Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • 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
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—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
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—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
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/77—Internalization into the cell

Definitions

• the present invention relates to antibodies that bind c-Met and their use in treating conditions and disorders in which pathogenesis is mediated by this receptor.
• c-Met a member of the tyrosine kinase superfamily, is the receptor for Hepatocyte Growth Factor (HGF). Binding of HGF to c-Met leads to receptor dimerization or multimerization, phosphorylation of multiple tyrosine residues in the intracellular region, catalytic activation, and downstream signaling.
• HGF Hepatocyte Growth Factor
• c-Met activation enhances cellular proliferation, migration, morphogenesis, survival (including protection from apoptosis), and protease synthesis, characteristics that are associated with invasive cell phenotype and poor clinical outcomes and drug resistance in cancer patients.
• the c-Met signaling pathway is one of the most frequently dysregulated pathways in human cancers, and occurs in virtually all types of solid tumors.
• PCT International Publication WO 09/007427 discloses murine and CDR-grafted, humanized c-Met antibodies.
• Murine antibody 224Gl 1 disclosed therein did not bind the c-Met Sema domain. Further functional properties of the humanized IgGl derivative of this murine antibody, denoted h224Gl 1 Mab, are reported in Abstracts nos.
• PCT International Publication WO 05/016382 also discloses c-Met antibodies but does not identify the epitope(s) to which the antibodies bind.
• An epitope mapping example is provided, however, the reported results merely indicate that six c-Met antibodies bind to a common epitope while a seventh c-Met antibody binds a distinct epitope. The particular epitopes to which these c-Met antibodies bind is not provided.
• There exists a need for antagonist antibodies to human c-Met, binding of which to the ⁇ -chain of human c-Met facilitates internalization of the receptor from the cell surface, in the presence and/or absence of HGF.
• antagonist antibodies to human c-Met which binding to the ⁇ -chain of human c-Met facilitates internalization of the receptor from the cell surface in cells comprising c-Met variants containing gain of function mutations.
• antagonist antibodies to human c-Met which induce c-Met degradation and reduction of phosphorylated c-Met.
• Such antagonist activities could decrease the number of available binding sites for HGF on tumor cell surfaces, and terminate the pathway activation caused by overexpression, amplification, or mutation of c-Met.
• antagonist antibodies should inhibit HGF binding to c-Met and HGF-induced c-Met activation, and induce little or no agonist activity themselves.
• Antibody compounds of the present invention meet these needs. They bind to epitopes in the ⁇ -chain of the human c-Met Sema domain, inhibiting HGF -binding to c- Met and receptor activation, while inducing little or no agonist activity.
• the antibodies of the present invention also induce internalization of the receptor in the presence or absence of HGF and also in cells comprising c-Met variants containing gain of function mutations. They induce degradation of c-Met and induce reduction of phosphorylated human c-Met, and inhibit HGF-dependent and HGF-independent proliferation of tumor cells that express this receptor. In view of these properties, these antibody compounds should be therapeutically useful in treating cancers mediated by c-Met via a variety of different mechanisms.
• the present antibody compounds possess a number of other desirable properties. They exhibit high affinity (KQ) to c-Met, block HGF-mediated c-Met phosphorylation and downstream signaling, cellular proliferation, and cellular migration; and induce only weak phosphorylation of c-Met while inducing little or no HGF-like biological agonist activities such as induction of tumor cell proliferation, motility, invasion, tubulogenesis, angiogenesis, or anti-apoptotic effects. They inhibit both ligand (HGF)-dependent and ligand-independent c-Met pathway activation.
• antibody compounds of the present invention preferentially bind human c-Met extracellular domain (ECD) compared to the ECDs of the closely related receptors RON and PlexinA2, and do not cause "shedding" of the c-Met ECD. Accordingly, the present invention provides:
• a monoclonal antibody, or antigen-binding fragment thereof that: a) binds an epitope within the ⁇ -chain of human c-Met, and b) induces internalization of cell surface human c-Met.
• the monoclonal antibody or antigen-binding fragment thereof comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• the gain of function mutation can be c- Met kinase domain mutation Ml 149T or juxtamembrane domain mutation R988C.
