EP1590431A2 - Molecules de liaison au facteur de necrose tumorale alpha - Google Patents

Molecules de liaison au facteur de necrose tumorale alpha

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Publication number
EP1590431A2
EP1590431A2 EP04700833A EP04700833A EP1590431A2 EP 1590431 A2 EP1590431 A2 EP 1590431A2 EP 04700833 A EP04700833 A EP 04700833A EP 04700833 A EP04700833 A EP 04700833A EP 1590431 A2 EP1590431 A2 EP 1590431A2
Authority
EP
European Patent Office
Prior art keywords
tnf
seq
binding molecule
sequence
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04700833A
Other languages
German (de)
English (en)
Other versions
EP1590431A4 (fr
Inventor
Jeffry D. Watkins
Alain P. Vasserot
David Marquis
William D. Huse
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Applied Molecular Evolution Inc
Original Assignee
Applied Molecular Evolution Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/338,627 external-priority patent/US20040131613A1/en
Priority claimed from US10/338,552 external-priority patent/US7101978B2/en
Application filed by Applied Molecular Evolution Inc filed Critical Applied Molecular Evolution Inc
Publication of EP1590431A2 publication Critical patent/EP1590431A2/fr
Publication of EP1590431A4 publication Critical patent/EP1590431A4/fr
Withdrawn legal-status Critical Current

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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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]

Definitions

  • the present invention relates to TNF- ⁇ binding molecules and nucleic acid sequences encoding TNF- ⁇ binding molecules.
  • the present invention relates to TNF- ⁇ binding molecules with a high binding affinity, a high association rate, a low dissociation rate with regard to human TNF- ⁇ , and that are capable of preventing TNF- ⁇ mediated cytotoxicity at low concentrations.
  • the TNF- ⁇ binding molecules of the present invention comprise light and/or heavy chain variable regions with fully human frameworks (e.g. human germline frameworks).
  • Tumor necrosis factor alpha is a cytokine produced by numerous cell types, including monocytes and macrophages, that was originally identified based on its ability to induce the necrosis of certain mouse tumors. Subsequently, a factor termed cachectin, associated with cachexia, was shown to be identical to TNF- ⁇ . TNF- ⁇ has been implicated in the pathophysiology of a variety of other human diseases and disorders, including shock, sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease.
  • hTNF- ⁇ human TNF- ⁇
  • therapeutic strategies have been designed to inhibit or counteract hTNF- ⁇ activity.
  • antibodies that bind to, and neutralize, hTNF- ⁇ have been sought as a means to inhibit hTNF- ⁇ activity.
  • Some of the earliest of such antibodies were mouse monoclonal antibodies (mAbs), secreted by hybridomas prepared from lymphocytes of mice immunized with hTNF- ⁇ (see e.g., U.S. Pat. No. 5,231,024 to Moeller et al).
  • mouse anti-hTNF- ⁇ antibodies often displayed high affinity for hTNF- ⁇ and were able to neutralize hTNF- ⁇ activity
  • their use in vivo has been limited by problems associated with the administration of mouse antibodies to humans' such as a short serum half-life, an inability to trigger certain human effector functions, and elicitation of an unwanted immune response against the mouse antibody in a human (the "human anti-mouse antibody” (HAMA) reaction).
  • HAMA human anti-mouse antibody
  • murine anti-hTNF- ⁇ antibodies have been genetically engineered to be more "human-like.”
  • chimeric antibodies in which the variable regions of the antibody chains are murine-derived and the constant regions of the antibody chains are human-derived, have been prepared (e.g., U.S. Pat. 5,698,195, herein incorporated by reference).
  • humanized antibodies in which the hypervariable domains and a number of the framework residues of the antibody variable regions are murine-derived but the remainder of the variable regions and the antibody constant regions are human-derived, have also been prepared (e.g. U.S. Pat.
  • TNF- ⁇ binding molecules with a high binding affinity, a high association rate, a low dissociation rate and improved neutralization properties with regard to human TNF- ⁇ , as well as TNF- ⁇ binding molecules with significantly reduced immunogenicity in humans.
  • the present invention provides TNF- ⁇ binding molecules and nucleic acid sequences encoding TNF- ⁇ binding molecules.
  • the present invention provides TNF- ⁇ binding molecules with a high binding affinity, a high association rate, a low dissociation rate with regard to human TNF- ⁇ , and enhanced neutralization properties in vitro and in vivo.
  • the TNF- ⁇ binding molecules of the present invention comprise light and/or heavy chain variable regions with fully human frameworks.
  • the TNF- ⁇ binding molecules of the present invention comprise light and/or heavy chain variable regions with germline frameworks (e.g. human germline frameworks).
  • the present invention provides compositions comprising a peptide, or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 11, 13, and 15.
  • the peptide further comprises one or more amino acid sequences selected from SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55.
  • the present invention provides compositions comprising a peptide, or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 21, 25, and 27.
  • the peptide further comprises one or more amino acid sequences selected from SEQ ID NOs: 9, 11, 13, 15, 17, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53 and 55.
  • the present invention provides compositions comprising a peptide, or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid sequence shown in SEQ ID NO: 33.
  • the peptide further comprises one or more amino acid sequences selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55.
  • the present invention provides compositions comprising a peptide, or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 35, 37, and 39.
  • the peptide further comprises one or more amino acid sequences selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 43, 45, 47, 49, 51, 53, and 55.
  • the present invention provides compositions comprising a peptide, or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid sequence selected from SEQ ID NOs: 45 and 49. h other embodiments, the peptide further comprises one or more amino acid sequences selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 53.
  • the present invention provides compositions comprising a peptide, or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid shown in SEQ ID NO: 53.
  • the peptide further comprises one or more amino acid sequences selected from SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 55.
  • the present invention provides compositions comprising a peptide, or a nucleic acid sequence encoding a peptide, wherein the peptide comprises an amino acid sequences selected from SEQ ID NOs:87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 and 109.
  • the TNF- ⁇ binding molecule is a TNF- ⁇ binding peptide or polypeptide.
  • the TNF- ⁇ binding peptide comprises an anti-TNF- ⁇ antibody or anti-TNF- ⁇ antibody fragment (e.g., Fab, F(ab') 2 , etc), hi other embodiments, the peptide comprises a light and/or heavy chain variable region.
  • the light chain variable region and/or heavy chain variable region comprises a framework region.
  • at least FRL1, FRL2, FRL3, or FRL4 is fully human.
  • at least FRH1, FRH2, FRH3, or FRH4 is fully human.
  • At least FRL1, FRL2, FRL3, or FRL4 is a germline sequence (e.g. human germline). In other embodiments, at least FRH1, FRH2, FRH3, or FRH4 is a germline sequence (e.g. human germline).
  • the framework region is a fully human framework region (e.g. the human framework regions shown in Figures 5 and 6). hi some embodiments, the framework region comprises SEQ ID NO: 57, 58, 59, 60 or combinations thereof. In other embodiments, the framework region comprises SEQ ID NO: 65, 66, 67, 68, or combinations thereof.
  • the present invention provides compositions comprising an oligonucleotide, wherein the oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NO: 12, 14, or 16.
  • the oligonucleotide further comprises one or more nucleic acid sequences selected from SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56.
  • the present invention provides compositions comprising an oligonucleotide, wherein the oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 22, 26, and 28.
  • the oligonucleotide further comprises one or more nucleic acid sequences selected from SEQ ID NOs: 10, 12, 14, 16, 18, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, and 54.
  • the present invention provides compositions comprising an oligonucleotide, wherein the oligonucleotide comprises a nucleic acid sequence shown in SEQ ID NO:34.
  • the oligonucleotide further comprises one or more nucleic acid sequences selected from SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, 50, 52 and 54.
  • the present invention provides compositions comprising an oligonucleotide, wherein the oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 36, 38, and 40. h additional embodiments, the oligonucleotide further comprises one or more nucleic acid sequences selected from SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 44, 46, 48, 50, 52, and 54.
  • the present invention provides compositions comprising an oligonucleotide, wherein the oligonucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 46 and 50.
  • the oligonucleotide further comprises one or more nucleic acid sequences selected from SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 54.
  • the present invention provides compositions comprising an oligonucleotide, wherein the oligonucleotide comprises a nucleic acid sequence shown in SEQ ID NO:54.
  • the oligonucleotide further comprises one or more nucleic acid sequences selected from SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.
  • the oligonucleotide further comprises a vector sequence
  • the oligonucleotide further comprises a nucleic acid sequence encoding an antibody variable region framework.
  • the framework is fully human
  • the oligonucleotide further comprises a nucleic acid sequence encoding an antibody constant region.
  • the oligonucleotide comprises a framework region selected from SEQ ID NO: 61, 62, 63, 64, 69, 70, 71, 72, or combinations thereof, hi certain embodiments, the human germline light and/or heavy chain frameworks shown in Figure 15 are employed (i.e.
  • the present invention provides compositions comprising a T ⁇ F- ⁇ binding molecule, or an oligonucleotide encoding a T ⁇ F- ⁇ binding molecule, wherein the T ⁇ F- ⁇ binding molecule comprises: i) a CDRL3 sequence comprising SEQ ID NO: 33, and ii) a CDRH3 comprising SEQ ID NO: 53.
  • the TNF- ⁇ molecule comprises the hul Fab (e.g., an IgG with the variable regions present in the hul Fab fragment).
  • the TNF- ⁇ binding molecule further comprises iii) a CDRLl sequence comprising SEQ ID NO: 11, and iv) a CDRL2 sequence comprising SEQ ID NO:21.
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 37, and vi) a CDRH2 sequence comprising SEQ ID NO: 49.
  • the TNF- ⁇ binding molecule comprises the 2C6K Fab (e.g., an IgG with the variable regions present in the 2C6K Fab fragment ).
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 39, and vi) a CDRH2 sequence comprising SEQ ID NO: 49.
  • the TNF- ⁇ binding molecule comprises the 2C6P Fab (e.g., an IgG with the variable regions present in the 2C6P Fab fragment).
  • the TNF- ⁇ binding molecule further comprises: iii) a CDRLl sequence comprising SEQ ID NO: 11, and iv) a CDRL2 sequence comprising SEQ ID NO: 25.
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 37, and vi) a CDRH2 sequence comprising SEQ ID NO: 55.
  • the TNF- ⁇ binding molecule comprises the 2E7K Fab (e.g., an IgG with the variable regions present in the 2E7K Fab fragment).
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 39, and vi) a CDRH2 sequence comprising SEQ ID NO: 55.
  • the TNF- ⁇ binding molecule comprises the 2E7P Fab (e.g., an IgG with the variable regions present in the 2E7P Fab fragment).
  • the TNF- ⁇ binding molecule further comprises: iii) a CDRLl sequence comprising SEQ ID NO: 13, and iv) a CDRL2 sequence comprising SEQ ID NO: 27.
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 37, and vi) a CDRH2 sequence comprising SEQ ID NO: 55.
  • the TNF- ⁇ binding molecule comprises the A9K Fab (e.g., an IgG with the variable regions present in the A9K Fab fragment).
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 39, and vi) a CDRH2 sequence comprising SEQ ID NO: 55.
  • the TNF- ⁇ binding molecule comprises the A9P Fab (e.g., an IgG with the variable regions present in the A9P Fab fragment).
  • the TNF- ⁇ binding molecule further comprises: iii) a CDRLl sequence comprising SEQ ID NO: 15, and iv) a CDRL2 sequence comprising SEQ ID NO: 25.
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 37, and vi) a CDRH2 sequence comprising SEQ ID NO: 45.
  • the TNF- ⁇ binding molecule comprises the A10K Fab (e.g., an IgG with the variable regions present in the AIOK Fab fragment), hi other embodiments, the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 39, and vi) a CDRH2 sequence comprising SEQ ID NO: 45. In particular embodiments, the TNF- ⁇ binding molecule comprises the A10P Fab (e.g., an IgG with the variable regions present in the A10P Fab fragment).
