen-binding fragment thereof) described herein covalently linked to a maytansinoid compound described herein through the e-amino group of one or more lysine residues located on the MET-binding agent through the e-amino group of one or more lysine residues located on the MET-binding agent (e.g., anti- cMET antibody or antigen-binding fragment thereof or through the thiol group of one or more cysteine residues located on the MET-binding agent (e.g., anti-cMET antibody or an antigen-binding fragment thereof).
  • the immunoconjugate is represented by formula (I) described above.
  • the immunoconjugate of the present invention is represented by formula (I) described above, wherein R x , R y , R x and R y are all H; and 1 and k are each independently an integer an integer from 2 to 6; and the remaining variables are as described above for formula (I).
  • the immunoconjugate of the present invention is represented by formula (I) described above, wherein A is a peptide containing 2 to 5 amino acid residues; and the remaining variables are as described above for formula (I) in the first embodiment or the I st specific embodiment.
  • A is a peptide cleavable by a protease.
  • A is a peptide having an amino acid that is covalent linked with -NH-CR R -S-Li-D selected from the group consisting of Ala, Arg,
  • the amino acid connected to -NH-CR R -S-Li-D is an L amino acid.
  • the immunoconjugate of the present invention is represented by formula (I) described above, wherein A is selected from the group consisting of Gly-Gly-Gly, Ala- Val, Val-Ala, D- Val- Ala, Val-Cit, D-Val-Cit, Val- Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Ala, Phe-N9-tosyl-Arg, Phe- N9-nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val- Ala- Val, Ala- Ala- Ala, D-Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-D-Ala, Al
  • the immunoconjugate of the present invention is represented by formula (I) described above, wherein R and R are both H; and the remaining variables are as described for formula (I) in the first embodiment or the I st , 2 nd , or 3 rd specific embodiment.
  • the immunoconjugate of the present invention is represented by formula (I) described above, wherein D is represented by the following formula:
  • the immunoconjugate of the present invention is represented by the following formula:
  • H is the anti-cMET antibody or antigen-binding fragment thereof connected to the L 2 group through a Lys amine group;
  • R 3 and R 4 are each independently H or Me
  • nl, rl, sl and tl are each independently an integer from 1 to 6;
  • n2, r2, s2 and t2 are each independently an integer from 1 to 7;
  • t3 is an integer from 1 to 12;
  • Di is represented by the following formula:
  • the immunoconjugate of the present invention is represented by the following formula:
  • nl and m3 are each independently an integer from 2 to 4;
  • r2 is an integer from 2 to 5;
  • A is Ala- Ala- Ala, Ala-D-Ala-Ala, Ala- Ala, D-Ala-Ala, Val-Ala, D- Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.
  • A is Ala- Ala- Ala, Ala-D-Ala-Ala, Ala- Ala, D-Ala-Ala, Val-Ala, D- Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly.
  • A is L-Ala-D-Ala-L-Ala.
  • the immunoconjugate of the present invention is represented by the following formula:
  • A is Ala- Ala- Ala, Ala-D-Ala-Ala, Ala- Ala, D-Ala-Ala, Val-Ala, D-Val-Ala, D-Ala-Pro, or D-Ala-tBu-Gly, and
  • A is L-Ala-D-Ala-L-Ala.
  • Di is represented by the following formula:
  • the immunoconjugate of the present invention is represented by the following formula:
  • Di is represented by the following formula:
  • the immunco the immunoconjugate of the present invention is represented by the following formula:
  • CBA is an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to an epitope in the extracellular region of human cMET, wherein said antibody or antigen-binding fragment thereof comprises light chain complementary determining regions LC CDR1, LC CDR2, and LC CDR3 and heavy chain complementary determining regions HC CDR1, HC CDR2, and HC CDR3 having the sequences of SEQ ID NOs:4, 5, and 7 and SEQ ID NOs:l3, 14, and 15, respectively;
  • q 1 or 2;
  • the isolated monoclonal antibody, or antigen-binding fragment thereof comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) having sequences of SEQ ID NO:32 and SEQ ID NO:36, respectively.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:54, respectively.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:8l, respectively.
  • the immunoconjugate of the present invention is represented by the following formula:
  • CBA an isolated monoclonal antibody, or antigen-binding fragment thereof, that specifically binds to an epitope in the extracellular region of human cMET, wherein said antibody or antigen-binding fragment thereof comprises light chain complementary determining regions LC CDR1, LC CDR2, and LC CDR3 and heavy chain complementary determining regions HC CDR1, HC CDR2, and HC CDR3 having the sequences of SEQ ID NOs:4, 5, and 7 and SEQ ID NOs:l3, 14, and 15, respectively;
  • q is an integer from 1 or 10;
  • the isolated monoclonal antibody, or antigen-binding fragment thereof comprises a light chain variable domain (VL) and a heavy chain variable domain (VH) having sequences of SEQ ID NO:32 and SEQ ID NO:36, respectively.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:53, respectively.
  • the isolated monoclonal antibody comprises a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:82, respectively.
  • the immunoconjugate of the present invention comprises an anti-cMET antibody coupled to a maytansinoid compound DM21C (also referred to as Mal- LDL-DM or MalC5-LDL-DM or compound l7a) represented by the following structural formula:
  • anti-cMET antibody comprising a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:8l, respectively; and Di is represented by the following formula:
  • the immunoconjugate is represented by the following structural formula:
  • CBA is an anti-cMET antibody connected to the maytansinoid compound through a Cys thiol group, wherein the anti-cMET antibody comprising a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:8l, respectively;
  • DAR is in the range of 1.5 to 2.2, 1.7 to 2.2 or 1.9 to 2.1. In some embodiment, the DAR is 1.7, 1.8, 1.9, 2.0 or 2.1.
  • the immunoconjugate of the present invention comprises an anti-cMET antibody coupled to a maytansinoid compound DM21L (also referred to as LDL-DM or compound l4c) represented by the following structural formula:
  • the anti- cMET antibody has a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:82, respectfully.
  • the conjugate is referenced herein as hucMet27Gvl.3Hinge28-sGMBS-LDL-DM.
  • the conjugate can also be referred to as hucMet27Gvl.3Hinge28-GMBS-LDL-DM, which can be used interchangeably with hucMet27 Gv 1.3Hinge28- sGMB S -LDL-DM .
  • GMBS and sulfo-GMBS (or sGMBS) linkers are known in the art and can be presented by the following structural formula:
  • the immunoconjugate is represented by the following structural formula: wherein:
  • CBA is an anti-cMET antibody connected to the maytansinoid compound through a Lys amine group, wherein the anti-cMET antibody comprising a light chain and a heavy chain having the sequences of SEQ ID NO:49 and SEQ ID NO:82, respectively;
  • q is an integer from 1 or 10.