• any of the foregoing antibodies or antigen-binding fragments thereof wherein the antibody or antigen-binding fragment thereof induces at least 40% internalization of cell surface human c-Met in cells. Any of the foregoing antibodies or antigen-binding fragments thereof wherein the antibody or antigen-binding fragment thereof induces at least 45% internalization of cell surface human c-Met in cells. Any of the foregoing antibodies or antigen-binding fragments thereof wherein the antibody or antigen-binding fragment thereof induces at least 50% internalization of cell surface human c-Met in cells. Any of the foregoing antibodies or antigen-binding fragments thereof wherein the antibody or antigen-binding fragment thereof induces at least 55% internalization of cell surface human c-Met in cells.
• the monoclonal antibody or antigen-binding fragment thereof which induces reduction of total c-Met in hepatocyte growth factor- independent tumor cells comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof which induces reduction of total c-Met in hepatocyte growth factor-independent tumor cells comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof which bind to an amino acid sequence within the epitope characterized by 121 VVDTYYDDQL 130 (SEQ ID NO:77), 131 ISCGSVNRGTCQRHVFPHNHTADIQS 156 (SEQ ID NO:78), 179 ALGAKVLSSVKDRFINF 195 (SEQ ID NO:79), and 2 16 VRRLKETKDGFM 227 (SEQ ID NO:80), inclusive.
• any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof excluding those comprising a light chain having the amino acid sequence shown in SEQ ID NO:29 and a heavy chain having the amino sequence shown in SEQ ID NO:41, wherein the epitope comprises one or more amino acid residues within (SEQ ID NO: 86) inclusive.
• any one of the foregoing monoclonal antibodies, or antigen-binding fragments thereof, that binds human c-Met which comprises three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), wherein said three LCDRs and said three HCDRs are selected from the group consisting of: a) LCDRl comprising the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49);
• LCDR2 comprising the amino acid sequence GTSXiLX 2 S (SEQ ID NO:87), wherein X 1 is Y or R, and X 2 is A or R;
• LCDR3 comprising the amino acid sequence QQWSSYPYS (SEQ ID NO:51); HCDRl comprising the amino acid sequence GYTFTSRYIH (SEQ ID NO:51);
• HCD R2 comprising the amino acid sequence
• WIYPVTGDTYYX 7 EX 8 FKG (SEQ ID NO: 90), wherein X 7 is N, I, or R, and X 8 is K or P; and HCDR3 comprising the amino acid sequence GYGAFX 9 Y (SEQ ID NO: 90), wherein X 7 is N, I, or R, and X 8 is K or P; and HCDR3 comprising the amino acid sequence GYGAFX 9 Y (SEQ ID NO: 90), wherein X 7 is N, I, or R, and X 8 is K or P; and HCDR3 comprising the amino acid sequence GYGAFX 9 Y (SEQ ID NO: 90), wherein X 7 is N, I, or R, and X 8 is K or P; and HCDR3 comprising the amino acid sequence GYGAFX 9 Y (SEQ ID NO: 90), wherein X 7 is N, I, or R, and X 8 is K or P; and HCDR3 comprising the amino acid sequence GYGA
• LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:54); LCDR3 comprising the amino acid sequence X 4 X5YX6GYPLT (SEQ ID NO:54);
• HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCD R2 comprising the amino acid sequence RVNPX 1 0RX 11 X 12 TTYNQKFEG (SEQ ID NO:92), wherein X 10 is N or Y, X 11 is G or R, and X 12 is G or S; and
• HCDR3 comprising the amino acid sequence X 13 NX 14 LDY (SEQ ID NO:93), wherein X 13 is T or A, and X 14 is W or I; wherein said monoclonal antibody or antigen-binding fragment thereof binds an epitope within the ⁇ -chain of said human c-Met and induces internalization of cell surface human c-Met.