  • the TNF- ⁇ binding molecule further comprises: iii) a CDRLl sequence comprising SEQ ID NO: 9, and iv) a CDRL2 sequence comprising SEQ ID NO: 19.
  • the TNF- ⁇ binding molecule further comprises: v) a CDRHl sequence comprising SEQ ID NO: 35, and vi) a CDRH2 sequence comprising SEQ ID NO:43.
  • the TNF- ⁇ binding molecule further comprises a light chain variable region, wherein the light chain variable region comprises a framework region.
  • the light chain variable region comprises light chain framework regions selected from SEQ ID NOs: 57, 58, 59, 60, and combinations thereof, h preferred embodiments, the framework region is a fully human framework, hi particularly preferred embodiments, the framework region is a germline framework (e.g. human or other animal germline framework).
  • the TNF- ⁇ binding molecule further comprises a heavy chain variable region, wherein the heavy chain variable region comprises a framework region.
  • the heavy chain variable region comprises a heavy chain framework regions selected from SEQ ID NOs: 65, 66, 67, 68, and combinations thereof, hi preferred embodiments, the framework is a fully human framework.
  • the framework region is a germline framework (e.g.
  • the present invention provides compositions comprising a light chain variable region, or a nucleic acid sequence encoding a light chain variable region, wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 1 and 5.
  • the present invention provides compositions comprising an oligonucleotide encoding a light chain variable region, wherein the oligonucleotide comprises SEQ ID NO: 2 or SEQ ID NO: 6.
  • the present invention provides compositions comprising a heavy chain variable region, or a nucleic acid sequence encoding a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 3 and 7.
  • the present invention provides compositions comprising an oligonucleotide encoding a heavy chain variable region, wherein the oligonucleotide comprises SEQ ID NO: 4 or SEQ ID NO: 8.
  • the present invention provides a computer readable medium that encodes a representation of a nucleic acid or amino acid sequence selected from SEQ ID NOs: 1-108, or the complement thereof, hi certain embodiments, the representation of these sequences, when delivered to a computer processor, may be displayed to a user (e.g., over the internet).
  • the present invention provide the complement of the CDR encoding nucleic acid sequences described herein (e.g.
  • the present invention provides nucleic acid sequences that will hybridize (under low, medium or high stringency conditions) to the CDR encoding nucleic acid sequences described herein (e.g.
  • the present invention provides the complement of the nucleic acid sequences described herein (see e.g. Tables 1-3 and 6). In some embodiments, the present invention provides sequences that hybridize under high, medium or low stringency with the nucleic acid sequences described herein (see e.g. Tables 1-3, and 6). In certain embodiments, the present invention provides compositions comprising a
  • TNF- ⁇ binding molecule that has an association rate constant (k on ) for human TNF- ⁇ of 3.0 x 10 6 M “1 s “1 or greater.
  • the association rate constant is 4.0 x 10 6 M “1 s “1 or greater (e.g., about 4.1 x 10 6 M “1 s “1 , about 5.5 x 10 6 M “1 s “ ⁇ or about 7.0 x 10 6 M “1 s "1 ).
  • the association rate constant is between about 3.0 x 10 M “ s " and about 7.5 x 10 6 M “1 s “1 (e.g., between about 3.5 x 10 6 M “1 s “1 and about 7.0 x 10 6 M “1 s “1 , or between about 4.0 x 10 6 M “1 s “1 and about 6.0 x 10 6 M “1 s “1 ).
  • the association rate constant is determined by a kinetic exclusion assay (See, e.g., Chiu et al., (2001) Anal. Chem., 73:5477-5484; Blake, et al., (1996) Journal of Biological Chemistry, 271 :27677-27685; Hongo, et al., (2000)
  • the kinetic exclusion assay is performed with a KiiiExA instrument (e.g., KinExATM3000 from Sapidyne Instruments, Boise, Idaho), or similar device.
  • KiiiExA instrument e.g., KinExATM3000 from Sapidyne Instruments, Boise, Idaho
  • the TNF- ⁇ binding molecule has a binding affinity (K d ) for human TNF- ⁇ of about 7.5 x 10 "12 M or less, hi other embodiments, the binding affinity is
  • the binding affinity is about 3.0 x 10 " M or less, hi some embodiments, the binding affinity is 2.2 x 10 "12 M or less. In yet other embodiments, the binding affinity is between about 7.2 x 10 "12 M and about 2.0 x 10 "12 M (e.g., between about 6.0 x 10 "12 M and about 3.0 x 10 "12 M, or between about 5.0 x 10 "12 M
  • the binding affinity is determined by a kinetic exclusion assay (e.g., with a KinExA instrument).
  • the TNF- ⁇ binding molecule has a dissociation rate constant (k off ) for human TNF- ⁇ of about 1.0 x 10 "4 s "1 or less, hi some embodiments, the dissociation rate constant is about 1.0 x 10 "5 s "1 or less, about 9.0 x 10 " s "1 or less, or about f ⁇ 1
  • the dissociation rate constant is between about 1.0 x 10 "4 s “1 and about 8.0 x 10 "6 s “1 (e.g., between about 1.0 x 10 "5 s “1 and about 6.0 x 10 "6 s “1 ).
  • the dissociation rate is determined by a kinetic exclusion assay.
  • the present invention provides compositions comprising a TNF- ⁇ binding molecule that neutralizes human TNF- ⁇ cytotoxicity in an in vitro, cell- based assay (see, e.g., Example 2) with an EC 50 of 1.0 x 10 "10 or less (e.g., 1.0 x 10 "10 - 1.0 x 10 " ).
  • the TNF- ⁇ binding molecule neutralizes human TNF- ⁇ cytotoxicity in an in vitro, cell-based assay with an EC 50 of 7.0 x 10 "11 or less (e.g., 7.0 x 10 "
  • the TNF- ⁇ binding molecule neutralizes human
  • TNF- ⁇ cytotoxicity in an in vitro, cell-based assay with an EC 50 of 2.0 x 10 "12 or less e.g.,
  • the TNF- ⁇ binding molecule neutralizes human TNF- ⁇ cytotoxicity in an in vitro, cell-based assay with an EC 50 of 3.0 x 10 "12 or less (e.g., 3.0 x 10 "12 - 2.0 x 10 "12 ).
  • the neutralization assay is performed by treating L929 mouse fibroblasts with the molecules of the present invention and subsequently determining an effective concentration capable of preventing TNF- ⁇ mediated cell death.
  • the TNF- ⁇ binding molecule comprises a light chain variable region and a heavy chain variable region
  • the light chain variable region comprises a fully human framework (see, e.g., Figure 5).
  • the light chain variable region comprises a germline framework (e.g. human germline framework).
  • the heavy chain variable region comprises a fully human framework (see, e.g., Figure 6).
  • the heavy chain variable region comprises a germline framework (e.g. human germline framework)
  • the human germline light and/or heavy chain frameworks shown in Figure 15 are employed (i.e. IGKNl-39 for the light chain, and IGNH3-72 for the heavy chain).
  • the T ⁇ F- ⁇ binding molecule comprises a Fab or F(ab') 2 -
  • the T ⁇ F- ⁇ binding molecule comprises a Fab, and further comprises one or more constant regions (e.g., CH2 and/or CH3, see Figure 13).
  • the T ⁇ F- ⁇ binding molecule comprises an antibody (e.g., an antibody comprising a fully human framework with synthetic CDR sequences), hi certain embodiments, 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.
  • the Fc region is an isotype selected from IgM, IgA, IgG, IgE, or other isotype.
  • the present invention provides compositions comprising a T ⁇ F- ⁇ binding molecule, or a nucleic acid sequence encoding a T ⁇ F- ⁇ binding molecule, wherein said T ⁇ F- ⁇ binding molecule comprises at least one of the following CDR sequences; i) a CDRLl sequence comprising SEQ ID ⁇ O:93; ii) a CDRL2 sequence comprising SEQ ID NO:95; iii) a CDRL3 sequence comprising SEQ ID NO: 97; iv) a
  • TNF- ⁇ binding molecule comprises at least two, or at least three, or at least four, or at least five of the CDR sequences, hi some embodiments, the TNF- ⁇ binding molecule comprises all six of the CDR sequences, hi certain embodiments, the TNF- ⁇ binding molecule comprises a heavy chain sequences as shown in SEQ ID NO:109 (See Figure 15). h other embodiments, the TNF- ⁇ binding molecule comprises a light chain sequences as shown in SEQ ID NO: 110 (See Figure 15). hi preferred embodiments, the TNF- ⁇ binding molecule is AME 3-2 and comprises both SEQ ID NO: 109 (heavy chain) and SEQ ID NO: 110 (light chain).
  • amino acid modification(s) are introduced into the CH2 domain of an Fc region of a TNF- ⁇ binding molecule.
  • Useful amino acid positions for modification in order to generate a variant IgG Fc region with altered Fc gamma receptor (Fc ⁇ R) binding affinity or activity include any one or more of the following amino acid positions: 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300 301, 303, 305, 307, 309, 331,333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region of a TNF- ⁇ binding molecule.
  • the parent Fc region used as the template to generate such variants comprises a human IgG Fc region.
  • Fc region variants with improved binding to one or more Fc ⁇ Rs may also be made.
  • Such Fc region variants may comprise an amino acid modification at any one or more of the following amino acid positions: 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398 or 430 of the Fc region of a TNF- ⁇ binding molecule.
  • the present invention provides TNF- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant binds an Fc ⁇ R with higher affinity than said parent polypeptide, and/or interacts with an Fc ⁇ R with a higher assay signal, and/or mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of effector cells, and comprises at least one amino acid modification at position 300 in the Fc region, h certain embodiments, the amino acid modification is Y300I. In other embodiments, the amino acid modification is Y300L.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present invention provides TNF- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant binds an Fc gamma receptor El (Fc ⁇ RHI) with higher affinity, or the variant interacts with an Fc ⁇ RUI with a higher assay signal, than the parent polypeptide, and/or mediates antibody- dependent cell-mediated cytotoxicity (ADCC) in the presence of effector cells, and comprises at least one amino acid modification at position 295 in the Fc region, ha certain embodiments, the amino acid modification is Q295K or Q295L.
  • Fc ⁇ RHI Fc gamma receptor El
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • the present invention provides TNF- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant binds an Fc ⁇ RIH with higher affinity, or the variant interacts with an Fc ⁇ Ri ⁇ with a higher assay signal, than the parent polypeptide, and/or mediates antibody-dependent cell-mediated cytotoxicity (ADCC) in the presence of effector cells, and comprises at least one amino acid modification at position 294 in the Fc region, hi certain embodiments, the amino acid modification is E294N.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present invention provides TNF- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant has a binding affinity, or assay signal, for Fc ⁇ R ⁇ or Fc ⁇ RDb that is approximately 0.25 or less as measured in an ELISA Fc ⁇ R binding assay, hi certain embodiments, the variant comprises at least one amino acid modification at position 296 in the Fc region, hi particular embodiments, the amino acid modification at position 296 is Y296P. h other embodiments, the variant comprises at least one amino acid modification at position 298 in the Fc region. In some embodiments, the amino acid modification at position 298 is S298P.
  • the present invention provides TNF- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant binds an Fc ⁇ RDI with greater affinity, and Fc ⁇ RIIb with less affinity, than the parent polypeptide and the variant comprises a S298N amino acid modification in the Fc region, h some embodiments, the present invention provides compositions comprising a variant of a parent polypeptide having an Fc region, wherein the variant interacts with an Fc ⁇ Ri ⁇ with a higher assay signal, and Fc ⁇ RIIb with lower assay signal, than the parent polypeptide and the variant comprises a S298N amino acid modification in the Fc region.
  • the present invention provides TNF- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant binds an Fc ⁇ RlH with greater affinity, and Fc ⁇ RIIb with less affinity, than the parent polypeptide and the variant comprises a S298N amino acid modification in the Fc region
  • the present invention provides compositions comprising a variant of a parent polypeptide having an Fc region, wherein the variant interacts with an Fc ⁇ Ri ⁇ with a higher assay signal, and Fc ⁇ RIIb with a lower assay signal, than the parent polypeptide and the variant comprises a S298N amino acid modification in the Fc region.