  • DAR is in the range of 3.0 to 4.0, 3.2 to 3.8, or 3.4 to 3.7. In some embodiments, the DAR is 3.2, 3.3, 3.4, 3.5, 3.5, 3.7, or
  • compositions comprising immunoconjugates of the first embodiment, or the I st , 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , l0 th , I I th , 12 th , 13 th , 14 th or 15 th specific embodiment
  • the average number of the cytotoxic agent per antibody molecule i.e ., average value of q
  • DAR Drug- Antibody Ratio
  • DAR is in the range of 1.0 to 5.0, 1.0 to 4.0, 1.5 to 4.0, 2.0 to 4.0, 2.5 to 4.0, 1.0 to 3.4, 1.0 to 3.0, 3.0 to 4.0, 3.lto 3.5, 3.4 to 3.6, 1.5 to 2.5, 2.0 to 2.5, 1.7 to 2.3, or 1.8 to 2.2. In some embodiments, the DAR is less than 4.0, less than 3.8, less than 3.6, less than 3.5, less than 3.0 or less than 2.5.
  • the DAR is in the range of 3.1 to 3.4. In some embodiments, the DAR is in the range of 3.3 to 3.7. In some embodiments, the DAR is in the range of 3.5 to 3.9. In some embodiments, the DAR is 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7 or 3.8. In some embodiments, the DAR is in the range of 1.8 to 2.0. In some embodiments, the DAR is in the range of 1.7 to 1.9. In some embodiments, the DAR is in the range of 1.9 to 2.1. In some embodiments, the DAR is 1.9, 2.0 or 2.1.
  • the DAR is in the range of 1.5 to 2.5, 1.8 to 2.2, 1.1 to 1.9 or 1.9 to 2.1. In some embodiments, the DAR is 1.8,
  • linkers are bifunctional linkers.
  • the term“bifunctional linker” refers to modifying agents that possess two reactive groups; one of which is capable of reacting with a cell binding agent while the other one reacts with the maytansinoid compound to link the two moieties together.
  • Such bifunctional crosslinkers are well known in the art (see, for example, Isalm and Dent in Bioconjugation chapter 5, r218-363, Groves Dictionaries Inc. New York, 1999).
  • SMCC N- succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxylate
  • SIAB /V-succinimidyl-4-(iodoacetyl)-aminobenzoate
  • Other bifunctional crosslinking agents that introduce maleimido groups or haloacetyl groups on to a cell binding agent are well known in the art (see US Patent Publication Nos. 2008/0050310, 20050169933, available from Pierce Biotechnology Inc. P.O.
  • BMPEO bis- maleimidopolyethyleneglycol
  • BMPS N-(b- maleimidopropyloxy)succinimide ester
  • GMBS g-maleimidobutyric acid N-succinimidyl ester
  • EMCS e-maleimidocaproic acid N-hydroxysuccinimide ester
  • NHS HBVS
  • N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l- carboxy-(6-amidocaproate) which is a“long chain” analog of SMCC (LC-SMCC), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid
  • BMDB l,4-bismaleimidyl-2,3-dihydroxybutane
  • BMH bis-maleimidohexane
  • BMOE bis-maleimidoethane
  • sulfo-SMCC sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate
  • sulfo-SIAB sulfo-SIAB
  • m- maleimidobenzoyl-N-hydroxysulfosuccinimide ester sulfo-MBS
  • N- (y-maleimidobutryloxy)sulfosuccinimide ester sulfo-GMBS or sGMBS
  • N-(e- maleimidocaproyloxy)sulfosuccimido ester sulfo-EMCS
  • N-(K- maleimidoundecanoyloxy)sulfosuccinimide ester sulfo-KM
  • Heterobifunctional crosslinking agents are bifunctional crosslinking agents having two different reactive groups. Heterobifunctional crosslinking agents containing both an amine-reactive N- h ydro x y s ucc i n i m i dc group (NHS group) and a carbonyl-reactive hydrazine group can also be used to link the cytotoxic compounds described herein with a cell-binding agent (e.g ., antibody).
  • a cell-binding agent e.g ., antibody
  • heterobifunctional crosslinking agents examples include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH) and succinimidyl hydrazinium nicotinate hydrochloride (SHNH).
  • Conjugates bearing an acid-labile linkage can also be prepared using a hydrazine-bearing benzodiazepine derivative of the present invention.
  • bifunctional crosslinking agents examples include
  • succinimidyl-p-formyl benzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).
  • Bifunctional crosslinking agents that enable the linkage of cell binding agent with cytotoxic compounds via disulfide bonds are known in the art and include /V-succinimidyl-3- (2-pyridyldithio)propionate (SPDP), /V-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N- succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), /V-succinimidyl-4-(2-pyridyldithio)2- sulfo butanoate (sulfo-SPDB or sSPDB) to introduce dithiopyridyl groups.
  • SPDP 2-pyridyldithio)propionate
  • SPP /V-succinimidyl-4-(2-pyridyldithio)pentanoate
  • SPDB N- succinimidyl-4-
  • bifunctional crosslinking agents that can be used to introduce disulfide groups are known in the art and are disclosed in U.S. Patents 6,913,748, 6,716,821 and US Patent Publications 20090274713 and 20100129314, all of which are incorporated herein by reference.
  • crosslinking agents such as 2-iminothiolane, homocysteine thiolactone or S- acetylsuccinic anhydride that introduce thiol groups can also be used.
  • the present invention provides the maytansinoid compounds that can be used for making the immunoconjugates of the present invention.
  • the maytansinoid compound is represented by the following formula:
  • L 2 is represented by the following structural formulas: wherein:
  • R x , R y , R x and R y are independently H, -OH, halogen, - 0-(Ci_ 4 alkyl), -S0 3 H, -NR 4 oR 4i R 42 + , or a C alkyl optionally substituted with -OH, halogen, -S0 3 H or NR 4 oR 4i R 42 + , wherein R 40 , R 41 and R 42 are each independently H or a Ci_ 4 alkyl;
  • 1 and k are each independently an integer from 1 to 10;
  • A is an amino acid or a peptide comprising 2 to 20 amino acids
  • R 1 and R 2 are each independently H or a Ci_ 3 alkyl
  • Li is represented by the following formula:
  • q is an integer from 1 to 20.
  • the maytansinoid of the present invention is represented by the following formula:
  • A’ is an amino acid or a peptide comprising 2 to 20 amino acids ( i.e ., A-NH 2 );
  • R 1 and R 2 are each independently H or a Ci_ 3 alkyl
  • q is an integer from 1 to 20.
  • the maytansinoid of the present invention is represented by the following formula:
  • R x and R y are independently H, -OH, halogen, -0-(Ci_ 4 alkyl), - S0 3 H, -NR 4 oR 4i R 42 + , or a Ci_ 4 alkyl optionally substituted with -OH, halogen, SO 3 H or NR 4O R 4 IR4 2 + , wherein R 4 o, R 4 I and R 42 are each independently H or a Ci_ 4 alkyl;
  • k is an integer from 1 to 10
  • A is an amino acid residue or a peptide comprising 2 to 20 amino acid residues;
  • R 1 and R 2 are each independently H or a Ci_ 3 alkyl
  • q is an integer from 1 to 20.
  • the variables are as described in the first embodiment, or in the I st , 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , 10 th or 1 I th specific embodiment in the first embodiment.