• any one of the foregoing monoclonal antibodies, or antigen-binding fragments thereof, that binds human c-Met which comprises three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), wherein the antibody comprises three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), wherein
• LCDRl comprises the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49); LCDR2 comprises the amino acid sequence GTSX 1 LX 2 S (SEQ ID NO:87), wherein X 1 is Y or R, and X 2 is A or R;
• LCDR3 comprises the amino acid sequence QQWSSYPYS (SEQ ID NO:51); HCDRl comprises the amino acid sequence GYTFTSRYIH (SEQ ID NO:59); HCDR2 comprises the amino acid sequence WIYPVTGDTYYX 7 EX 8 FKG (SEQ ID NO:90), wherein X 7 is N, I, or R, and X 8 is K or P; and HCDR3 comprises the amino acid sequence GYGAFX 9 Y (SEQ ID NO:91), wherein
• X 9 is Y or F. Any one of the foregoing monoclonal antibodies, or antigen-binding fragments thereof, that binds human c-Met, which comprises three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), wherein LCDRl comprises the amino acid sequence SVSSSVX3SIYLH (SEQ ID NO:88); wherein X 3 is S or R;
• LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO: 54);
• LCDR3 comprises the amino acid sequence X 4 X 5 YX 6 GYPLT (SEQ ID NO:89), wherein X 4 is I or Q, X 5 is Q or V, and Xe is S or R;
• HCDRl comprises the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCDR2 comprises the amino acid sequence RVNPX 1 0RX 11 X 12 TTYNQKFEG (SEQ ID NO:92), wherein Xi 0 is N or Y, Xn is G or R, and X 12 is G or S; and HCDR3 comprises the amino acid sequence Xi 3 NXi 4 LDY (SEQ ID NO:93), wherein Xi 3 is T or A, and Xi 4 is W or I.
• any one of the foregoing monoclonal antibodies, or antigen-binding fragments thereof, that binds human c-Met which comprises three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), and wherein said three LCDRs and said three HCDRs are selected from the group consisting of: a) LCDRl comprising the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49);
• LCDR2 comprising the amino acid sequence GTSYLAS (SEQ ID NO:50); LCDR3 comprising the amino acid sequence QQWSSYPYS (SEQ ID NO:50);
• HCDRl comprising the amino acid sequence GYTFTSRYIH (SEQ ID NO:59);
• HCD R2 comprising the amino acid sequence WIYPVTGDTYYNEKFKG (SEQ ID NO: 60);
• HCDR3 comprising the amino acid sequence GYGAFYY (SEQ ID NO:61); b) LCDRl comprising the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49);
• LCDR2 comprising the amino acid sequence GTSYLAS (SEQ ID NO:50);
• LCDR3 comprising the amino acid sequence QQWSSYPYS (SEQ ID N0:51);
• HCDRl comprising the amino acid sequence GYTFTSRYIH (SEQ ID NO:59); HCD R2 comprising the amino acid sequence
• WIYPVTGDTYYIEKFKG (SEQ ID NO: 62).
• HCDR3 comprising the amino acid sequence GYGAFFY (SEQ ID NO:63); c) LCDRl comprising the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49);
• LCDR2 comprising the amino acid sequence GTSRLRS (SEQ ID NO:52);
• LCDR3 comprising the amino acid sequence QQWSSYPYS (SEQ ID N0:51); HCDRl comprising the amino acid sequence GYTFTSRYIH (SEQ ID NO:
• HCD R2 comprising the amino acid sequence WIYPVTGDTYYREPFKG (SEQ ID NO: 64), and
• HCDR3 comprising the amino acid sequence GYGAFYY (SEQ ID N0:61);
• LCDRl comprising the amino acid sequence SVSSSVSSIYLH (SEQ ID NO:53);
• LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:54);
• LCDR3 comprising the amino acid sequence IQYSGYPLT (SEQ ID NO:55); HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCD R2 comprising the amino acid sequence RVNPNRGGTTYNQKFEG (SEQ ID NO: 66), and HCDR3 comprising the amino acid sequence TNWLDY (SEQ ID NO: 66).
• LCDRl comprising the amino acid sequence SVSSSVSSIYLH (SEQ ID NO:53); LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:53);
• LCDR3 comprising the amino acid sequence QVYSGYPLT (SEQ ID NO:56);
• HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCD R2 comprising the amino acid sequence RVNPNRRGTTYNQKFEG (SEQ ID NO:68);
• HCD R3 comprising the amino acid sequence ANWLDY (SEQ ID NO:69).
• LCDRl comprising the amino acid sequence SVSSSVRSIYLH (SEQ ID NO:57);
• LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:54); LCDR3 comprising the amino acid sequence QVYRGYPLT (SEQ ID NO:54);
• HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCD R2 comprising the amino acid sequence RVNPYRGSTTYNQKFEG (SEQ ID NO: 70);
• HCDR3 comprising the amino acid sequence ANILDY (SEQ ID NO:71); and wherein said monoclonal antibody or antigen-binding fragment thereof binds an epitope within the ⁇ -chain of said human c-Met and induces internalization of cell surface human c-Met.