  • the present invention provides T ⁇ F- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant binds an Fc ⁇ Ri ⁇ with greater affinity, and Fc ⁇ RIIb with less affinity, than the parent polypeptide and the variant comprises a S298D amino acid modification in the Fc region.
  • the present invention provides compositions comprising a variant of a parent polypeptide having an Fc region, wherein the variant interacts with an Fc ⁇ RIH with a higher assay signal, and Fc ⁇ RIIb with a lower assay signal, than the parent polypeptide and the variant comprises a S298D amino acid modification in the Fc region.
  • the present invention provides T ⁇ F- ⁇ binding molecules comprising a variant of a parent polypeptide having an Fc region, wherein the variant has a binding affinity, or assay signal, for Fc ⁇ R ⁇ or Fc ⁇ RIIb that is approximately 0.25 or less as measured in an ELISA Fc ⁇ R binding assay, and wherein the variant comprises at least one amino acid modification at position 298 of the Fc region, h particular embodiments, the amino acid modification at position 298 is S298P.
  • polypeptide variants described above may be subjected to further modifications, depending on the desired or intended use of the polypeptide.
  • modifications may involve, for example, further alteration of the amino acid sequence (substitution, insertion and/or deletion of amino acid residues), fusion to heterologous polypeptide(s) and/or covalent modifications.
  • further modifications may be made prior to, simultaneously with, or following, the amino acid modification(s) disclosed above which result in an alteration of Fc receptor binding and/or ADCC activity.
  • the starting polypeptide of particular interest may be one that binds to Clq and displays complement dependent cytotoxicity (CDC).
  • Amino acid substitutions described herein may serve to alter the ability of the starting polypeptide to bind to Clq and/or modify its complement dependent cytotoxicity function (e.g., to reduce and preferably abolish these effector functions).
  • polypeptides comprising substitutions at one or more of the described positions with improved Clq binding and/or complement dependent cytotoxicity (CDC) function are contemplated herein.
  • the starting polypeptide may be unable to bind Clq and/or mediate CDC and may be modified according to the teachings herein such that it acquires these further effector functions.
  • 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 WO0042072, which is hereby incorporated by reference.
  • Fc mutations can also be introduced in the TNF- ⁇ binding molecules of the present invention to alter their interaction with the neonatal Fc receptor (FcRn) and improve their pharmacokinetic properties.
  • FcRn neonatal Fc receptor
  • Another type of amino acid substitution serves to alter the glycosylation pattern of the Fc region of a TNF- ⁇ binding molecule. This may be achieved, for example, by deleting one or more glycosylation site(s) found in the polypeptide, and/or adding one or more glycosylation sites that are not present in the polypeptide.
  • Glycosylation of an Fc region is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the peptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation 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 sites to the Fc region of a TNF- ⁇ binding molecule is conveniently accomplished by altering the amino acid sequence such that it contains 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 Asn 297 of the heavy chain. The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original polypeptide (for O-linked glycosylation sites).
  • the present invention provides methods of treating a TNF- ⁇ mediated disease comprising the administration of the TNF- ⁇ binding molecules of the present invention to a subject (e.g., a human).
  • a subject e.g., a human
  • the administration is under conditions such that the symptoms of the TNF- ⁇ mediated disease are reduced or eliminated.
  • the TNF- ⁇ mediated disease is selected from sepsis, an autoimmune disease, rheumatoid arthritis, allergy, multiple sclerosis, autoimmune uveitis, nephrotic syndrome, an infectious disease, a malignancy, transplant rejection, graft- versus-host disease, systemic lupus erythematosus, thyroidosis, scleroderma, diabetes mellitus, Graves' disease, a pulmonary disorder, a bone disorder, an intestinal disorder, a cardiac disorder, cachexia, circulatory collapse, shock resulting from acute or chronic bacterial infections, acute and chronic parasitic and/or infectious diseases, chronic inflammatory pathologies, vascular inflammatory pathologies, sarcoidosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, disseminated intravascular coagulation, atherosclerosis, Kawasaki's pathology, neurodegenerative diseases, demyelinating diseases, multiple sclerosis, acute transverse myelitis,
  • the TNF- ⁇ mediated disease is selected from the group consisting of juvenile and adult rheumatoid
  • the TNF- ⁇ binding molecule is administered in combination with an immunosuppressant such as methotrexate.
  • an immunosuppressant such as methotrexate.
  • the TNF- ⁇ binding molecule is administered intravenously.
  • kits comprising: a) a TNF- ⁇ binding molecule of the present invention; and b) instructions for using the TNF- ⁇ binding molecule to treat a disease in a subject or instructions for employing the TNF- ⁇ binding molecule for scientific research or diagnostic purposes (e.g., for performing ELISA assays, etc.).
  • the present invention provides cell lines stably or transiently transfected with nucleic acid sequences encoding the TNF- ⁇ binding molecules of the present invention.
  • Figure 1 A shows the amino acid sequence of the light chain variable region of hul (SEQ ID NO: 1)
  • Figure IB shows the nucleic acid sequence of the light chain variable region of hul (SEQ ID NO: 2).
  • Figure 2A shows the amino acid sequence of the heavy chain variable region of hul (SEQ ID NO: 3), and Figure 2B shows the nucleic acid sequence of the heavy chain variable region of hul (SEQ ID NO: 4).
  • Figure 3 A shows the amino acid sequence of the light chain variable region of AIOK (SEQ ID NO: 5)
  • Figure 3B shows the nucleic acid sequence of the light chain variable region of AIOK (SEQ ID NO: 6).
  • Figure 4A shows the amino acid sequence of the heavy chain variable region of AIOK (SEQ ID NO: 7), and Figure 4B shows the nucleic acid sequence of the heavy chain variable region of AIOK (SEQ ID NO: 8).
  • Figure 5 A shows the amino acid sequence of a fully human light chain framework region with interspersed CDRs. The four framework sub-regions are labeled as follows: FRLl (SEQ ID NO: 57), FRL2 (SEQ ID NO: 58), FRL3 (SEQ ID NO: 59), and FRL4 (SEQ ID NO: 60).
  • Figure 5B shows the nucleic acid sequence of a fully human light chain framework region with interspersed CDRs. The four framework sub-regions are labeled as follows: FRLl (SEQ ID NO: 61), FRL2 (SEQ ID NO: 62), FRL3 (SEQ ID NO: 63), and FRL4 (SEQ ID NO: 64).
  • Figure 6 A shows the amino acid sequence of a fully human heavy chain framework region with interspersed CDRs.
  • the four framework sub-regions are labeled as follows:
  • FRH1 (SEQ ID NO: 65), FRH2 (SEQ ID NO: 66), FRH3 (SEQ ID NO: 67), and FRH4 (SEQ ID NO: 68).
  • Figure 6B shows the nucleic acid sequence of a fully human heavy chain framework region with interspersed CDRs. The four framework sub-regions are labeled as follows: FRH1 (SEQ ID NO: 69), FRH2 (SEQ ID NO: 70), FRH3 (SEQ ID NO: 71), and FRH4 (SEQ ID NO: 72).
  • Figure 7 shows the results of an ELISA described in Example 1.
  • Figure 8 shows the results of an L929 cell protection assay described in Example 2.
  • Figure 9 shows the results of an L929 cell protection assay described in Example 2.
  • Figure 10 shows the results of an L929 cell protection assay described in Example 2.
  • Figure 11 shows the results of an in vivo TNF- ⁇ neutralizing assay described in Example 3.
  • Figure 12 shows the effect of a TNF- ⁇ binding molecule in reducing progression of TNF- ⁇ mediated polyarthritis in mice, as described in Example 3.
  • Figure 13 shows a schematic representation of an IgG molecule with the various regions and sections labeled.
  • the CDRs and framework regions (FR) of one of the two variable region light chains, and one of the two variable region heavy chains, are also labeled.
  • Figure 14 shows the nucleic acid sequence of a human CL sequence, and a human CHI sequence, that were used in the construction of the Fab fragments described in Example 1.
  • Figure 15 shows the complete light and heavy chain amino acid sequences for AME
  • antibody is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCNR or NH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3 (see
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or NL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL (see Figure 13).
  • the NH and NL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each variable region (NH or NL) contains 3 CDRs, designated CDR1, CDR2 and CDR3 (see Figures 5, 6, and 13).
  • Each variable region also contains 4 framework sub-regions, designated FR1, FR2, FR3 and FR4 (see Figures 5, 6, and 13).
  • antibody fragments refers to a portion of an intact antibody.
  • antibody fragments include, but are not limited to, linear antibodies, single-chain antibody molecules, Fab and F(ab') 2 fragments, and multispecific antibodies formed from antibody fragments.
  • the antibody fragments preferably retain at least part of the heavy and/or light chain variable region.
  • complementarity determining region and “CDR” refer to the regions that are primarily responsible for antigen-binding. There are three CDRs in a light chain variable region (CDRLl, CDRL2, and CDRL3), and three CDRs in a heavy chain variable region (CDRHl, CDRH2, and CDRH3).
  • residues that make up these six CDRs have been characterized by Kabat and Chothia as follows: residues 24-34 (CDRLl), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable region and 31-35 (CDRHl), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable region; Kabat et al., (1991) Sequences of Proteins of hnmunological Interest, 5th Ed.
  • CDR complementarity determining region
  • CDRLl residues 24-34
  • CDRL2 residues 24-56
  • CDRL3 residues 89-97
  • CDRL3 residues 89-97
  • 26-35 residues 50-65
  • CDRH3 residues 95-102
  • framework refers to the residues of the variable region other than the CDR residues as defined herein.
  • framework sub- regions that make up the framework: FR1, FR2, FR3, and FR4 (See Figures 5, 6, and 13).
  • L or “H” maybe added to the sub-region abbreviation (e.g., "FRLl” indicates framework sub-region 1 of the light chain variable region).
  • the numbering of framework residues is according to Kabat.
  • the term “fully human framework” means a framework with an amino acid sequence found naturally in humans.
  • Fully human frameworks include, but are not limited to, KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences of Proteins of hnmunological Interest, US Department of Health and Human Services, NIH, USA; and Wu et al, (1970) J. Exp. Med. 132, 211- 250, both of which are herein inco ⁇ orated by reference).
  • the terms “subject” and “patient” refer to any animal, such as a mammal like a dog, cat, bird, livestock, and preferably a human (e.g., a human with a TNF- ⁇ mediated disease).
  • the terms “nucleic acid sequence encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides dete ⁇ nines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
  • DNA molecules are said to have "5' ends” and "3' ends” because mononucleotides are reacted to make oligonucleotides or polynucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage.
  • an end of an oligonucleotide or polynucleotide is referred to as the "5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring and as the "3' end” if its 3' oxygen is not linked to a 5' phosphate of a subsequent mononucleotide pentose ring.
  • a nucleic acid sequence even if internal to a larger oligonucleotide or polynucleotide, also may be said to have 5' and 3' ends, hi either a linear or circular DNA molecule, discrete elements are referred to as being "upstream” or 5' of the "downstream” or 3' elements.
  • This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA strand.
  • the promoter and enhancer elements that direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.
  • the term “codon” or “triplet” refers to a group of three adjacent nucleotide monomers which specify one of the naturally occurring amino acids found in polypeptides. The term also includes codons which do not specify any amino acid.
  • an oligonucleotide having a nucleotide sequence encoding a polypeptide means a nucleic acid sequence comprising the coding region of a particular polypeptide.
  • the coding region may be present in a cDNA, genomic DNA, or RNA form.
  • the oligonucleotide or polynucleotide maybe single-stranded (i.e., the sense strand) or double- stranded.
  • Suitable control elements such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript.
  • the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.