  • the maytansinoid compound is represented by the following formula:
  • immunoconjugates comprising a MET-binding agent covalently linked to a maytansinoid compound described herein can be prepared according to any suitable methods known in the art.
  • the immunoconjugates of the first embodiment can be prepared by a first method comprising the steps of reacting the MET-binding agent (e.g ., anti- cMET antibody or an antigen-binding fragment thereof) with the maytansinoid compound of formula (II) described in the second embodiment.
  • the MET-binding agent e.g ., anti- cMET antibody or an antigen-binding fragment thereof
  • the immunoconjugates of the first embodiment can be prepared by a second method comprising the steps of:
  • the immunoconjugates of the first embodiment can be prepared by a third method comprising the steps of:
  • the linker compound is represented by any one of the formula (alL) - (alOL):
  • X is halogen; J D - SH, or -SSR d ; R d is phenyl, nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl or nitropyridyl; R g is an alkyl; and U is -H or S0 3 H or a pharmaceutically acceptable salt thereof.
  • the linker compound is GMBS or sulfo-GMBS (or sGMBS) represented by represented by formula (a9L), wherein U is -H or SO3H or a pharmaceutically acceptable salt thereof.
  • the immunoconjugate of the present invention is represented by the following formula:
  • the immunoconjugate can be prepared by the second, third or fourth method described above, wherein the linker compound is GMBS or sulfo-GMBS represented by represented by formula (a9L), wherein U is -H or S0 3 H or a pharmaceutically acceptable salt thereof; and the maytansinoid compound is represented by formula (D-l) described above.
  • the immunoconjugate of formula (1-1) is prepared by reacting the maytansinoid compound of formula (D-l) with the linker compound GMBS or sulfo-GMBS to form a maytansinoid-linker compound, followed by reacting the anti-cMET antibody or antigen-binding fragment thereof with the maytansinoid-linker compound.
  • the maytansinoid linker compound is not purified before reacting with the anti-cMET antibody or an antigen-binding fragment thereof.
  • the immunoconjugate is represented by the following formula:
  • the immunoconjugate can be prepared by the second, third or fourth method described above, wherein the linker compound is GMBS or sulfo-GMBS represented by represented by formula (a9L), wherein U is -H or SO 3 H or a pharmaceutically acceptable salt thereof; and the maytansinoid compound is represented by formula (D-2) described above.
  • the immunoconjugate of formula (1-2) is prepared by reacting the maytansinoid compound of formula (D-2) with the linker compound GMBS or sulfo-GMBS to form a maytansinoid-linker compound, followed by reacting the anti-cMET antibody or antigen-binding fragment thereof with the maytansinoid-linker compound.
  • the maytansinoid linker compound is not purified before reacting with the anti-cMET antibody or an antigen-binding fragment thereof.
  • the immunoconjugate is represented by the following formula:
  • the immunoconjugate is prepared according to the first method described above by reacting the anti-cMET antibody or an antigen-binding fragment thereof with the
  • the immunoconjugate is represented by the following formula:
  • the immunoconjugate is prepared according to the first method described above by reacting the anti-cMET antibody or an antigen-binding fragment thereof with the
  • the immunoconjugate is represented by the following formula:
  • the immunoconjugate is prepared according to the first method described above by reacting the anti-cMET antibody or an antigen-binding fragment thereof with the maytansinoid compound of formula (D-5) described above.
  • the immunoconjugate is represented by the following formula:
  • the immunoconjugate is prepared according to the first method described above by reacting the anti-cMET antibody or an antigen-binding fragment thereof with the maytansinoid compound of formula (D-6) described above.
  • the immunoconjugates prepared by any methods described above is subject to a purification step.
  • the immunoconjugate can be purified from the other components of the mixture using tangential flow filtration (TFF), non- adsorptive chromatography, adsorptive chromatography, adsorptive filtration, selective precipitation, or any other suitable purification process, as well as combinations thereof.
  • THF tangential flow filtration
  • the immunoconjugate is purified using a single purification step (e.g., TFF).
  • the conjugate is purified and exchanged into the appropriate formulation using a single purification step (e.g., TFF).
  • the immunoconjugate is purified using two sequential purification steps.
  • the immunoconjugate can be first purified by selective precipitation, adsorptive filtration, absorptive chromatography or non-absorptive chromatography, followed by purification with TFF.
  • purification of the immunoconjugate enables the isolation of a stable conjugate comprising the cell-binding agent chemically coupled to the cytotoxic agent.
  • TFF systems Any suitable TFF systems may be utilized for purification, including a Pellicon type system (Millipore, Billerica, Mass.), a Sartocon Cassette system (Sartorius AG, Edgewood, N.Y.), and a Centrasette type system (Pall Corp., East Hills, N.Y.)
  • Pellicon type system Millipore, Billerica, Mass.
  • Sartocon Cassette system Sartorius AG, Edgewood, N.Y.
  • Centrasette type system Pall Corp., East Hills, N.Y.
  • Any suitable adsorptive chromatography resin may be utilized for purification.
  • Preferred adsorptive chromatography resins include hydroxyapatite chromatography, hydrophobic charge induction chromatography (HCIC), hydrophobic interaction
  • hydroxyapatite resins include ceramic hydroxyapatite (CHT Type I and Type II, Bio-Rad Faboratories, Hercules, Calif.), HA Ultrogel hydroxyapatite (Pall Corp., East Hills, N.Y.), and ceramic fluoroapatite (CFT Type I and Type II, Bio-Rad Laboratories, Hercules, Calif.).
  • An example of a suitable HCIC resin is MEP Hypercel resin (Pall Corp., East Hills, N.Y.).
  • HIC resins examples include Butyl-Sepharose, Hexyl-Sepharose, Phenyl-Sepharose, and Octyl Sepharose resins (all from GE Healthcare, Piscataway, N.J.), as well as Macro-prep Methyl and Macro-Prep t-Butyl resins (Biorad Laboratories, Hercules, Calif.).
  • suitable ion exchange resins include SP- Sepharose, CM-Sepharose, and Q-Sepharose resins (all from GE Healthcare, Piscataway, N.J.), and Unosphere S resin (Bio-Rad Laboratories, Hercules, Calif.).
  • suitable mixed mode ion exchangers include Bakerbond ABx resin (JT Baker, Phillipsburg N.J.)
  • suitable IMAC resins include Chelating Sepharose resin (GE Healthcare, Piscataway, N.J.) and Profinity IMAC resin (Bio-Rad Laboratories, Hercules, Calif.).
  • suitable dye ligand resins include Blue Sepharose resin (GE Healthcare, Piscataway, N.J.) and Affi-gel Blue resin (Bio-Rad Laboratories, Hercules, Calif.).
  • suitable affinity resins include Protein A Sepharose resin (e.g., MabSelect, GE Healthcare, Piscataway, N.J.), where the cell-binding agent is an antibody, and lectin affinity resins, e.g., Lentil Lectin Sepharose resin (GE Healthcare, Piscataway, N.J.), where the cell-binding agent bears appropriate lectin binding sites.