• any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof comprising three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), wherein said three LCDRs and said three HCDRs are selected from the group consisting of: a) LCDRl comprising the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49); LCDR2 comprising the amino acid sequence GTSYLAS (SEQ ID NO:
• LCDR3 comprising the amino acid sequence QQWSSYPYS (SEQ ID NO:51);
• HCDRl comprising the amino acid sequence GYTFTSRYIH (SEQ ID NO:59);
• HCD R2 comprising the amino acid sequence WIYPVTGDTYYNEKFKG (SEQ ID NO: 60);
• HCD R3 comprising the amino acid sequence GYGAFYY (SEQ ID NO:61);
• LCDRl comprising the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49);
• LCDR2 comprising the amino acid sequence GTSYLAS (SEQ ID NO:50); LCDR3 comprising the amino acid sequence QQWSSYPYS (SEQ ID NO:50);
• HCDRl comprising the amino acid sequence GYTFTSRYIH (SEQ ID NO:59;
• HCD R2 comprising the amino acid sequence WIYPVTGDTYYIEKFKG (SEQ ID NO:62);
• HCDR3 comprising the amino acid sequence GYGAFFY (SEQ ID NO:63); c) LCDRl comprising the amino acid sequence SVSSSISSTNLH (SEQ ID NO:49);
• LCDR2 comprising the amino acid sequence GTSRLRS (SEQ ID NO:52);
• LCDR3 comprising the amino acid sequence QQWSSYPYS (SEQ ID N0:51);
• HCDRl comprising the amino acid sequence GYTFTSRYIH (SEQ ID NO:59); HCD R2 comprising the amino acid sequence
• HCDR3 comprising the amino acid sequence GYGAFYY (SEQ ID N0:61).
• any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof comprising three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), wherein said three LCDRs and said three HCDRs are selected from the group consisting of: a) LCDRl comprising the amino acid sequence SVSSSVSSIYLH (SEQ ID NO:53); LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:53);
• LCDR3 comprising the amino acid sequence IQYSGYPLT (SEQ ID NO:55);
• HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCD R2 comprising the amino acid sequence RVNPNRGGTTYNQKFEG (SEQ ID NO:66);
• HCD R3 comprising the amino acid sequence TNWLDY (SEQ ID NO:67);
• LCDRl comprising the amino acid sequence SVSSSVSSIYLH (SEQ ID NO:53); LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:54);
• LCDR3 comprising the amino acid sequence QVYSGYPLT (SEQ ID NO:56); HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:56);
• HCD R2 comprising the amino acid sequence RVNPNRRGTTYNQKFEG (SEQ ID NO:68);
• HCD R3 comprising the amino acid sequence ANWLDY (SEQ ID NO:69).
• LCDRl comprising the amino acid sequence SVSSSVRSIYLH (SEQ ID NO:57);
• LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:54);
• LCDR3 comprising the amino acid sequence QVYRGYPLT (SEQ ID NO:58);
• HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:65); HCD R2 comprising the amino acid sequence
• RVNPYRGSTTYNQKFEG (SEQ ID NO: 70);
• HCDR3 comprising the amino acid sequence ANILDY (SEQ ID N0:71). Any one of the foregoing monoclonal antibodies, or antigen-binding fragments thereof comprising three light chain complementarity determining regions (LCDRs) and three heavy chain complementarity determining regions (HCDRs), wherein:
• LCDRl comprising the amino acid sequence SVSSSVSSIYLH (SEQ ID NO:53);
• LCDR2 comprising the amino acid sequence STSNLAS (SEQ ID NO:54); LCDR3 comprising the amino acid sequence IQYSGYPLT (SEQ ID NO:54);
• HCDRl comprising the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCD R2 comprising the amino acid sequence RVNPNRGGTTYNQKFEG (SEQ ID NO:66); and HCDR3 comprising the amino acid sequence TNWLDY (SEQ ID NO:66).