  • oligonucleotide and “polynucleotide.” Both terms simply refer to molecules composed of nucleotides. Likewise, there is no size distinction between the terms “peptide” and “polypeptide.” Both terms simply refer to molecules composed of amino acid residues.
  • peptide and “polypeptide.” Both terms simply refer to molecules composed of amino acid residues.
  • complementary or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “5'-A-G-T 3”, is complementary to the sequence "3-T-C- A-5'”.
  • Complementarity may be "partial", in which only some of the nucleic acids' bases are matched according to the base pairing rules, or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization. This is of particular importance in amplification reactions, as well as in detection methods that depend upon binding between nucleic acids.
  • the term "the complement of a given sequence is used in reference to the sequence that is completely complementary to the sequence over its entire length. For example, the sequence 5'-A-G-T-A-3' is "the complement" of the sequence 3'-T-C-A-T- 5'.
  • the present invention also provides the complement of the sequences described herein (e.g., the complement of the nucleic acid sequences in SEQ ID NOs: 1-84).
  • the term "homology" when in reference to nucleic acid sequences) refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity).
  • a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid and is referred to using the functional term "substantially homologous”.
  • inhibittion of binding when used in reference to nucleic acid binding, refers to inhibition of binding caused by competition of homologous sequences for binding to a target sequence.
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
  • a partial degree of complementarity e.g., less than about 30% identity
  • low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and the length and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions, hi addition, the art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
  • high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the T m of the formed hybrid, and the G:C ratio within the nucleic acids.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. Those skilled in the art will recognize that “stringency” conditions may be altered by varying the parameters just described either individually or in concert. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences (e.g., hybridization under "high stringency” conditions may occur between homologs with about 85-100%) identity, preferably about 70-100% identity).
  • nucleic acid base pairing will occur between nucleic acids with an intermediate frequency of complementary base sequences (e.g., hybridization under "medium stringency” conditions may occur between homologs with about 50-70% identity).
  • intermediate stringency e.g., hybridization under "medium stringency” conditions may occur between homologs with about 50-70% identity.
  • conditions of "weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less.
  • High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA, followed by washing in a solution comprising 0. IX SSPE, 1.0% SDS at 42 C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent 50X Denhard
  • “Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE, 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA, followed by washing in a solution comprising 1.0X SSPE, 1.0%) SDS at 42 C when a probe of about 500 nucleotides in length is employed.
  • Low stringency conditions comprise conditions equivalent to binding or hybridization at 42 C in a solution consisting of 5X SSPE, 0.1% SDS, 5X Denhardt's reagent and 100 g/ml denatured salmon spe ⁇ n DNA, followed by washing in a solution comprising 5X SSPE, 0.1 % SDS at 42 C when a probe of about 500 nucleotides in length is employed.
  • PCR polymerase chain reaction
  • the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • PCR polymerase chain reaction
  • PCR With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; inco ⁇ oration of biotinylated primers followed by avidin-enzyme conjugate detection; inco ⁇ oration of 32p-l a beled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
  • any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process are, themselves, efficient templates for subsequent PCR amplifications.
  • isolated when used in relation to a nucleic acid, as in "an isolated oligonucleotide” or “isolated polynucleotide” or “isolated nucleic acid sequence encoding a TNF- ⁇ binding molecule” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is present in a form or setting that is different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.
  • an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the polypeptide where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • the isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single-stranded or double-stranded form.
  • oligonucleotide or polynucleotide When an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to express a protein, the oligonucleotide or polynucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide or polynucleotide maybe single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide maybe double- stranded).
  • portion when used in reference to a nucleotide sequence (as in “a portion of a given nucleotide sequence”) refers to fragments of that sequence.
  • the fragments may range in size from ten nucleotides to the entire nucleotide sequence minus one nucleotide (e.g., 10 nucleotides, 20, 30, 40, 50, 100, 200, etc.).
  • portion when in reference to an amino acid sequence (as in “a portion of a given amino acid sequence”) refers to fragments of that sequence.
  • the fragments may range in size from six amino acids to the entire amino acid sequence minus one amino acid (e.g., 6 amino acids, 10, 20, 30, 40, 75, 200, etc.).
  • the term "purified” or “to purify” refers to the removal of contaminants from a sample.
  • TNF- ⁇ specific antibodies may be purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of immunoglobulins that do not bind to the same antigen.
  • the removal of non- immunoglobulin proteins and/or the removal of immunoglobulins that do not bind the particular antigen results in an increase in the percentage of antigen specific immunoglobulins in the sample.
  • recombinant antigen-specific polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percentage of recombinant antigen-specific polypeptides is thereby increased in the sample.
  • recombinant DNA molecule refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques.
  • recombinant protein or “recombinant polypeptide” as used herein refers to a protein molecule that is expressed from a recombinant DNA molecule.
  • vector is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another.
  • vehicle is sometimes used interchangeably with “vector.”
  • expression vector refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • host cell refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells such as PER.C6TM (Crucell, The Netherlands) and CHO cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
  • bacterial cells such as E. coli, yeast cells, mammalian cells such as PER.C6TM (Crucell, The Netherlands
  • CHO cells avian cells, amphibian cells, plant cells, fish cells, and insect cells
  • host cells may be located in a transgenic animal.
  • transfection and “transformation” as used herein refer to the introduction of foreign DNA into cells (e.g., eukaryotic and prokaryotic cells). Transfection may be accomplished by a variety of means known to the art including calcium phosphate- DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retro viral infection, and biolistics.
  • stable transfection or "stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell.
  • stable transfectant refers to a cell that has stably integrated foreign DNA into the genomic DNA.
  • transient transfection or “transiently transfected” refers to the introduction of foreign DNA into a cell where the foreign DNA fails to integrate into the genome of the transfected cell.
  • the foreign DNA persists in the nucleus of the transfected cell for several days. During this time, the foreign DNA is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes.
  • transient transfectant refers to cells that have taken up foreign DNA but have failed to integrate this DNA.
  • computer memory and “computer memory device” refer to any storage media readable by a computer processor.
  • Examples of computer memory include, but are not limited to, RAM, ROM, computer chips, digital video discs (DVDs), compact discs (CDs), hard disk drives (HDD), and magnetic tapes.
  • computer readable medium refers to any device or system for storing and providing information (e.g., data and instructions) to a computer processor.
  • Examples of computer readable media include, but are not limited to, DVDs, CDs, hard disk drives, magnetic tapes and servers for streaming media over networks.
  • computer readable medium encodes a representation of a nucleic acid or amino acid sequence
  • processor and "central processing unit” or “CPU” are used interchangeably and refer to a device that is able to read a program from a computer memory (e.g., ROM or other computer memory) and perform a set of steps according to the program.
  • a computer memory e.g., ROM or other computer memory
  • Fc region refers to a C-terminal region of an immunoglobulin heavy chain (e.g., as shown in Figure 13).
  • the "Fc region” may be a native sequence Fc region or a variant Fc region (e.g., with increased or decreased effector functions).
  • an Fc region may possess "effector functions" that are responsible for activating or diminishing a biological activity (e.g., in a subject).
  • effector functions include, 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.
  • Such 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.).
  • an "isolated" peptide, polypeptide, or protein is one that has been identified and separated and/or recovered from a component of its natural environment.
  • Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes, hi certain embodiments, the isolated polypeptide is purified (1) to greater than 95% by weight of polypeptides as determined by the Lowry method, and 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 under reducing or nonreducing conditions using Coomassie blue, or silver stain.
  • An isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, an isolated polypeptide will be prepared by a least one purification step.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those aheady with the disorder as well as those in which the disorder is to be prevented.
  • nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation, hi preferred embodiments, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers, for example, do not have to be contiguous. Linking may be accomplished, for example, by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.
  • the phrase "under conditions such that the symptoms are reduced” refers to any degree of qualitative or quantitative reduction in detectable symptoms of any TNF- ⁇ mediated disease, including but not limited to, a detectable impact on the rate of recovery from disease (e.g., rate of weight gain), or the reduction of at least one of the symptoms normally associated with the particular disease (e.g., if the TNF- ⁇ mediated disease were Crohn's disease, a reduction in at least one of the following symptoms: abdominal pain, diarrhea, rectal bleeding, weight loss, fever, loss of appetite, dehydration, anemia, distention, fibrosis, inflamed intestines and malnutrition).
  • human TNF- ⁇ (abbreviated herein as hTNF- ⁇ , or simply hTNF), as used herein, is intended to refer to a human cytokine that exists as a 17 kDa secreted form and a 26 kDa membrane associated form, the biologically active form of which is composed of a trimer of noncovalently bound 17 kDa molecules.
  • hTNF- ⁇ The structure of hTNF- ⁇ is described further in, for example, Jones, et al. (1989) Nature, 338:225-228.
  • human TNF- ⁇ is intended to include recombinant human TNF- ⁇ (rhTNF- ⁇ ), which can be prepared by standard recombinant expression methods or purchased commercially.
  • affinity refers to the equilibrium dissociation constant (expressed in units of concentration) associated with each TNF- ⁇ binding molecule — TNF- ⁇ protein complex.
  • the binding affinity is directly related to the ratio of the off-rate constant (generally reported in units of inverse time, e.g., seconds "1 ) to the on-rate constant (generally reported in units of concentration per unit time, e.g., molar/second).
  • the binding affinity may be determined by, for example, a kinetic exclusion assay or surface plasmon resonance.
  • dissociation dissociation rate
  • k 0ff as used herein, are intended to refer to the off rate constant for dissociation of a TNF- ⁇ binding molecule from the antibody/antigen complex.
  • association means association of a TNF- ⁇ binding molecule with an antigen to form an antibody/antigen complex.
  • EC 50 as used herein, are intended to refer to the concentration of a TNF- ⁇ binding molecule capable of interacting with, and neutralizing sufficient quantities of TNF- ⁇ molecules to produce an effect on approximately 50%) of the treated cells.
  • the EC 50 is the concentration necessary to protect approximately 50% of the cells from TNF- ⁇ mediated cytotoxicity. h these assays, the concentration of TNF- ⁇ used is empirically determined in order to exert a cytotoxic effect on > than 90%> of the cell population.
  • effective (EC 50 ) and inhibitory (IC 50 ) concentration are examples of concentration of TNF- ⁇ binding molecule capable of interacting with, and neutralizing sufficient quantities of TNF- ⁇ molecules to produce an effect on approximately 50%) of the treated cells.
  • the concentration of TNF- ⁇ used is empirically determined in order to exert a cytotoxic effect on > than 90%> of the cell population.
  • IC 50 inhibitory
  • the present invention provides TNF- ⁇ binding molecules and nucleic acid sequences encoding TNF- ⁇ binding molecules.
  • the present invention provides TNF- ⁇ binding molecules with a high binding affinity, a high association rate, a low dissociation rate with regard to human TNF- ⁇ and a low EC 50 value (e.g., in an L929 cell assay).
  • the TNF- ⁇ binding molecules of the present invention comprise light and/or heavy chain variable regions with fully human frameworks.
  • the TNF- ⁇ binding molecules of the present invention comprise light and/or heavy chain variable regions with human germline frameworks.
  • the description of the invention is divided into the following sections below for convenience: I. TNF- ⁇ Binding Molecules; ⁇ . Generating TNF- ⁇ Binding Molecules; III. Therapeutic Formulations and Uses; and IV. Additional TNF- ⁇ Binding Molecule Uses.
  • the present invention provides TNF- ⁇ binding molecules with desirable characteristics, hi particular, in some embodiments, the TNF- ⁇ binding molecules have a high binding affinity (K d ) with regard to human TNF- ⁇ . In other embodiments, the TNF- ⁇ binding molecules have a high association rate constant (k on ) with regard to human TNF- ⁇ . In certain embodiments, the TNF- ⁇ binding molecules have a low dissociation rate (k 0ff ) with regard to human TNF- ⁇ .