  • an antibody specific to the cell-binding agent may be used.
  • Such an antibody can be immobilized to, for instance, Sepharose 4 Fast Flow resin (GE Healthcare, Piscataway, N.J.).
  • suitable reversed phase resins include C4, C8, and C18 resins (Grace Vydac, Hesperia, Calif.).
  • Any suitable non-adsorptive chromatography resin may be utilized for purification.
  • suitable non-adsorptive chromatography resins include, but are not limited to, SEPHADEXTM G-25, G-50, G-100, SEPHACRYLTM resins (e.g., S-200 and S-300), SUPERDEXTM resins (e.g., SUPERDEXTM 75 and SUPERDEXTM 200), BIO-GEL® resins (e.g., P-6, P-10, P-30, P-60, and P-100), and others known to those of ordinary skill in the art.
  • the antibodies and/or fragments of the present invention can be employed in many known diagnostic and research applications.
  • Antibodies and or fragments of the present invention may be used, for example, in the purification, detection, and targeting of MET, included in both in vitro and in vivo diagnostic methods.
  • the antibodies and/or fragments may be used in immunoassays for qualitatively and quantitatively measuring levels of MET expressed by cells in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988), incorporated by reference herein in its entirety.
  • the antibodies of the present invention may be used in, for example, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987)).
  • the present invention also provides the above anti-MET peptides and antibodies, detectably labeled, as described below, for use in diagnostic or prognostic or patient stratification methods for detecting MET in patients known to be or suspected of having a MET-mediated condition.
  • Anti-MET peptides and/or antibodies of the present invention are useful for immunoassays which detect or quantitate MET, or anti-MET antibodies, in a sample.
  • An immunoassay for MET typically comprises incubating a biological sample in the presence of a detectably labeled high affinity anti-MET peptide and/or antibody of the present invention capable of selectively binding to MET, and detecting the labeled peptide or antibody which is bound in a sample.
  • an anti-MET peptide or antibody or fragment thereof can be added to nitrocellulose, or another solid support which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support can then be washed with suitable buffers followed by treatment with the detectably labeled MET- specific peptide or antibody or fragment thereof.
  • the solid phase support can then be washed with the buffer a second time to remove unbound peptide or antibody or fragment thereof.
  • the amount of bound label on the solid support can then be detected by known method steps.
  • solid phase support or “carrier” is intended any support capable of binding peptide, antigen or antibody or fragment thereof.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. Those skilled in the art will know many other suitable carriers for binding antibody or fragment thereof, peptide or antigen, or can ascertain the same by routine experimentation.
  • Detectably labeling a MET-specific peptide and/or antibody or fragment thereof can be accomplished by linking to an enzyme for use in an enzyme immunoassay (EIA), or enzyme-linked immunosorbent assay (ELISA).
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Enzymes which can be used to detectably label the MET-specific antibodies or fragment thereof of the present invention 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.
  • MET-specific antibodies and/or fragment thereof By radioactively labeling the MET-specific antibodies and/or fragment thereof, it is possible to detect MET through the use of a radioimmunoassay (RIA) (see, for example, Work, et ah, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N.Y. (1978)).
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are: H, I, I, S, 14 C, and, preferably, 125 I.
  • a fluorescent compound it is also possible to label the MET-specific antibodies and or fragments thereof with a fluorescent compound.
  • fluorescent labeled antibody When the fluorescent labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the MET-specific antibodies or fragments thereof can also be detectably labeled using fluorescence-emitting metals such as or others of the lanthanide series. These metals can be attached to the MET-specific antibody or fragment thereof using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine- tetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediamine- tetraacetic acid
  • the MET-specific antibodies or fragments thereof also can be detectably labeled by coupling to a chemiluminescent compound.
  • the presence of the chemiluminescently labeled 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.
  • a bioluminescent compound can be used to label the MET- specific antibody, fragment or derivative thereof of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Detection of the MET-specific antibody, fragment or derivative thereof can be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material.
  • the detection can be accomplished by colorometric methods which employ a substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • the MET which is detected by the above assays can be present in a biological sample.
  • a biological sample such as, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like.
  • the invention is not limited to assays using only these samples, it being possible for one of ordinary skill in the art to determine suitable conditions which allow the use of other samples.
  • In situ detection can be accomplished by removing a histological specimen from a patient, and providing the combination of labeled antibodies of the present invention to such a specimen.
  • the antibody or fragment thereof is preferably provided by applying or by overlaying the labeled antibody or fragment thereof to a biological sample.
  • the antibody or fragment thereof of the present invention can be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
  • an immunometric assay also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled antibody or fragment thereof is bound to a solid support that is insoluble in the fluid being tested and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the MET from the sample by formation of a binary solid phase antibody-MET complex.
  • the solid support is washed to remove the residue of the fluid sample, including unreacted MET, if any, and then contacted with the solution containing a known quantity of labeled antibody (which functions as a "reporter molecule").
  • a known quantity of labeled antibody which functions as a "reporter molecule"
  • the solid support is washed a second time to remove the unreacted labeled antibody or fragment thereof.
  • This type of forward sandwich assay can be a simple "yes/no" assay to determine whether MET is present or can be made quantitative by comparing the measure of labeled antibody or fragment thereof with that obtained for a standard sample containing known quantities of MET.
  • Such "two-site” or “sandwich” assays are described by Wide (Radioimmune Assay Method, Kirkham, ed., Livingstone, Edinburgh, 1970, pp. 199-206).
  • a simultaneous assay involves a single incubation step wherein the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.
  • a combination of antibodies of the present invention specific for separate epitopes can be used to construct a sensitive three-site immunoradiometric assay.
  • the antibodies or fragments thereof of the invention also are useful for in vivo imaging, wherein an antibody or fragment thereof labeled with a detectable moiety such as a radio-opaque agent or radioisotope is administered to a subject, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is assayed.
  • a detectable moiety such as a radio-opaque agent or radioisotope
  • the antibody or fragment thereof may be labeled with any moiety that is detectable in a host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • the label can be any detectable moiety that is capable of producing, either directly or indirectly, a detectable signal.
  • the label may be a biotin label, an enzyme label (e.g., luciferase, alkaline phosphatase, beta-galactosidase and horseradish peroxidase), a radio-label (e.g., H, C, P, S, and I), a fluorophore such as fluorescent or
  • chemiluminescent compound e.g., fluorescein isothiocyanate, rhodamine
  • an imaging agent e.g., Tc-m99 and indium ( m In)
  • a metal ion e.g., gallium and europium
  • any method known in the art for conjugating the antibody or fragment thereof to the label may be employed, including those exemplary methods described by Hunter, et ah, 1962, Nature 144:945; David et ah, 1974, Biochemistry 13:1014; Pain et ah, 1981, J. Immunol. Meth. 40:219; Nygren, J., 1982, Histochem. and Cytochem. 30:407.
  • the antibodies or fragments thereof of the invention also are useful as affinity purification agents.
  • the antibodies for example, are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art.