• LCDRl comprises the amino acid sequence SVSSSVSSIYLH (SEQ ID NO:
• LCDR2 comprises the amino acid sequence STSNLAS (SEQ ID NO:54);
• LCDR3 comprises the amino acid sequence QVYSGYPLT (SEQ ID NO:56);
• HCDRl comprises the amino acid sequence GYTFTDYYMH (SEQ ID NO:65);
• HCDR2 comprises the amino acid sequence RVNPNRRGTTYNQKFEG (SEQ ID NO: 68); and HCDR3 comprises the amino acid sequence ANWLDY (SEQ ID NO: 68).
• LCVR light chain variable region
• HCVR heavy chain variable region
• any one of the foregoing monoclonal antibodies, or antigen-binding fragments thereof comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR comprises SEQ ID NO:94 and said HCVR comprises SEQ ID NO:96.
• LCVR light chain variable region
• HCVR heavy chain variable region
• any one of the foregoing monoclonal antibodies, or antigen-binding fragments thereof comprising a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein said LCVR comprises SEQ ID NO:95 and said HCVR comprises SEQ ID NO:97.
• LCVR light chain variable region
• HCVR heavy chain variable region
• LCVR and said HCVR comprise amino acid sequences which are selected from the group consisting of: a) LCVR is SEQ ID NO : 1 and HCVR is SEQ ID NO: 13; b) LCVR is SEQ ID NO:2 and HCVR is SEQ ID NO: 14; c) LCVR is SEQ ID NO: 3 and HCVR is SEQ ID NO: 15; d) LCVR is SEQ ID NO:4 and HCVR is SEQ ID NO: 16; e) LCVR is SEQ ID NO:5 and HCVR is SEQ ID NO: 17; and f) LCVR is SEQ ID NO:6 and HCVR is SEQ ID NO: 18.
• LCVR and said HCVR comprise amino acid sequences selected from the group consisting of: a) LCVR is SEQ ID NO: 1 and HCVR is SEQ ID NO: 13; b) LCVR is SEQ ID NO:2 and HCVR is SEQ ID NO: 14; and c) LCVR is SEQ ID NO: 3 and HCVR is SEQ ID NO: 15.
• LCVR and said HCVR comprise amino acid sequences selected from the group consisting of: a) LCVR is SEQ ID NO:4 and HCVR is SEQ ID NO: 16; b) LCVR is SEQ ID NO:5 and HCVR is SEQ ID NO: 17; and c) LCVR is SEQ ID NO: 6 and HCVR is SEQ ID NO: 18.
• any one of the foregoing monoclonal antibodies wherein said antibody comprises a light chain and a heavy chain wherein the light chain and the heavy chain comprise amino acid sequences which are selected from the group consisting of: a) light chain is SEQ ID NO:25 and heavy chain is SEQ ID NO:37; b) light chain is SEQ ID NO:26 and heavy chain is SEQ ID NO:38; c) light chain is SEQ ID NO:27 and heavy chain is SEQ ID NO:39; d) light chain is SEQ ID NO:28 and heavy chain is SEQ ID NO:40; e) light chain is SEQ ID NO:29 and heavy chain is SEQ ID NO:41; and f) light chain is SEQ ID NO:30 and heavy chain is SEQ ID NO:42.
• any one of the foregoing monoclonal antibodies wherein said antibody comprises a light chain and a heavy chain wherein the light chain and the heavy chain comprise amino acid sequences which are selected from the group consisting of a) light chain is SEQ ID NO:25 and heavy chain is SEQ ID NO:37; b) light chain is SEQ ID NO:26 and heavy chain is SEQ ID NO:38; and c) light chain is SEQ ID NO:27 and heavy chain is SEQ ID NO:39.
• any one of the foregoing monoclonal antibodies wherein said antibody comprises two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 28 and each heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• any one of the foregoing monoclonal antibodies wherein said antibody comprises two light chains and two heavy chains, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 29 and each heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• a monoclonal antibody or antigen-binding fragment thereof that competes with any of the foregoing c-Met monoclonal antibodies or antigen-binding fragments thereof for binding to c-Met.
• Such competing monoclonal antibody or antigen-binding fragment thereof can bind to the same epitope of c-Met as any one of the foregoing c-Met monoclonal antibodies or antigen-binding fragments thereof.