  • the TNF- ⁇ binding molecules have a low effective concentration (EC 50 ) in cell-based assays
  • the TNF- ⁇ binding molecules of the present invention have a high binding affinity, a high association rate, a low dissociation rate and are effective at a low concentrations. While not necessary to practice or understand the invention, it is believed that the TNF- ⁇ binding molecules of the present invention, with high binding affinity, a high association rate, a low dissociation rate and a low EC 50 , are particularly well suited for therapeutic use in humans (e.g., for treating TNF- ⁇ mediated diseases).
  • the TNF- ⁇ binding molecules of the present invention do not bind murine TNF- ⁇ . In other embodiments, the TNF- ⁇ binding molecules of the present invention do not bind rat, pig, or rhesus macaque TNF- ⁇ .
  • the TNF- ⁇ binding molecules of the present invention comprise a light and/or heavy chain variable region, preferably having a fully human framework. In particularly preferred embodiments, the TNF- ⁇ binding molecules of the present invention comprise a light and/or heavy chain variable region, preferably having a human germline framework. While not necessary to practice or understand the invention, it is believed that the TNF- ⁇ binding molecules of the present invention (see, e.g. the Examples below) will illicit very little or no immunogenic response when administered to a human (e.g. to treat a disease).
  • the present invention provides numerous CDRs useful for generating TNF- ⁇ binding molecules.
  • one or more of the CDRs shown can be combined with a framework sub-region (e.g., a fully human FR1, FR2, FR3, or FR4) in order to generate a TNF- ⁇ binding peptide, or a nucleic acid sequence encoding a TNF- ⁇ binding peptide.
  • the CDRs shown in the Tables below may be combined, for example, such that three CDRs are present in a light chain variable region, and/or three CDRs are present in a heavy chain variable region.
  • the CDRs shown below may be inserted into a human framework (by recombinant techniques) into the light and heavy chain frameworks shown in Figures 5 and 6 in order to generate TNF- ⁇ binding molecules or nucleic acid sequences encoding TNF- ⁇ binding molecules.
  • the CDRLl shown in Figure 5A could be replaced by SEQ ID NO: 9, 11, 13, 15, or 17 as shown in Table 1.
  • the CDRLl shown in Figure 5B could be replaced by SEQ ID NO: 10, 12, 14, 16, or 18 and shown in Table 1. This same procedure may be used with all of the CDRs shown in Tables 1-3 and 6. These three tables are show immediately below.
  • CDRs include Kabat and Chothia residues. ** Changes from hul are designated by the hul amino acid, followed by the position, and then the new amino acid (e.g., S3 IL is a change from S at position 31 to L).
  • ***"Xaa” indicates this position can be any amino acid, and "n” indicates this position can be any nucleotide.
  • CDRs include both Kabat and Chothia residues. ** Changes from hul are designated by the hul amino acid, followed by the position, and then the new amino acid (e.g., T28K is a change from T at position 28 to K).
  • ***"Xaa” indicates this position can be any amino acid, and "n” indicates this position can be any nucleotide.
  • CDRs include both Kabat and Chothia residues.
  • ***"Xaa” indicates this position can be any amino acid, and "n” indicates this position can be any nucleotide.
  • CDRs include both Kabat and Chothia residues.
  • the present invention also provides sequences that are substantially the same as the CDR sequences (both amino acid and nucleic acid) shown in the above Tables
  • one or two amino acid may be changed in the sequences shown in the Tables.
  • a number of nucleotide bases maybe changed in the sequences shown in the Tables.
  • Changes to the amino acid sequence may be generated by changing the nucleic acid sequence encoding the amino acid sequence.
  • a nucleic acid encoding a variant of a given CDR may be prepared by methods known in the art using the guidance of the present specification for particular sequences.
  • Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (see, e.g., Carter et al., (1985) Nucleic Acids Res. 13: 4431-4443 and Kunkel et. al., (1987) Proc. Natl. Acad. Sci. USA 82: 488-492, both of which are hereby inco ⁇ orated by reference).
  • the starting DNA is altered by first hybridizing an oligonucleotide encoding the desired mutation to a single strand of such starting DNA. After hybridization, a DNA polymerase is used to synthesize an entire second strand, using the hybridized oligonucleotide as a primer, and using the single strand of the starting DNA as a template. Thus, the oligonucleotide encoding the desired mutation is inco ⁇ orated in the resulting double-stranded DNA.
  • PCR mutagenesis is also suitable for making amino acid sequence variants of the starting CDR (see, e.g., Nallette et. al, (1989) Nucleic Acids Res. 17: 723-733, hereby inco ⁇ orated by reference).
  • primers that differ slightly in sequence from the corresponding region in a template DNA can be used to generate relatively large quantities of a specific DNA fragment that differs from the template sequence only at the positions where the primers differ from the template.
  • the starting material is the plasmid (or other vector) comprising the starting CDR DNA to be mutated.
  • the codon(s) in the starting DNA to be mutated are identified.
  • the plasmid DNA is cut at these sites to linearize it.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures, wherein the two strands of the oligonucleotide are synthesized separately and then hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 5' and 3' ends that are compatible with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • This plasmid now contains the mutated DNA sequence.
  • the desired amino acid sequence encoding a polypeptide variant can be determined, and a nucleic acid sequence encoding such amino acid sequence variant can be generated synthetically.
  • Conservative modifications in the amino acid sequences of the CDRs may also be made.
  • Naturally occurring residues are divided into classes based on common side-chain properties:
  • hydrophobic norleucine, met, ala, val, leu, ile
  • the present invention also provides the complement of the nucleic acid sequences shown in Tables 1-3 and 6, as well as nucleic acid sequences that will hybridize to these nucleic acid sequences under low, medium, and high stringency conditions.
  • the CDRs of the present invention may be employed with any type of framework.
  • the CDRs are used with fully human frameworks, or framework sub-regions.
  • the frameworks are human germline sequences.
  • Figures 5 and 6 Another example is shown in Figure 15.
  • Figure 15 Another example is shown in Figure 15.
  • Other fully human frameworks or framework sub-regions may also be employed.
  • the NCBI web site contains the sequences for the currently known human framework regions.
  • human NH sequences include, but are not limited to, VH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8, VH2- 26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, NH3-48, NH3-49, NH3-53, VH3-64, NH3- 66, NH3-7, NH3-72, NH3-73, VH3-74, VH3-9, VH4-28, VH4-31, VH4-34, VH4-39, VH4- 4, VH4-59, NH4-61, NH5-51, NH6-1, and NH7-81, which are provided in Matsuda et al., V
  • human NK sequences include, but are not limited to, Al, A10, Al 1, A14, A17, A18, A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, LI, L10, Lll, L12, L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4/18a, L5, L6, L8, L9, Ol, Oil, 012, O14, 018, 02, 04, and 08, which are provided in Kawasaki et al., (2001) Eur.
  • human NL sequences include, but are not limited to, Nl-11, Nl-13, Nl-16, Nl-17, Nl-18, Nl-19, Nl-2, Nl-20, Nl-22, Nl-3, Nl-4, Nl-5, Vl-7, Nl-9, N2-1, N2-11, N2-13, N2-14, N2-15, V2-17, N2-19, N2-6, N2-7, N2-8, N3-2, N3-3, N3-4, N4-1, N4-2, N4-3, N4-4, N4-6, N5-1, N5-2, N5-4, and N5-6, which are provided in Kawasaki et al., (1997) Genome Res. 7:250-261, herein inco ⁇ orated by reference.
  • Fully human frameworks can be selected from any of these functional germline genes. Generally, these frameworks differ from each other by a limited number of amino acid changes. These frameworks may be used with the CDRs described herein. For example, L6, a frequently used gene among the Nk3 sub-family could be selected as an alternate framework for the light chain of the T ⁇ F- ⁇ binding molecules of the present invention.
  • human frameworks which may be used with the CDRs of the present invention include, but are not limited to, KOL, ⁇ EWM, REI, EU, TUR, TEI, LAY and POM (See, e.g., Kabat et al., (1991) Sequences of Proteins of hnmunological Interest, US Department of Health and Human Services, NH, USA; and Wu et al., (1970), J. Exp. Med. 132:211-250, both of which are herein inco ⁇ orated by reference).
  • genes encoding the variable regions can be selected from a collection of commonly occurring, functional germline genes, and genes encoding NH and NL regions can be further selected to match known associations between specific heavy and light chains of immunoglobulin molecules.
  • the T ⁇ F- ⁇ binding molecules of the present invention comprise antibodies or antibody fragments (e.g., comprising one or more of the CDRs described herein).
  • An antibody, or antibody fragment, of the present invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
  • a host cell may be transfected with one or more recombinant expression vectors carrying D ⁇ A fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, secreted into the medium in which the host cell is cultured, from which medium the antibody can be recovered.
  • Standard recombinant D ⁇ A methodologies may be used to obtain antibody heavy and light chain genes, inco ⁇ orate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, ⁇ .Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al., all of which are herein inco ⁇ orated by reference.
  • DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of germline light and heavy chain variable sequences using the polymerase chain reaction (PCR). Germline DNA sequences for human heavy and light chain variable region genes are known in the art (see above).
  • these sequences can be mutated to encode one or more of the CDR amino acid sequences disclosed herein (see, e.g., Tables 1-3 and 6).
  • the amino acid sequences encoded by the germline NH and NL D ⁇ A sequences may be compared to the CDRs sequence(s) desired to identify amino acid residues that differ from the germline sequences.
  • the appropriate nucleotides of the germline D ⁇ A sequences are mutated such that the mutated germline sequence encodes the selected CDRs (e.g., the six CDRs that are selected from Tables 1-3 and 6), using the genetic code to determine which nucleotide changes should be made.
  • Mutagenesis of the germline sequences may be carried out by standard methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are inco ⁇ orated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis.
  • the variable region is synthesized de novo (e.g., using a nucleic acid synthesizer).
  • D ⁇ A fragments encoding the desired NH and NL segments are obtained (e.g., by amplification and mutagenesis of germline NH and NL genes, or synthetic synthesis, as described above), these D ⁇ A fragments can be further manipulated by standard recombinant D ⁇ A techniques, for example to convert the variable region genes to full- length antibody chain genes, to Fab fragment genes or to a scFv gene, hi these manipulations, a NL- or NH-encoding D ⁇ A fragment is operably linked to another D ⁇ A fragment encoding another polypeptide, such as an antibody constant region or a flexible linker.
  • the isolated D ⁇ A encoding the NH region can be converted to a full-length heavy chain gene by operably linking the NH-encoding D ⁇ A to another D ⁇ A molecule encoding heavy chain constant regions (CHI, CH2 and CH3).
  • CHI heavy chain constant regions
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E. A., et al., (1991) Sequences of Proteins of hnmunological Interest, Fifth Edition, U.S. Department of Health and Human Services, ⁇ Q ⁇ Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be, for example, an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGl or IgG4 constant region.
  • the NH-encoding D ⁇ A can be operably linked to another D ⁇ A molecule encoding only the heavy chain CHI constant region.
  • the isolated D ⁇ A encoding the NL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the NL-encoding D ⁇ A to another D ⁇ A molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E. A., et al., (1991) Sequences of Proteins of immunological Interest, Fifth Edition, U.S. Department of Health and Human Services. ⁇ 1H Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but most preferably is a kappa constant region.
  • the NH- and NL-encoding D ⁇ A fragments may be operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly 4 -Ser) 3 , such that the NH and NL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. ⁇ atl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554), all of which are herein inco ⁇ orated by reference).
  • a flexible linker e.g., encoding the amino acid sequence (Gly 4
  • DNAs encoding partial or full-length light and heavy chains, obtained as described above may be inserted into expression vectors such that the genes are operably linked to transcriptional and translational control sequences
  • operably linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are generally chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes may be inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • the expression vector Prior to insertion of the light or heavy chain sequences, the expression vector may already carry antibody constant region sequences.
  • one approach to converting the VH and VL sequences to full- length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment(s) within the vector and the VL segment is operably linked to the CL segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • promoters e.g., promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1 90), herein inco ⁇ orated by reference. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma virus.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634.665 and 5,179,017, all by Axel et al.).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neomycin gene (for G418 selection).