  • MET may be isolated and purified from a biological sample.
  • the immunoconjugates of the present invention have the ability to bind MET present on the surface of a cell and mediate cell killing.
  • the immunoconjugates of the present invention comprising a cytotoxic payload, e.g., a indolinobenzodiazepine DNA-alkylating agent, are internalized and mediate cell killing via the activity of the cytotoxic payload e.g., a benzodiazepine, e.g., an indolinobenzodiazepine DNA-alkylating agent.
  • a cytotoxic payload e.g., a indolinobenzodiazepine DNA-alkylating agent
  • Such cell killing activity may be augmented by the immunoconjugate inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • inhibitors and“inhibiting” should be understood to include any inhibitory effect on cell growth, including cell death.
  • the inhibitory effects include temporary effects, sustained effects and permanent effects.
  • the therapeutic applications of the present invention include methods of treating a subject having a disease.
  • the diseases treated with the methods of the present invention are those characterized by the expression (e.g., cMET overexpression in the presence or absence of gene amplification) and/or activation of MET (e.g., in the presence or absence of gene amplification).
  • Such diseases include for example, glioblastoma, pancreatic cancer, gastric cancer, prostate cancer, ovarian cancer, breast cancer, hepatocellular carcinoma (HCC), melanoma, osteosarcoma, and colorectal cancer (CRC), lung cancer including small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), kidney cancer, renal cancer, esophageal cancer and thyroid cancer.
  • SCLC small-cell lung cancer
  • NSCLC non-small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • kidney cancer renal cancer
  • renal cancer esophageal cancer and thyroid cancer.
  • immunoconjugates of the present invention may be useful in the treatment of non-small-cell lung cancer (squamous cell, adenocarcinoma, or large-cell undifferentiated carcinoma), colorectal cancer (adenocarcinoma, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, primary colorectal lymphoma,
  • non-small-cell lung cancer squamous cell, adenocarcinoma, or large-cell undifferentiated carcinoma
  • colorectal cancer adenocarcinoma, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, primary colorectal lymphoma
  • the present invention also includes therapeutic applications of the antibodies or conjugates of the present invention wherein the antibodies or conjugates may be administered to a subject, in a pharmaceutically acceptable dosage form. They can be administered intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, subcutaneous, parenteral, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. They may also be administered by intratumoral, peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e ., compositions that are suitable for administration to a subject or patient) that can be used in the preparation of unit dosage forms.
  • pharmaceutical compositions e.g., impure or non-sterile compositions
  • pharmaceutical compositions i.e ., compositions that are suitable for administration to a subject or patient
  • compositions comprise a prophylactically or therapeutically effective amount of the immunoconjugates of the present invention, or a combination of such agents and a pharmaceutically acceptable carrier.
  • compositions of the invention comprise a prophylactically or therapeutically effective amount of immunoconjugates of the present invention and a pharmaceutically acceptable carrier.
  • the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S.
  • compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with an immunoconjugates of the present invention, alone or with such pharmaceutically acceptable carrier.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • kits that can be used in the above methods.
  • a kit can comprise any of the immunoconjugates of the present invention.
  • compositions of the present invention may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder by administering to a subject a therapeutically effective amount an immunoconjugate of the invention.
  • such compositions are substantially purified (i.e ., substantially free from substances that limit its effect or produce undesired side effects).
  • the subject is an animal, preferably a mammal such as non-primate (e.g ., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey such as, a cynomolgus monkey, human, etc.).
  • the subject is a human.
  • Methods of administering an immunoconjugate of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes).
  • parenteral administration e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous
  • epidural e.g., intranasal and oral routes
  • mucosal e.g., intranasal and oral routes.
  • the immunoconjugates of the present invention are administered intramuscularly, intravenously, or subcutaneously.
  • the compositions may be administered by any convenient route, for example, by infusion or bolus injection, and may be
  • Administration can be systemic or local.
  • the invention also provides that preparations of the immunoconjugates of the present invention are packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of the molecule.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of the molecule.
  • such molecules are supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject.
  • the immunoconjugates of the present invention are supplied as a dry sterile lyophilized powder in a hermetically sealed container.
  • the lyophilized preparations of the immunoconjugates of the present invention should be stored at between 2°C and 8°C in their original container and the molecules should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted.
  • such molecules are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the molecule, fusion protein, or conjugated molecule.
  • such immunoconjugates when provided in liquid form are supplied in a hermetically sealed container.
  • an“therapeutically effective amount” of a pharmaceutical As used herein, an“therapeutically effective amount” of a pharmaceutical
  • composition is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results such as decreasing symptoms resulting from the disease, attenuating a symptom of infection (e.g., viral load, fever, pain, sepsis, etc.) or a symptom of cancer (e.g., the proliferation, of cancer cells, tumor presence, tumor metastases, etc.), thereby increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication such as via targeting and/or internalization, delaying the progression of the disease, and / or prolonging survival of individuals.
  • beneficial or desired results including, without limitation, clinical results such as decreasing symptoms resulting from the disease, attenuating a symptom of infection (e.g., viral load, fever, pain, sepsis, etc.) or a symptom of cancer (e.g., the proliferation, of cancer cells, tumor presence, tumor metastases, etc.), thereby increasing the quality of life of those suffering from the disease, decreasing the dose of
  • a therapeutically effective amount can be administered in one or more of
  • a therapeutically effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to reduce the proliferation of (or the effect of) viral presence and to reduce and /or delay the development of the viral disease, either directly or indirectly.
  • compositions of the invention may be administered locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • an implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • care must be taken to use materials to which the molecule does not absorb.
  • compositions of the invention can be delivered in a vesicle, in particular a liposome ( See Langer (1990)“New Methods Of Drug Delivery,” Science 249:1527-1533); Treat et al, in LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez- Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327).
  • Cell lines used herein were grown in the appropriate media, for example DMEM or RPMI-1640 media supplemented with 10% fetal bovine serum, 2 mM glutamine and 1% penicillin-streptomycin (all reagents from Invitrogen) at 37°C in a humidified 5% C0 2 incubator unless otherwise indicated. Cells were passaged twice per week and maintained between 0.2 to 1 x 10 6 cells/ml.
  • An expression plasmid pSRa-MET was constructed that contained the MET extracellular and transmembrane domain sequence flanked by Kpnl and Xhol restriction sites that allowed expression of a truncated version of human MET which corresponds to the first 1077 amino acids of the 1390 amino acid protein described by GenBank Protein ID 188595716.
  • This truncated version does not contain the intracellular receptor kinase domain which comprises the receptor autophosphorylation site and the adaptor protein docking site. However, it does contain the entire extracellular portion of MET including the ligand-binding site.
  • mice 300-19 cells, a pre-B cell line derived from a Balb/c mouse (Reth et ah, Nature , 317:353-355 (1985)), was transfected with this expression plasmid to stably express high levels of truncated human MET on the cell surface and used for immunization of Balb/c VAF mice. Mice were first immunized subcutaneously with 10 pg of recombinant human
  • HGFR/cMET-Fc chimeric protein (R&D systems; 358-MT/CF) in complete Freund’s adjuvant (CFA) followed by the same antigen in incomplete Freund’s adjuvant (IFA) two weeks later.