• the monoclonal antibody or antigen-binding fragment thereof competes with an antibody which comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof competes with an antibody which comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• the monoclonal antibody or antigen-binding fragment thereof which induces reduction of total human c-Met and phosphorylated human c-Met in hepatocyte growth factor-independent tumor cells that constitutively overexpress said human c-Met comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof which induces reduction of total human c-Met and phosphorylated human c-Met in hepatocyte growth factor- independent tumor cells that constitutively overexpress said human c-Met comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• the monoclonal antibody or antigen-binding fragment thereof which induces reduction of total human c-Met and phosphorylated human c-Met in hepatocyte growth factor-independent tumor cells that constitutively phosphorylate said human c-Met.
• the monoclonal antibody or antigen- binding fragment thereof which induces reduction of total human c-Met and phosphorylated human c-Met in hepatocyte growth factor-independent tumor cells that constitutively phosphorylate said human c-Met comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof which induces reduction of total human c-Met and phosphorylated human c-Met in hepatocyte growth factor-independent tumor cells that constitutively phosphorylate said human c-Met comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• the monoclonal antibody or antigen-binding fragment thereof which does not induce shedding of human c-Met extracellular domain comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof which does not induce shedding of human c-Met extracellular domain comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• the monoclonal antibody or antigen-binding fragment thereof which does not protect tumor cells expressing human c-Met from staurosporine-induced apoptosis comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof which does not induce HGF-like biological agonist activities include tumor cell proliferation, tumor cell motility, tumor cell invasion, tubulogenesis, angiogenesis, and anti-apoptotic effects.
• the monoclonal antibody or antigen-binding fragment thereof which does not induce HGF-like biological agonist activities comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• a pharmaceutical composition comprising any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier, diluent, or excipient.
• the monoclonal antibody or antigen-binding fragment thereof for use in therapy comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof for use in therapy comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• the monoclonal antibody or antigen-binding fragment thereof for use in treating a cancer in a human comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof for use in treating a cancer in a human comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• the monoclonal antibody or antigen-binding fragment thereof for use in treating a cancer in a human in combination with another therapeutic comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 28 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 40.
• the monoclonal antibody or antigen-binding fragment thereof for use in treating a cancer in a human in combination with another therapeutic comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 29 and the heavy chain comprises the amino acid sequence of SEQ ID NO: 41.
• a pharmaceutical composition comprising any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof and a pharmaceutically acceptable carrier, diluent, or excipient.
• a method of treating a cancer comprising administering to a human patient in need thereof an effective amount of any one of the foregoing monoclonal antibodies or antigen-binding fragments thereof.
• a full-length antibody as it exists naturally is an immunoglobulin molecule comprising 2 heavy (H) chains and 2 light (L) chains interconnected by disulfide bonds.
• the amino terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein.
• the carboxy -terminal portion of each chain defines a constant region primarily responsible for effector function.
• Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino- terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
• the 3 CDRs of the light chain are referred to as "LCDRl, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain are referred to as "HCDRl, HCDR2, and HCDR3.”
• the CDRs contain most of the residues which form specific interactions with the antigen.
• the numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions are in accordance with the well-known Kabat numbering convention.
• Light chains are classified as kappa or lambda, and are characterized by a particular constant region as known in the art.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the isotope of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively.
• IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4. Each heavy chain type is characterized by a particular constant region with a sequence well known in the art.
• the term "monoclonal antibody” refers to an antibody that is derived from a single copy or clone including, for example, any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
• Mabs of the present invention preferably exist in a homogeneous or substantially homogeneous population. Complete Mabs contain 2 heavy chains and 2 light chains.
• Antigen-binding fragments of such monoclonal antibodies include, for example, Fab fragments, Fab' fragments, F(ab') 2 fragments, single chain Fv fragments, and one-armed antibodies comprising a light chain and a heavy chain.
• Monoclonal antibodies and antigen-binding fragments thereof of the present invention can be produced, for example, by recombinant technologies, phage display technologies, synthetic technologies, e.g., CDR-grafting, or combinations of such technologies, or other technologies known in the art.
• Antibody compounds refers to Mabs and Fabs disclosed herein. Additional antibody compounds exhibiting similar functional properties according to the present invention can be generated by conventional methods. For example, mice can be immunized with human c-Met or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding and functional properties similar to or the same as the antibody compounds disclosed herein can be assessed by the methods disclosed in Examples 2-19, below.
• Antigen-binding fragments can also be prepared by conventional methods.