  • the expression vector(s) encoding the heavy and light chains may be transfected into a host cell by standard techniques.
  • the various fonns of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the invention include PER.C6TM cells (Crucell, The Netherlands), Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A. Sha ⁇ (1982) Mol. Biol. 159:601-621), NSO myeloma cells, COS cells and SP2 cells.
  • PER.C6TM cells Cell, The Netherlands
  • Chinese Hamster Ovary (CHO cells) including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R. J. Kaufman and
  • the antibodies When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are generally produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It will be understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain of an antibody of this invention. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to hTNF- ⁇ . The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.
  • bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than hTNF- ⁇ by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operably linked to enhancer/promoter regulatory elements (e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an S V40 enhancer/AdMLP promoter regulatory element) to drive high levels of transcription of the genes.
  • enhancer/promoter regulatory elements e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory element or an S V40 enhancer/AdMLP promoter regulatory element
  • the recombinant expression vector may also carry a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
  • the antibodies and antibody fragments of the present invention are produced in transgenic animals.
  • transgenic sheep and cows may be engineered to produce the antibodies or antibody fragments in their milk (see, e.g., Pollock DP, et al., (1999) Transgenic milk as a method for the production of recombinant antibodies. J. Immunol. Methods 231 : 147-157, herein inco ⁇ orated by reference).
  • the antibodies and antibody fragments of the present invention may also be produced in plants (see, e.g., Larrick et al., (2001) Production of secretory IgA antibodies in plants. Biomol. Eng. 18:87-94, herein inco ⁇ orated by reference). Additional methodologies and purification protocols are provided in Humphreys et al., (2001) Therapeutic antibody production technologies: molecules applications, expression and purification, Curr. Opin. Drug Discov. Devel. 4: 172-185, herein inco ⁇ orated by reference, hi certain embodiments, the antibodies or antibody fragments of the present invention are produced by transgenic chickens (see, e.g., US Pat. Pub. Nos. 20020108132 and 20020028488, both of which are herein inco ⁇ orated by reference).
  • the TNF- ⁇ binding molecules of the present invention are useful for treating a subject having a pathology or condition associated with abnormal levels of a substance reactive with a TNF- ⁇ binding molecule, such as TNF- ⁇ in excess of, or less than, levels present in a normal healthy subject, where such excess or diminished levels occur in a systemic, localized or particular tissue type or location in the body.
  • tissue types can include, but are not limited to, blood, lymph, CNS, liver, kidney, spleen, heart muscle or blood vessels, brain or spinal cord, white matter or gray matter, cartilage, ligaments, tendons, lung, pancreas, ovary, testes, and prostate.
  • TNF- ⁇ concentrations relative to normal levels can also be localized to specific regions or cells in the body, such as joints, synovium, nerve blood vessel junctions, bones, specific tendons or ligaments, or sites of infection, such as bacterial or viral infections.
  • TNF-related pathologies include, but are not limited to, the following:
  • A acute and chronic immune and autoimmune pathologies, such as systemic lupus erythematosus (SLE), rheumatoid arthritis, thyroidosis, graft- versus-host disease, scleroderma, diabetes mellitus, Graves' disease, and the like;
  • SLE systemic lupus erythematosus
  • rheumatoid arthritis thyroidosis
  • graft- versus-host disease graft- versus-host disease
  • scleroderma diabetes mellitus
  • Graves' disease and the like
  • C inflammatory diseases, such as chronic inflammatory pathologies and vascular inflammatory pathologies, including chronic inflammatory pathologies such as, but not limited to, sarcoidosis, inflammatory bowel disease (i.e., ulcerative colitis and Crohn's disease) and vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, and Kawasaki's pathology;
  • (D) neurodegenerative diseases including, but not limited to, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders such as lesions of the corticospinal system; disorders of the basal ganglia; hyperkinetic movement disorders such as Huntington's disease and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; progressive supranuclear palsy; cerebellar and spinocerebellar disorders, such as structural lesions of the cerebellum; spinocerebellar degenerations (spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Shy-Drager syndrome, , Dejerine-Sottas, and Machado-Joseph diseases)); and systemic disorders (Refsum's disease, , ataxia, telangiec
  • E malignant pathologies involving TNF-secreting tumors or other malignancies involving TNF, such as, but not limited to leukemias (acute, chronic myelocytic, chronic lymphocytic and/or myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodgkin's lymphomas, such as malignant lymphomas (Burkitt's lymphoma or mycosis fungoides)); and
  • TNF- ⁇ pathologies include, but are limited to, psoriasis, psoriatic arthritis, Wegener's granulomatosis, ankylosing spondylitis, heart failure, reperfusion injury, chronic obstructive pulmonary disease, pulmonary fibrosis, and hepatitis C infection. See, e.g., Berkow et al., eds., The Merck Manual, 16th edition, chapter 11, pp 1380-1529, Merck and Co., Rahway, N.J., 1992, herein inco ⁇ orated by reference.
  • TNF- ⁇ binding molecules of the present invention may be administered by any suitable means, including parenteral, non-parenteral, subcutaneous, topical, intraperitoneal, intrapulmonary, intranasal, and intralesional administration (e.g., for local immunosuppressive treatment).
  • Parenteral infusions include, but are not limited to, intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration.
  • TNF- ⁇ binding molecules are suitably administered by pulse infusion, particularly with declining doses.
  • the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Dosage regimens may be adjusted to provide the optimum desired response (e g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • the dosages of the TNF- ⁇ binding molecules of the present invention are generally dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody fragment (or other TNF- ⁇ binding molecule of the invention) is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the present invention.
  • a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1.0 to 5, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form, is effective to obtain desired results.
  • treatment of TNF-related pathologies in humans or animals can be provided as a daily dosage of a TNF- ⁇ binding molecule of the present invention of 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 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, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
  • Dosage forms (compositions) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
  • the TNF- ⁇ binding molecules of the invention can be inco ⁇ orated into pharmaceutical compositions suitable for administration to a subject.
  • the pharmaceutical composition may comprise a TNF- ⁇ binding molecule (e.g. an antibody or antibody fragment) and a pharmaceutically acceptable carrier.
  • a TNF- ⁇ binding molecule e.g. an antibody or antibody fragment
  • a pharmaceutically acceptable carrier e.g. an antibody or antibody fragment
  • pharmaceutically acceptable carrier includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the TNF- ⁇ binding molecules.
  • compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, Hposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, Hposomes and suppositories.
  • Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies.
  • Therapeutic compositions typically are sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (i.e., antibody or antibody fragment) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterile filtration.
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze- drying that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged abso ⁇ tion of injectable compositions can be brought about by including in the composition an agent that delays abso ⁇ tion, for example, monostearate salts and gelatin.
  • the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art (see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).
  • the TNF- ⁇ binding molecules of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or inco ⁇ orated directly into the subject's diet.
  • the compounds may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Supplementary active compounds can also be inco ⁇ orated into the compositions, hi certain embodiments, a TNF- ⁇ binding molecule (e.g. an antibody or antibody fragment) of the invention is coformulated with and/or coadministered with one or more additional therapeutic agents that are useful for treating disorders in which TNF- ⁇ activity is detrimental.
  • a TNF- ⁇ binding molecule of the invention may be coformulated and/or coadministered with one or more antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules), one or more cytokines, soluble TNF- ⁇ receptor (see e.g., PCT Publication No.
  • TNF- ⁇ binding molecules of the invention may be used in combination with one or more of the foregoing therapeutic agents.
  • Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with the various monotherapies.
  • Non-limiting examples of therapeutic agents for rheumatoid arthritis with which the TNF- ⁇ binding molecules of the invention can be combined include, but are not limited to the following: non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNF- ⁇ antibody; Celltech/Bayer); cA2 (chimeric anti-TNF- ⁇ antibody; Centocor); 75 kDa TNFR- IgG (75 kDa TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rheumatism
  • Anti-Tac humanized anti-IL-2R. alpha; Protein Design Labs/Roche
  • IL-4 anti-inflammatory cytokine; DNAX/Schering
  • IL-10 SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering
  • IL-10 and/or IL-4 agonists e.g., agonist antibodies
  • IL-1 RA IL-1 receptor antagonist
  • Synergen/Amgen TNF-bp/s-TNFR (soluble TNF binding protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39 No. 9 (supplement), S284; Heart and Circulatory Physiology (1995) Vol. 268, pp.
  • R973401 phosphodiesterase Type IV inliibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39. No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 3, No. 9 (supplement), S81); Iloprost (see e.g., Arthritis & Rheumatism (1996) Vol. 39. No. 9 (supplement), S82); methotrexate: thalidomide (see e.g., Arthritis & Rheumatism (1996) Vol. 9, No.
  • thalidomide-related drugs e.g., Celgen
  • leflunomide anti-inflammatory and cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39 No. 9 (supplement), S131; Inflammation Research (1996) Vol. 4, pp. 103-107
  • tranexamic acid inhibitor of plasminogen activation; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S284)
  • T614 cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39 No.
  • ICE inhibitor inhibitor of the enzyme interleukin-1 beta converting enzyme
  • zap-70 and/or Ick inliibitor inhibitor of the tyrosine kinase zap-70 or Ick
  • VEGF inhibitor and/or VEGF-R inhibitor inhibitors of vascular endothelial cell growth factor or vascular endothelial cell growth factor receptor; inhibitors of angiogenesis
  • corticosteroid anti-inflammatory drugs e.g., SB203580
  • TNF-convertase inhibitors anti-IL-12 antibodies
  • interleukin-11 see e.g., Arthritis & Rheumatism (1996) Vol. 39, No.
  • glycosaninoglycan polysulphate glycosaninoglycan polysulphate
  • minocycline anti-IL-2R antibodies
  • marine and botanical lipids fish and plant seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am.
  • Non-limiting examples of therapeutic agents for inflammatory bowel disease with which the TNF- ⁇ binding molecules of the invention can be combined include, but are not limited to, the following: budenoside; epidermal growth factor; corticosteroids; cyclosporin, sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine; metronidazole; hpoxygenase inhibitors; mesalamine; olsalazine; balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptor antagonists; anti-IL-lbeta monoclonal antibodies; anti-IL-6 monoclonal antibodies; growth factors; elastase inhibitors; pyridinyl-imidazole compounds; CDP-571/BAY-10-3356 (humanized anti-TNF- ⁇ antibody; Celltech/Bayer); cA2 (chimeric anti-TNF- ⁇ antibody; Centocor); 75 kDa
  • Non-limiting examples of therapeutic agents for multiple sclerosis with which the TNF- ⁇ binding molecules of the invention can be combined include, but are not limited to, the following: corticosteroids; prednisolone; ' methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine; tizanidine; interferon- betala (Avonex.TM.; Biogen); interferon-betalb (Betaseron.TM.; Chiron/Berlex); Copolymer 1 (Copl; Copaxone.TM.; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine; CDP-571/BAY-10-3356 (humanized anti- TNF- ⁇ antibody; Celltech Bayer); cA2 (chimeric anti-TNF- ⁇ antibody; Centocor); 75 kDa TNFR-IgG (75 kD
  • Non-limiting examples of therapeutic agents for sepsis with which the TNF- ⁇ binding molecules of the invention can be combined include, but are not limited to, the following: hypertonic saline solutions; antibiotics; intravenous gamma globulin; continuous hemofiltration; carbapenems (e.g., meropenem); antagonists of cytokines such as TNF- ⁇ , IL-6 and/or IL-8; CDP-571/BAY-10-3356 (humanized anti-TNF- ⁇ antibody; Celltech/Bayer); cA2 (chimeric anti-TNF- ⁇ antibody; Centocor); 75 kDa TNFR-IgG (75 kDa TNF receptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rlieumatism (1994) Vol.