  • CFA complete Freund’s adjuvant
  • IFA incomplete Freund’s adjuvant
  • mice were then boosted with three immunizations of 5xl0 6 MET-expressing 300-19 cells per mouse every 2 weeks by standard immunization protocols known to those of skill, for example, such as those used at ImmunoGen, Inc. Immunized mice were boosted one more time with 5xl0 6 MET-expressing 300-19 cells per mouse three days before being sacrificed for hybridoma generation.
  • Spleens from mice was collected according to standard animal protocols, such as, for example grinding tissue between two sterile, frosted microscopic slides to obtain a single cell suspension in RPMI-1640 medium.
  • the spleen cells were centrifuged, pelleted, washed, and fused with a murine myeloma, such as, for example P3X63Ag8.653 cells (Kearney et ah, J. Immunol., 123:1548-1550 (1979)) using polyethylene glycol-l500 (Roche 783 641).
  • the fused cells were resuspended in RPMI-1640 selection medium containing hypoxanthine-aminopterin-thymidine (HAT) (Sigma H-0262) and selected for growth in 96-well flat-bottomed culture plates (Corning-Costar 3596, 200 pF of cell suspension per well) at 37°C with 5% carbon dioxide (C0 2 ). After 5 days of incubation, 100 pF of culture supernatant were removed from each well and replaced with 100 pF of RPMI-1640 medium containing hypoxanthine-thymidine (HT) supplement (Sigma H-0137). Incubation at 37°C with 5% C0 2 was continued until hydridoma clones were ready for antibody screening.
  • Other techniques of immunization and hybridoma production can also be used, including those described in Fangone et al. (Eds.,“Immunochemical
  • the cells were centrifuged, pelleted, washed, and incubated for 1 h with 100 pL of PE-conjugated goat anti-mouse IgG-antibody (such as obtainable from, for example Jackson Laboratory) at 6 pg/mL in FACS buffer.
  • the cells were centrifuged, pelleted again, washed with FACS buffer and resuspended in 200 pL of PBS containing 1% formaldehyde.
  • Cells were acquired using a FACSCalibur flow cytometer with the HTS multiwell sampler or a FACS array flow cytometer and analyzed using CellQuest Pro (all from BD Biosciences, San Diego, US).
  • Hybridoma clones identified as secreting anti-MET antibodies were expanded and grown to collect antibody-containing supernatant for additional screening.
  • the amino acid sequence for the HC and LC variable region of the 224G11 antibody was derived from WO 2009007427 (Goetsch L.) using SEQ ID NO: 18 for the HC variable region and SEQ ID NO:2l for the LC variable region.
  • the amino acid sequence for the HC and LC variable region of the 5D5 antibody was derived from US07476724 using SEQ ID NOs 187 to 193 for the HC variable region and SEQ ID NOs 179 to 185 for the LC variable region.
  • variable region sequences for both antibodies were codon-optimized and synthesized by Blue Heron Biotechnology.
  • the sequences are flanked by restriction enzyme sites for cloning in-frame with the respective constant sequences in single chain mammalian expression plasmids. Cloning, expression and purification was carried out as described above.
  • a Fab preparation was isolated from the whole IgG using the Pierce ® Fab preparation kit (Thermo Fisher Scientific, Waltham, MA). Briefly, 0.5 ml of purified 5D5 IgG at a concentration of 4.7 mg/ml were buffer exchanged to the Fab digestion buffer containing 20 mM cysteine, pH 7.0, and mixed with 30 pg (0.88 BAEE unit) of immobilized papain that was equilibrated in the same digestion buffer. The digestion reaction was incubated for 6 hours with an end-over-end mixer at 37°C to maintain constant mixing of resin.
  • the digestion was then stopped by removing the IgG digest from the resin by centrifugation at 5000 x g.
  • the digested antibody solution was then incubated with pre-packed immobilized Protein A column that was equilibrated in phosphate buffered saline (PBS) for 10 mins.
  • PBS phosphate buffered saline
  • the Fab fragment was collected as the flowthrough fraction while the Fc fragments and undigested IgG bound to the column.
  • the 5D5 Fab fragment was then buffer exchanged into PBS using an Amicon centrifugal filter unit (Millipore, Billerica, MA). Fab purity was assessed with size exclusion
  • WO 2009007427 was used in comparison and it resulted in % inhibition of HGF binding to BxPC3 and MKN45 cells of 50% and 67%, respectively.
  • Several of the isolated hybridoma clones had more potent activity as compared to 224G11.
  • Hybridoma clones such as 247.7, 247.22, 247.26, 247.32, 247.33, 247.48, 248.51, 248.61, 248.62, 248.66, 248.67, 248.69, 248.71, 248.74, 248.76, 248.78, 248.81, 248.83, 248.90, 248.91, 248.92, and 248.96 resulted in at least 80% inhibition of HGF binding to both BxPC3 and MKN45.
  • Hybridoma clones 247.22, 247.48, and 248.69 are parental clones for hybridomas 247.22.2, 247.48.38 and 248.69.4, respectively, as described below in this and subsequent Examples.
  • WST-8 is reduced by dehydrogenases in living cells to an orange formazan product that is soluble in tissue culture medium. The amount of formazan produced is directly proportional to the number of living cells.
  • WST-8 was added to 10% of the final volume and plates were incubated at 37°C in a humidified 5% C0 2 incubator for an additional 2-4 hours. Plates were analyzed by measuring the absorbance at 450 nm (A 450 ) in a multiwell plate reader. Controls included untreated cells incubated with HGF (0% inhibition) and untreated cells incubated without HGF (100% inhibition).
  • Desirable hybridoma clones were subcloned by limiting dilution. Hybridoma supernatant from subclones were screened again for binding to MET-expressing cell by flow cytometry as outlined above. One or two subclones from each parental hybridoma clone, which showed the same reactivity against MET as the parental clone by flow cytometry, was chosen for subsequent analysis.
  • Hybridoma supernatant from positive subclones was tested for inhibition of HGF binding to MKN45 and BxPC3 cells as outlined above. Percent inhibition was determined for each sample. Typically, subclones showed substantial inhibition of HGF binding to MKN45 and BxPC3 cells as expected.
  • Antibodies were purified from hybridoma subclone supernatants using standard methods such as, for example, Protein A or G chromatography.
  • Antibody was eluted with 0.1 M acetic acid buffer containing 0.15 M NaCl, pH 2.8, using a flow rate of 0.5 mL/min. Fractions of approximately 0.25 mL were collected and neutralized by the addition of 1/10 volume of 1M Tris/HCl, pH 8.0. The peak fraction(s) was dialyzed overnight twice against lx PBS and sterilized by filtering through a 0.2 pm filter membrane. Purified antibody was quantified by absorbance at A280. [361] Protein A purified fractions were further polished using ion exchange chromatography (IEX) with quaternary ammonium (Q) chromatography for murine antibodies.