• human engineered antibodies refers to monoclonal antibodies and antigen-binding fragments in addition to the antibody compounds disclosed herein that have binding and functional properties according to the invention similar to those disclosed herein, and that have framework regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody.
• Framework region or “framework sequence” refers to any one of framework regions 1 to 4.
• Human engineered antibodies and antigen-binding fragments encompassed by the present invention include molecules wherein any one or more of framework regions 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human framework regions 1 to 4, is present.
• this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework region 1, 2, and 3, etc., are substantially or fully human.
• Substantially human frameworks are those that have at least about 80% sequence identity to a known human germline framework sequence.
• the substantially human frameworks have at least about 85%, about 90%, about 95%, or about 99% sequence identity to a known human germline framework sequence.
• Fully human frameworks are those that are identical to a known human germline framework sequence.
• Human framework germline sequences can be obtained from ImMunoGeneTics (IMGT) via their website http://imgt.cines.fr, or from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, ISBN 012441351.
• germline light chain frameworks can be selected from the group consisting of: All, A17, A18, A19, A20, A27, A30, LI, LlI, L12, L2, L5, L15, L6, L8, 012, 02, and 08
• germline heavy chain framework regions can be selected from the group consisting of: VH2-5, VH2-26, VH2-70, VH3-20, VH3-72, VHI-46, VH3-9, VH3-66, VH3-74, VH4-31, VHI-18, VHI-69, VI- 13-7, VH3-11, VH3- 15, VH3-21, VH3-23, VH3-3O, VH3-48, VH4-39, VH4-59, and VH5-5I.
• Human engineered antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present invention can be generated using several different methods.
• the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework.
• the sequence identity of the new framework will generally be at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to the sequence of the corresponding framework in the parent antibody compound.
• frameworks having fewer than 100 amino acid residues one, two, or three amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody.
• the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. ScL USA 88:2869.
• Additional references describing methods useful in humanizing mouse antibodies include U.S. Patent Nos. 4,816,397; 5,225,539, and 5,693,761; computer programs ABMOD and ENCAD as described in Levitt (1983) J. MoI. Biol. 168:595-620; and the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536.
• the framework amino acid of the human germ-line sequence that is being used (the "acceptor framework") is replaced by a framework amino acid from a framework of the parent antibody compound (the "donor framework”):
• any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid in a three dimensional immunoglobulin model.
• Another approach to generating human engineered antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as or better than those of the parent antibody compounds.
• Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties.
• Single mutations producing improved properties can be combined to assess their effects in combination with one another.
• amino acid substitution within the frameworks is restricted to one, two, or three positions within any one or more of the 4 light chain and/or heavy chain framework regions disclosed herein.
• amino acid substitution within the CDRs is restricted to one, two, or three positions within any one or more of the 3 light chain and/or heavy chain CDRs. Combinations of the various changes within these framework regions and CDRs described above are also possible.
• epitope refers to a specific arrangement of amino acids located on a peptide or protein to which an antibody or antibody fragment binds.
• Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics.
• Epitopes can be linear, i.e., involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.
• the epitopes disclosed herein can consist of, consist essentially of, or comprise the amino acid sequences disclosed in Example 3.
• Monoclonal antibodies or antigen-binding fragments thereof that "compete” with the molecules disclosed herein are those that bind human c-Met at site(s) that are identical to, or overlapping with, the site(s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human c-Met can be bound to a solid support. Then, an antibody compound or antigen binding fragment thereof of the present invention and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such invention antibody compound are added. One of the two molecules is labeled.
• the labeled compound and the unlabeled compound bind to separate and discrete sites on c-Met, the labeled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be lowered. If the unlabeled compound is present in excess, very little, if any, labeled compound will bind.
• competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to c-Met by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%.
• Monoclonal antibodies or antigen-binding fragments thereof that bind substantially the same epitope(s) of c-Met as the monoclonal antibodies or antigen- binding fragments disclosed herein are those that bind human c-Met at site(s) that are overlapping with the site(s) at which the present molecules bind.
• Methods that facilitate identification of monoclonal antibodies or antigen-binding fragments thereof that bind substantially the same epitope of c-Met as the c-Met monoclonal antibodies or antigen- binding fragments disclosed herein are well known in the art and are described, for example, in PCT International Publication WO 00/64946.