  • Non-limiting examples of therapeutic agents for adult respiratory distress syndrome (ARDS) with which the TNF- ⁇ binding molecules of the invention can be combined include the following: anti-IL-8 antibodies; surfactant replacement therapy; CDP-571/BAY-10- 3356 (humanized anti-TNF- ⁇ antibody; Celltech/Bayer); cA2 (chimeric anti-TNF- ⁇ antibody; Centocor); 75 kDa TNFR-IgG (75 kDa TNF receptor-IgG fusion protein;
  • compositions of the invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antibody fragment of the invention.
  • 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 the antibody or antibody fragment may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody fragment to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody fragment are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • the present invention also provides TNF- ⁇ binding molecules (e.g., anti-TNF- ⁇ peptides and antibodies) detectably labeled, as described below, for use in diagnostic methods for detecting TNF- ⁇ in patients known to be or suspected of having a TNF- ⁇ - mediated disease.
  • TNF- ⁇ binding molecules e.g., anti-TNF- ⁇ peptides and antibodies
  • TNF- ⁇ binding molecules of the present invention such as anti-TNF- ⁇ peptides and/or antibodies are useful for immunoassays which detect or quantify TNF- ⁇ in a sample.
  • An immunoassay for TNF- ⁇ typically comprises incubating a biological sample in the presence of a detectably labeled high affinity anti-TNF- ⁇ peptide and/or antibody of the present invention capable of selectively binding to TNF- ⁇ , and detecting the labeled peptide or antibody which is bound in a sample.
  • Various clinical assay procedures are well known in the art, e.g., as described in hnmunoassays for the 80's, A. Voller et al., eds., University Park, 1981.
  • an anti-TNF peptide or antibody can be captured on nitrocellulose, or on any other solid support which is capable of immobilizing soluble proteins.
  • a TNF- ⁇ -containing sample is then added to the support which is subsequently washed with suitable buffers to remove unbound proteins.
  • a second, detectably labeled, TNF- ⁇ specific peptide or antibody is added to the solid phase support that can then be washed with the buffer a second time to remove unbound detectably labeled peptide or antibody.
  • the amount of bound label on the solid support can then be detected by known methods.
  • solid phase support or “carrier” is intended any support capable of binding peptide, antigen or antibody.
  • 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 pvuposes of the present invention.
  • the support material can have virtually any possible stractural configuration so long as the coupled molecule retains its ability to bind to TNF- ⁇ .
  • the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface can be flat such as a sheet, culture dish, test strip, microtiter plates, etc.
  • Preferred supports include polystyrene beads.
  • suitable carriers for binding antibody, peptide or antigen or can ascertain the same by routine experimentation.
  • Well known methods can be used to determine the binding activity of a given lot of anti-TNF- ⁇ peptide and/or antibody.
  • Detectably labeling a TNF- ⁇ binding molecule, such as a TNF-specific peptide and/or antibody can be accomplished by coupling 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 TNF- ⁇ binding molecules 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.
  • radioactively labeling the TNF- ⁇ binding molecules By radioactively labeling the TNF- ⁇ binding molecules, it is possible to detect TNF- ⁇ through the use of a radioimmunoassay (RIA) (see, for example, Work, et al., (1978) Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, N. Y.).
  • RIA radioimmunoassay
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • TNF- ⁇ binding molecules it is also possible to label the TNF- ⁇ binding molecules with a fluorescent compound.
  • fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the TNF- ⁇ binding molecules can also be detectably labeled using fluorescence- emitting metals such as Eu, or others of the lanthanide series. These metals can be attached to the TNF- ⁇ binding molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediamine-tetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediamine-tetraacetic acid
  • the TNF- ⁇ binding molecules also can be detectably labeled by coupling to a chemiluminescent compound.
  • the presence of the chemiluminescently labeled molecule is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound can be used to label the TNF- ⁇ binding molecules 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 pvuposes of labeling are luciferin, luciferase and aequorin.
  • Detection of the TNF- ⁇ binding molecules 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 to similarly prepared standards.
  • the TNF- ⁇ which is detected by the above assays can be present in a biological sample. Any sample containing TNF- ⁇ can be used.
  • the sample is a biological fluid such as, for example, blood, serum, lymph, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, a tissue extract or homogenate, and the like.
  • a biological fluid such as, for example, blood, serum, lymph, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, a tissue extract or homogenate, and the like.
  • the invention is not limited to assays using only these samples, as it is 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 TNF- ⁇ binding molecules of the present invention to such a specimen.
  • the TNF- ⁇ binding molecule is preferably provided by applying or by overlaying the labeled TNF- ⁇ binding molecule to a biological sample.
  • the TNF- ⁇ binding molecules of the present invention can be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay, hi a typical immunometric assay, a quantity of unlabeled TNF- ⁇ binding molecule (such as an anti- TNF- ⁇ antibody) 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.
  • an immunometric assay also known as a "two-site” or “sandwich” assay
  • a quantity of unlabeled TNF- ⁇ binding molecule such as an anti- TNF- ⁇ antibody
  • 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 TNF- ⁇ binding molecule (e.g. antibody) bound to the solid phase is first contacted with the sample being tested to extract the TNF- ⁇ from the sample by formation of a binary solid phase antibody-TNF- ⁇ complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted TNF- ⁇ , if any, and then contacted with the solution containing a known quantity of labeled antibody (which functions as a "reporter molecule").
  • TNF- ⁇ binding molecule e.g. antibody
  • This type of forward sandwich assay can be a simple "yes/no” assay to determine whether TNF- ⁇ is present or can be made quantitative by comparing the measure of labeled antibody with that obtained for a standard sample containing known quantities of TNF- ⁇ .
  • 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.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period, is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.
  • the TNF- ⁇ binding molecules of this invention attached to a solid support, can be used to remove TNF- ⁇ from fluids or tissue or cell extracts. In a preferred embodiment, they are used to remove TNF- ⁇ from blood or blood plasma products. In another preferred embodiment, the TNF- ⁇ binding molecules are advantageously used in extraco ⁇ oreal immunoadsorbent devices, which are known in the art (see, for example, Seminars in Hematology, 26 (2 Suppl. 1)(1989)). Patient blood or other body fluid is exposed to the attached TNF- ⁇ binding molecule, resulting in partial or complete removal of circulating TNF- ⁇ (free or in immune complexes), following which the fluid is returned to the body.
  • This immunoadso ⁇ tion can be implemented in a continuous flow arrangement, with or without inte ⁇ osing a cell centrifugation step. See, for example, Terman, et al., (1976) J. Immunol. 117:1971-1975.
  • This example describes the construction of anti-TNF- ⁇ Fab fragments with fully human framework regions and synthetic CDRs.
  • the nucleic acid sequences encoding the hul CDRs and framework regions are shown in SEQ ID NOs: 2 and 4, respectively.
  • the six hul CDRs employed were as follows: CDRLl (SEQ ID NO:10); CDRL2 (SEQ ID NO: 20); CDRL3 (SEQ ID NO: 34); CDRHl (SEQ ID NO: 36); CDRH2 (SEQ ID NO: 44); and CDRH3 (SEQ ID NO: 54).
  • libraries of synthetic CDRs were inserted into a deletion template as described below.
  • CDRs include residues that encompass both the Kabat and Chothia definitions (e.g., residues 26-35 for CDRHl).
  • the length of CDRH2 made it necessary to construct two separate libraries to cover the entire region.
  • Mutagenic oligonucleotides were annealed to a uridinylated phage template in which the corresponding hul CDR was deleted.
  • This template was composed of the hul light chain variable region sequence (SEQ ID NO: 2, with the appropriate hul CDR deleted) and the human CL sequence shown in SEQ ID NO: 85 as well as the hul heavy chain variable region sequence (SEQ ID NO: 4, with the appropriate hul CDR deleted) and the human CHI sequence shown in SEQ ID NO: 86.
  • Annealing was accomplished by incubating the reaction at 75 °C for 5 minutes followed by slow cooling to 20 °C over the course of 45 minutes. The annealed samples were placed on ice and T4 DNA polymerase and T4 DNA ligase were added to generate double stranded DNA and the reaction was incubated for 5 minutes at 4 °C followed by 90 minutes at 37 °C. The reaction was phenol extracted, ethanol precipitated and the DNA was electroporated into DH10B cells. XLl Blue cells were added to the reaction to allow phage amplification before the libraries were plated. Phage stocks were prepared by the addition of 8 ml of growth medium to the plates followed by incubation at 4 °C for a minimum of 4 hours.
  • the phage-containing medium was harvested and clarified by centrifugation and sodium azide (0.02%) was added as a preservative.
  • Initial screening of the anti-TNF- ⁇ Fab libraries was done by a plaque lift as described in Watkins, J.D. et al., (1998) Anal. Biochem., 256: 169-177, herein inco ⁇ orated by reference. Briefly, nitrocellulose filters were coated with goat anti-human kappa antibodies, blocked with 1% BSA and placed on plates containing plaques generated from the phage libraries for 16 hours at 22 °C. Filters were then rinsed in PBS and incubated with varying concentrations of biotinylated human TNF- ⁇ for varying lengths of time.
  • Positive plaques were picked with a Pasteur pipet and phages were eluted for 1 hour at 37 °C in 100 ⁇ l of 10 mM Tris HCl, pH 7.4 and 100 mM NaCl. High titer stocks were then obtained by standard amplification techniques. XLl Blue cells were grown to an OD 60 o of 1 and IPTG was added to a final concentration of 1 mM. Following infection of 15 ml of bacterial culture with 10 ⁇ l of high titer phage stock, cells were incubated at 37 °C for an hour and at 22 °C for 16 hours. Bacteria were harvested and washed with 30 mM Tris HCl, pH 8 and 150 mM NaCl.
  • the pellet was resuspended in 640 ⁇ l of 30 mM Tris HCl, pH 8, 2 mM EDTA and 20%> sucrose and incubated at 4 °C for 15 minutes. Lysed cells were pelleted and the supernatant containing the periplasmic content and Fab fragments was assayed in an ELISA format to determine approximate off-rates.
  • COSTAR #3366 microtiter plates were coated with 1 ⁇ g/ml of hTNF- ⁇ in carbonate buffer for 16 hours at 4 °C. The plates were washed 3 times with PBS containing 0.1 %> Tween 20 and blocked with 1% BSA for 1 hour at 22 °C.
  • Fab fragments released from the periplasmic space were serially diluted in PBS containing 0.05%> Tween 20 and added to the plates for 1 hour at 22 °C.
  • the plates were washed and incubated with goat anti-human kappa antibodies conjugated to alkaline phosphatase in PBS containing 0.05% Tween 20 for 1 hour at 22 °C.
  • the plates were then washed with PBS containing 0.1% Tween 20 and developed with standard colorimetric substrates for alkaline phosphatase (see, e.g., Watkins, J.D. et al., (1997) Anal. Biochem., 253: 37-45, herein inco ⁇ orated by reference).
  • ELISA assays were also used to determine approximate on-rates.
  • COSTAR #3366 microtiter plates were coated with 2 ⁇ g/ml of goat anti-human kappa antibodies in carbonate buffer for 16 hours at 4 °C.
  • Fab fragments released from the periplasmic space were serially diluted in PBS containing 0.05%> Tween 20 and added to wells and incubated for 1 hour at 22 °C. The plates were washed 3 times with PBS containing 0.1 %> Tween 20 and incubated with 5 nM biotinylated hTNF- ⁇ for 2 minutes at 22 °C.
  • binding kinetics of three of the Fab fragments described in Table 4 were determined in a kinetic exclusion assay. Affinity measurements were performed on a KinExATM3000 instrument (Sapidyne Instruments, Inc., Boise, Idaho) using Fab fragments (AIOK, A9K, and A10P) released from the periplasmic space whose concentration was determined by a quantitative ELISA. Briefly, antigen and antibody fragment were allowed to react in solution and the fraction of unbound reagent was determined by capture and measured on a bead support. Binding kinetics can be determined or calculated from the amount of unbound reagent.