  • IEX ion exchange chromatography
  • Q quaternary ammonium
  • samples from protein A purification were buffer exchanged into binding buffer (10 mM Tris, 10 mM sodium chloride, pH 8.0) and filtered through 0.22 pm filer.
  • the prepared sample was then loaded onto a Q fast flow resin (GE Lifesciences) that was equilibrated with binding buffer at a flow rate of 120 cm/hr. Column size was chosen to have sufficient capacity to bind all the MAb in the sample.
  • the column was then washed with binding buffer until a stable baseline was obtained with no absorbance at 280 nm.
  • Antibody was eluted by initiating a gradient from 10 mM to 500 mM sodium chloride in 20 column volume (CV). Peak fractions were collected based on absorbance measurement at 280 nm (A280). The percentage of monomer was assessed with size exclusion chromatography (SEC) on a TSK gel G3000SWXL, 7.8 x 300 mm with a SWXL guard column, 6.0 x 40 mm (Tosoh
  • the 247.22.2 and 247.27.16 antibodies were humanized following resurfacing methods previously described, such as, for example in Roguska et al., Proc. Natl. Acad. Sci., USA, 9l(3):969-973 (1994) and Roguska et al., Protein Eng. 9(l0):895-904 (1996), which are incorporated in their entirety herein by reference. Resurfacing generally involves
  • variable region surface residues in both light and heavy chains and replacing them with human equivalents.
  • Surface residue positions are defined as any position with its relative accessibility of 30% or greater (Pedersen et al., J. Mol. Biol., 235(3):959-973 (1994)).
  • Surface residues are aligned with human germline surface sequences to identify the most homologous human surface sequence and replacements with human equivalent residues are made based on these alignments.
  • LC CDR2 AATNLAD (SEQ ID NO:2)
  • LC CDR3 QHFWGTPYT (SEQ ID NOG)
  • HC CDR1 DYNMD (SEQ ID NOG)
  • HC CDR2 DLNPNNGATI (SEQ ID NO: 12)
  • HC CDR3 GNYY GNYYYLMD Y (SEQ ID NO: 10)
  • Murine HC CDR2 DLNPNNGATIYNOKFKG (SEQ ID NO:9)
  • Human HC CDR2 DLNPNNGATIYNEKFOG (SEQ ID NO:73)
  • Exemplary CDRs for 247.27.16 are defined as indicated in the table below. Table 9. Exemplary 247.27.16 (cMET-27) CDRs
  • HC CDR1 SYDMS (SEQ ID NO: 13)
  • HC CDR2 TINSNGVSIY (SEQ ID NO: 17)
  • Murine and human HC CDR2 TINS N G V S IY YPDSVKG (SEQ ID NO: 14)
  • the light and heavy chain CDR’s as defined for the resurfacing are given by way of example in Table 8 and Table 9.
  • the Rabat definition for heavy chain CDR2 is also given for both the murine and human sequence.
  • the underlined sequence marks the portion of the Rabat heavy chain CDR2 not considered a CDR for resurfacing.
  • the CDR3 of the 247.27.16 light chain contains a potential protease cleavage site. Therefore two alternate resurfaced versions LC CDR3 1.2 and LC CDR3 1.3 were generated to remove this site.
  • the murine CMET-27 antibody was humanized following complementary determining region (CDR) grafting procedures described in Jones et al., Nature 321: 604-608 (1986), Verhoeyen et al., Science 239: 1534-1536 (1988), U.S. Patent No. 5225539 A (1993), and U.S. Patent No. 5585089 A (1996).
  • CDR grafting consists of replacing the Fv framework regions (FRs) of a mouse antibody with human antibody Fv framework regions while preserving the mouse CDR residues.
  • Exemplary CDRs of the CMET-27 antibody following the Rabat numbering scheme and the Rabat CDR definitions are as indicated in Table 10.
  • the CDR-grafting process begins by selecting appropriate human acceptor frameworks, typically those derived from human antibody genes sharing the highest sequence homology to the parent murine antibody utilizing the interactive tool, DomainGapAlign, of the International ImMunoGeneTics information system® (IMGT, http://www.imgt.org/) as described in Ehrenmann et al, Nucleic Acids Res. 38: D301-307 (2010).
  • IMGT International ImMunoGeneTics information system®
  • the human germline sequences selected as the acceptor frameworks for the V L and V H domains of cMET-27 antibody were IGRV3-11*01 and IGHV3-48*03, respectively (FIGs. 3A and 3B and in Table 3).
  • VL domain (G68R) and the VH domain (W47L, S49A, and N73I) belong to the Vernier zone residues.
  • V L domain, and v2.2 contains backmutations in both V L and V H domains, retained parent binding to the cell line expressing human cMET antigen in direct FACS binding. It would be intuitive to pick vl.l as the final humanized version since it contains no backmutation thereby keeping the CDR grafted antibody as“human” as possible.
  • direct comparison of the transient expression titers of the four versions revealed that vl.l expressed at a low level, 6 mg/L (Table 11). Low yield from transient expression makes research material less accessible; additionally, based on our experience, low transient titer is indicative of the difficulty in obtaining high expressing stable cell lines.
  • VHGv3 removing the two low frequency backmutations was constructed (FIG. 4B).
  • anti-MET antibodies containing hinge modifications i.e., anti-MET antibodies comprising a light chain having the amino acid sequence of SEQ ID NO:49 and a heavy chain having the amino acid sequence of SEQ ID NO:77, SEQ ID NO:78; SEQ ID NO:79; SEQ ID NO:80, SEQ ID NO:8l; SEQ ID NO:82; SEQ ID NO:83 or SEQ ID NO:84
  • anti-MET antibody containing hinge modifications has a light chain having the amino acid sequence of SEQ ID NO:49 and a heavy chain having the amino acid sequence of SEQ ID NO:82.
  • variable region sequences for hu247.22.2 and hu247.27.16 were codon-optimized and synthesized by Blue Heron Biotechnology. The sequences are flanked by restriction enzyme sites for cloning in-frame with the respective constant sequences in single chain mammalian expression plasmids.
  • the light chain variable region is cloned into EcoRI and BsiWI sites in the LC expression plasmids.
  • the heavy chain variable region is cloned into the HindHI and Apa 1 sites in the HC expression plasmid. These plasmids can be used to express human antibodies in either transient or stable transfections in mammalian cells.
  • Transient transfections to express human antibodies in HEK-293T cells can be performed using a modified PEI procedure (Durocher, Y. et al., Nucleic Acids Res. 30(2):E9 (2002)). Supernatant can be purified by Protein A and polishing chromatography steps using standard procedures as described above for chimerized antibodies.
  • the NSCLC (105) and Gastric cancer samples (15) were purchased from Avaden
  • Exemplary antibodies were evaluated for potential induction of cell growth in the absence of HGF under serum-free conditions. Briefly, 3,000 NCI-H441 cells were plated in serum free media (SFM; 0.l%BSA in RPMI1640 medium). The following day cells were incubated with lnM of the indicated anti-cMet antibodies in SFM or 100 ng/mF of HGF at 37 °C in a humidified 5% C0 2 incubator for 4 days. Viability was tested using WST-8 which was added to 10% of the final volume and the samples were incubated at 37°C in a humidified 5% C0 2 incubator for an additional 2-4 hours.
  • SFM serum free media
  • WST-8 was added to 10% of the final volume and the samples were incubated at 37°C in a humidified 5% C0 2 incubator for an additional 2-4 hours.
  • hucMet27Gvl.3, and particularly hucMet27Gvl.3Hinge28 and hucMet27Gvl.3HingeIgG2Sl27C antibodies resulted in significantly less induction at lOug/mL than 5D5 and ARGX-l 11, with signal similar to ABT-700 and 5D5-F’ab.
  • ELISA-based assays were used to quantify downstream signaling events triggered by cMet activation.
  • NCI-H441 cells were plated in SFM. The next day cells were incubated with lnM of the indicated anti-cMet antibodies/ ADC in SFM or 100 ng/mL of HGF for 15 minutes. Samples were lysed and clarified lysates were assayed by ELISA for phophorylated-Erk and phosphorylated-Akt. Briefly, an immobilized capture antibody binds both phosphorylated and unphosphorylated of either Erk or Akt.
  • a biotinylated detection antibody is used to detect only phosphorylated protein, utilizing a standard HRP format.
  • hucMet27 antibodies While treatment with the agonistic cMet antibody 5D5 resulted in moderate phosphorylation of Erk, 5D5 induced elevated levels of phosphorylated Akt that mimic the activity of the native ligand, HGF.
  • hucMet27 antibodies and particularly hucMet27Gvl.3Hinge28 and hucMet27Gvl.3HingeIgG2Sl27C antibodies induced significantly lower levels of phosphorylated Erk and in particular phosphorylated Akt.
  • hucMET27 antibody showed similar levels of downstream signaling as compared to other cMET targeting antibodies with less agonistic activity compared to 5D5.
  • Maytansinol (5.0 g, 8.85 mmol) was dissolved in anhydrous DMF ( 125 mL) then cooled in an ice bath.
  • the N-carboxy anhydride of N-methyl alanine (5.7 g, 44.25 mmol), anhydrous DIPEA (7.70 mL, 44.25 mmol) and zinc trifluoromethane sulfonate (22.5 g, 62 mmol) were then added with magnetic stirring under an argon atmosphere.
  • the ice bath was removed and the reaction was allowed to warm with stirring. After 16 h, deionized water (10 mL) was added. After 30 min a 1:1 solution of saturated aqueous sodium bicarbonate :
  • concentration of the solution was estimated by dividing the mmoles of maytansinol used in the reaction (1.77 mmol) by the volume (150 mL) giving DM-H stock solution (0.06 mmol/mL). Aliqouts of the stock solution were immediately dispensed then used in reactions or stored in a -80 C freezer then thawed when needed.
  • Step 1 FMoc-F-Ala-D-Ala-OtBu (3c): FMoc-L-alanine (lOg, 32.1 mmol) and D-Ala-OtBu, HC1 (7.00 g, 38.5 mmol) were dissolved in CH2C12 (100 ml), treated with COMU (20.63 g, 48.2 mmol) and DIPEA (11.22 ml, 64.2 mmol). The reaction was allowed to proceed for under argon at room temperature.
  • reaction was allowed to proceed at room temerpature under argon atmosphere. After 4 hours the reaction showed completion by UPLC, diluted with toluene (25mL) and coevaporated 3x. to yield FMoc-L-Ala-D-Ala, assume 100% yield.
  • Step 5 FMoc-L-Ala-D-Ala-L-Ala-Gly-OtBu (7c) FMoc-LAla-D-ALa-OH (0.959 g, 2.508 mmol) and L-Ala-Gly-OtBu (0.718 g, 3.01 mmol) were dissolved in CH2C12 (10 ml), treated with COMU (1.181 g, 2.76 mmol) and DIPEA (0.876 ml, 5.02 mmol). The reaction was allowed to proceed under argon at room
  • FMoc-LAla-DAla-LAla-GlyOtBu 200mg, 0.353 mmol
  • TFA Water (95:5) (2 ml).
  • the reaction was allowed to proceed under argon at room temperature. After 1 hr the reaction showed completion by UPLC.
  • the crude product was diluted with toluene (lmL), coevaporated 2x with toluene to yield FMoc-L-Ala-D-Ala-L-Ala-Gly-OH, assume 100% yield.
  • the crude reaction was purified via Combiflash Rf 200i using C18 450g column with a flow rate of l25mL/min with deionized water containing 0.1% formic acid and acetonitrile as solvents using a gradient as follows (time in minutes, percent acetonitrile) (0,5) (8,50) (26, 55).
  • the desired product having a retention time of 11 minutes, product fractions were immediatley froze and lypholized to yield FMoc-L-Ala-D-Ala-L-Ala-CH2-OAc (843mg, 1.607 mmol, 31.0 % yield).
  • FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 )5-CO-DM 11a: To DM-H stock solution (8.2 mL, 0.49 mmol) was added FMoc-L-Ala-L-Ala-L-Ala-NH-CH 2 -S-(CH 2 ) 5 -COOH (300 mg, 0.49 mmol), EDC (94 mg, 0.490 mmol) and DIPEA (90 pL, 0.49 mmol). The reaction was allowed to proceed with magnetic stirring at room temperature under argon atmosphere for 2 h.

Abstract

Le MET est un récepteur tyrosine kinase situé à la surface des cellules tumorales. La présente invention concerne des anticorps, des formes et des fragments anti-MET ayant des propriétés physiques et fonctionnelles supérieures ; des immunoconjugués, des compositions, des réactifs de diagnostic, des procédés d'inhibition de la croissance, des méthodes thérapeutiques, des anticorps et des lignées cellulaires améliorés ; et des polynucléotides, des vecteurs et des constructions génétiques codant pour ceux-ci.
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US10792372B2 (en) 2017-02-28 2020-10-06 Immunogen, Inc. Maytansinoid derivatives with self-immolative peptide linkers and conjugates thereof
WO2022095934A1 (fr) * 2020-11-05 2022-05-12 上海津曼特生物科技有限公司 Anticorps anti-siglec-15 et son utilisation dans la préparation d'un médicament
WO2022169975A1 (fr) 2021-02-03 2022-08-11 Mythic Therapeutics, Inc. Anticorps et leurs utilisations
WO2024059263A1 (fr) * 2022-09-15 2024-03-21 Immunogen, Inc. Procédés de préparation de dérivés maytansinoïdes dotés de lieurs peptidiques auto-immolables

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US11135308B2 (en) 2017-02-28 2021-10-05 Immunogen, Inc. Maytansinoid derivatives with self-immolative peptide linkers and conjugates thereof
WO2022095934A1 (fr) * 2020-11-05 2022-05-12 上海津曼特生物科技有限公司 Anticorps anti-siglec-15 et son utilisation dans la préparation d'un médicament
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