  • binding of the unreacted, free Fab fragment to TNF- ⁇ coated beads can be measured on the instrument by detection with a labeled secondary antibody to the Fab fragment (e.g., a goat anti-IgG (heavy and light)).
  • a labeled secondary antibody to the Fab fragment e.g., a goat anti-IgG (heavy and light)
  • individual tubes containing a constant concentration of Fab fragment with decreasing concentrations of human TNF- ⁇ were incubated for 20 hours at 20 °C in PBS supplemented with 0.1%) BSA. A total of 13 tubes were used for each K d determination.
  • the Fab concentration was 10 pM with the human TNF- ⁇ concentration beginning at 160 pM and then serially diluted to 0.078 pM with the final tube containing no human TNF- ⁇ .
  • A9K incubation was set in a similar manner, with the Fab at a constant concentration of 20 pM and human TNF- ⁇ serially diluted beginning at 500 pM with a final concentration of 0.24 pM. After the incubation, the amount of unbound Fab in the equilibrated samples was determined on the KinExATM3000 instrument according to the manufacturer's instructions. K values were determined by the instrument software. The results are provided in Table 5.
  • This example describes various in vitro testing procedures used to identify particular characteristics of the eight anti-TNF- ⁇ binding molecules from Example 1.
  • the ability of these Fab fragments to prevent TNF- ⁇ -mediated cell death was assessed by evaluating their protective effects on L929 mouse fibroblasts.
  • L929 mouse fibroblast cells were distributed in individual wells in a 96-well plate at a density of 20,000 cells per well and maintained according to ATCC recommendations. After overnight incubation, Fab fragments released from the periplasmic space were serially diluted in culture medium, sterile filtered and 50 ⁇ l were added to the wells along with actinomycin D to a final concentration of 1 ⁇ g/ml.
  • hTNF- ⁇ was added to a final concentration of 80 pg/ml and the plates were placed in a humidified incubator at 37 °C in a 5%> CO 2 atmosphere. After overnight incubation, the protective effects of the various Fab fragments were assessed by monitoring cell survival using the Cell Titer 96 R AQueous One solution reagent (Promega, Madison WI) according to the manufacturer's instructions. The optical density of individual wells was measured at 490 mn and the results are shown in Figure 8.
  • each of the eight anti-TNF- ⁇ Fab fragments described in Table 4 was able to prevent cell death.
  • A10K and A10P were able to protect cells at the lowest concentration, reflecting improved binding properties for TNF- ⁇ .
  • the effective concentration (EC 50 ) was determined using the curve fitting function and EC 50 derivation capabilities of the SigmaPlot® software (SPSS Science, Chicago, IL). In the experiment shown in Figure 8, the EC 50 of the A10K Fab fragment was estimated at approximately 25 pM.
  • the following procedure may be employed.
  • the light chain variable region (VL) and the heavy chain variable region (VH) can be transferred from the A10K Fab expressing phage into, for instance, an expression vector containing both the heavy and light chain constant regions.
  • the variable regions can be cloned into two separate expression vectors, each vector eventually expressing only one of the two chains.
  • the NL region can be amplified by PCR using the following primers: NL 5' primer:
  • TGGCTCCCAGGTGCCAAATGTGAAATTGTGCTGACTCAG SEQ ID ⁇ O:109
  • VL 5' biotinylated primer Biotin-TGGCTCCCAGGTGCCAAATGT (SEQ ID NO: 110); and VL 3 ' primer: GACAGATGGTGCAGCCACAGT (SEQ ID NO: 111).
  • VH region can be amplified using the following primers (VH 5' primer: CTCTCCACAGGTGTCCACT CCCAGGTCCAACTGCAGGTC (SEQ ID NO: 112); NH 5' biotinylated primer: Biotin-CTCTCCACAGGTGTCCACTCC (SEQ ID ⁇ O:l 13); and VH 3' primer: GAAGACCGATGGGCCCTTGGT (SEQ ID NO: 114).
  • the biotinylated DNA strand is removed and the non-biotinylated strand is annealed to single stranded uridinylated template prepared from the expression vector at an annealing ratio of 20:1, followed by synthesis with T4 DNA polymerase in presence of T4 DNA ligase.
  • the uridinylated template is lost, yielding a double stranded plasmid with, for instance, the A10K IgG.
  • mammalian cells e.g., CHO-K1 cells
  • a pool of stable integrants can be selected by culturing the transfected cells in an appropriate medium and under appropriate selection.
  • the pool can be expanded, for instance in roller bottles, and maintained in selection.
  • the medium is usually changed once every other day and the conditioned medium containing the A10K IgG is used for IgG purification. This same procedure may be employed to generate a hul IgG or any other IgG molecule.
  • the L929 cell protection assay was used to characterize Al OK and hul both as Fab fragments and as intact IgG molecules, along with a control Fab and a control IgG.
  • the assay was performed as described in the previous section.
  • both TNF- ⁇ binding molecules either as Fab fragments or as intact IgGs, were able to protect cells from the detrimental effects of TNF- ⁇ .
  • the concentration of A10K necessary to achieve identical protection is significantly lower than that of hul, showing that the improvements in binding properties achieved by protein engineering translate into lower effective doses on cells.
  • the EC 50 of the A10K Fab fragment was estimated at approximately 67 pM.
  • Figure 10 shows a direct comparison between A10K and REMICADE (Oncology Supply, Dothan, Alabama), a standard of care in rheumatoid arthritis and Crohn's disease.
  • A10K is a more potent molecule and is more effective at neutralizing TNF- ⁇ .
  • the EC 50 of the A10K IgG molecule was estimated at approximately 2.7 pM.
  • This example describes two in vivo assays with the AIOK intact IgG. hi particular, this example describes the in vivo TNF- ⁇ neutralizing effects of AIOK in mice, as well as the protective effects of AIOK on TNF- ⁇ mediated polyarthritis in mice.
  • mice Female C3H/HeN mice ( ⁇ 9 weeks of age) were injected intraperitoneally with recombinant human TNF- ⁇ (5 ⁇ g/mouse) in combination with the sensitizing agent galactosamine (18 mg/mouse). h untreated mice, these conditions result in approximately 90 percent mortality within 48 hours.
  • the neutralizing effect of Al OK was evaluated by inj ecting mice with 0.8 and 4 mg/kg, intravenously, 24 hours prior to hTNF- ⁇ challenge.
  • A10K demonstrated a dose-dependent inhibition of mortality, with complete protection being observed at the higher dose investigated.
  • mice transgenic for human hTNF- ⁇ develop severe chronic arthritis by approximately 20 weeks of age (Keffer, J. et al., (1991) EMBO J., 10:4025- 4031 , herein inco ⁇ orated by reference).
  • a dose escalation study was performed on mice exhibiting very early stage joint swelling (9 weeks of age) to determine the effects of A10K on disease progression. Hind joint width was determined and animals were randomized into groups and balanced for initial joint size.
  • This example describes the construction of the AME-3-2 antibody. PCR primers were used to convert Al OK light chain framework into the human germline framework
  • IGKV1-39 also known as O2 or DP-K9 that appears frequently in the human population.
  • the heavy chain in AME-3-2 is IGVH3-72 (also known as 3-72 or DP-29).
  • IGVH3-72 also known as 3-72 or DP-29.
  • This construct was then mutagenized, screened, and tested in a similar fashion as described in Examples 1 and 2 above.
  • This procedure led to the identification of 6 new CDRs for AME 3-2 (shown in Figure 15): CDRLl - SEQ ID NO:93; CDRL2 - SEQ ID NO:95; CDRL3 - SEQ ID NO:97; CDRHl - SEQ ID NO:87; CDRH2 - SEQ ID NO:89; and CDRH3 - SEQ ID NO:91.
  • Figure 15 also shows the full light and heavy chains for the AME 3-2 antibody.
  • TNF- ⁇ Binding Molecules to Treat a TNF- ⁇ Mediated Disease
  • TNF- ⁇ binding molecules for the therapeutic and prophylactic treatment of a TNF- ⁇ mediated disease in a human patient (e.g., with juvenile and adult rheumatoid arthritis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, psoriasis, ankylosing spondylitis, Wegener's granulomatosis and sepsis).
  • a human patient e.g., with juvenile and adult rheumatoid arthritis, psoriatic arthritis, Crohn's disease, inflammatory bowel disease, ulcerative colitis, psoriasis, ankylosing spondylitis, Wegener's granulomatosis and sepsis.
  • a patient with a TNF- ⁇ mediated disease may be administered a single dose of an anti-TNF- ⁇ binding molecule, such as 2C6K, 2C6P, 2E7K, 2E7P, A9K, A9P, A10K, A10P, AME 3-2, or hul, intravenously at 1-15 milligrams per kilogram of patient body weight.
  • an anti-TNF- ⁇ binding molecule such as 2C6K, 2C6P, 2E7K, 2E7P, A9K, A9P, A10K, A10P, AME 3-2, or hul
  • This patient may also be given multiple doses over time (e.g. 3 mg/kg over multiple days or weeks).
  • Response to therapy may be monitored to determine the need for increased or reduced dosage and the need for repeat treatment. Additional guidance on response to therapy and dosage schedules is found in U.S. Patent 5,698,195, herein inco ⁇ orated by reference.
  • the patient may also be administered an immunosuppress

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Abstract

La présente invention concerne des molécules de liaison au facteur de nécrose tumorale alpha (TNF-α) et des séquences d'acide nucléique codantes pour les molécules de liaison au TNF-α. L'invention concerne notamment les molécules de liaison au TNF-α ayant une grande affinité de liaison, un taux d'association élevé, un faible taux de dissociation en ce qui concerne le TNF-α humain et aptes à neutraliser de faibles concentrations de TNF-α. Les molécules de liaison au TNF-α selon l'invention comprennent, de préférence, des domaines variables de lumière et/ou de chaîne lourde ayant des ossatures entièrement humaines (p.ex. les ossatures de lignées germales humaines).
EP04700833A 2003-01-08 2004-01-08 Molecules de liaison au facteur de necrose tumorale alpha Withdrawn EP1590431A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US10/338,627 US20040131613A1 (en) 2003-01-08 2003-01-08 TNF-alpha binding molecules
US338552 2003-01-08
US10/338,552 US7101978B2 (en) 2003-01-08 2003-01-08 TNF-α binding molecules
US338627 2003-01-08
PCT/US2004/000290 WO2004063335A2 (fr) 2003-01-08 2004-01-08 Molecules de liaison au facteur de necrose tumorale alpha

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CA2434668A1 (fr) * 2003-07-04 2005-01-04 Laurence Mulard Nouvelle approche pour concevoir des glycopeptides a base de o-specifique polysaccharide de shigella flexneri serotype 2a
CN102127167B (zh) * 2010-01-15 2013-06-19 苏州工业园区晨健抗体组药物开发有限公司 全人TNFa单克隆抗体及其制备与应用
CN102336834B (zh) * 2010-07-22 2014-01-01 苏州工业园区晨健抗体组药物开发有限公司 全人TNFα-Fab抗体及其PEG化抗体
BR112014009799A2 (pt) * 2011-10-24 2017-06-13 Abbvie Inc imunoligantes dirigidos conra tnf

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0626389A1 (fr) * 1990-12-21 1994-11-30 Celltech Therapeutics Limited Anticorps récombinants contre le TNF-alpha
WO1997029131A1 (fr) * 1996-02-09 1997-08-14 Basf Aktiengesellschaft ANTICORPS HUMAINS SE FIXANT AU FACTEUR NECROSANT DES TUMEURS DE TYPE $g(a)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0626389A1 (fr) * 1990-12-21 1994-11-30 Celltech Therapeutics Limited Anticorps récombinants contre le TNF-alpha
WO1997029131A1 (fr) * 1996-02-09 1997-08-14 Basf Aktiengesellschaft ANTICORPS HUMAINS SE FIXANT AU FACTEUR NECROSANT DES TUMEURS DE TYPE $g(a)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO2004063335A2 *

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