• c-Met or "human c-Met” refers to any human c-Met, as well as functionally active, mutated forms thereof.
• the structure of the c-Met is depicted schematically as:
• PSI Plexin, Semaphorins, and Integrins domain IPT: 4 Immunoglobulins, Plexins, and Transcription factor domains TM: Transmembrane region
• amino acids 1-24 comprise the signal sequence.
• the mature protein begins at amino acid 25 (E).
• the Sema domain consists of approximately 500 amino acid residues at the N-terminus of c-Met, and contains the ⁇ - chain (amino acid residues 25-307) and part of the ⁇ -chain (amino acid residues 308-519).
• the term "inhibit” means the ability to substantially antagonize, prohibit, prevent, restrain, slow, disrupt, eliminate, stop, reduce, or reverse the biological effects of c-Met.
• treating means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related symptoms, conditions, or disorders.
• Acute events and chronic conditions may be treated.
• an antibody or antigen-binding fragment thereof is administered at the onset of a symptom, disorder, condition, or disease, and is discontinued when the acute event ends.
• a chronic symptom, disorder, condition, or disease is treated over a more protracted time frame.
• an antibody compound of the present invention which, upon single or multiple dose administration to a patient, provides the desired treatment or prevention.
• Therapeutically effective amounts of the present antibody compounds can comprise an amount in the range of from about 0.1 mg/kg to about 20 mg/kg per single dose.
• a therapeutically effective amount for any individual patient can be determined by the health care provider by monitoring the effect of the antibody compounds on a biomarker, such as cell surface c-Met in tumor or non- tumor tissues, tumor regression, etc. Analysis of the data obtained by these methods permits modification of the treatment regimen during therapy so that optimal amounts of antibody compounds, whether employed alone or in combination with one another therapeutic agent, are administered, and so that the duration of treatment can be determined as well. In this way, the dosing/treatment regimen can be modified over the course of therapy so that the lowest amounts of antibody compounds used alone or in combination that exhibit satisfactory tumor reducing effectiveness are administered, and so that administration of such compounds is continued only so long as is necessary to successfully treat the patient.
• the antibody compounds of the present invention can be used as medicaments in human medicine, administered by a variety of routes. Most preferably, such compositions are for parenteral administration.
• Such pharmaceutical compositions can be prepared by methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 19 th ed. (1995), A. Gennaro et al, Mack Publishing Co., and comprise one or more antibody compounds disclosed herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
• tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by aberrant cell growth/proliferation.
• cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias.
• c-Met pathways can be induced by transcriptional up regulation, c-Met gene amplification, specific genetic alterations, or ligand-dependent autocrine or paracrine mechanisms.
• the most frequent cause of constitutive c-Met activation in human tumors is increased protein expression as a consequence of transcriptional upregulation, in the absence of gene amplification.
• amplification of the MET gene with consequent protein overexpression and constitutive kinase activation, has been reported in a number of human primary tumors, including gastric and oesophageal carcinomas, non-small-cell lung (NSCL) carcinomas, and medulloblastomas.
• HGF hypoxia-derived neurotrophic factor
• Missense c-MET mutations are found in all individuals with well-characterized hereditary papillary renal cell carcinomas (PRCC) and in a small subset (13%) of sporadic PRCC samples. Some of the mutations possess oncogenic potential due to increased kinase activity. Trisomy of chromosome 7, where both HGF and c-MET genes reside, occurs frequently in PRCC, and results in non-random duplication of the mutant c-MET allele. In addition, somatic c-MET mutations have been identified in other human cancers, including gastric, head and neck, liver, ovarian, non- small cell lung and thyroid cancers, as well as in metastases of some of these cancers.
• the c-MET gene is often amplified in breast, liver, brain, colorectal, gastric, lung and stomach cancers, which is correlated to disease progression in some patients.
• HGF/c-MET signaling has been documented in a wide range of human malignancies, including bladder, breast, cervical, colorectal, endometrial, esophageal, gastric, head and neck, kidney, liver, lung, nasopharyngeal, ovarian, pancreatic, prostate and thyroid cancers, as well as cholangiocarcinoma, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, Kaposi's sarcoma, leiomyosarcomas, and MFH/fibrosarcoma.
• abnormal HGF and/or c-Met expression has also been reported in hematological malignancies such as acute myelogenous leukemia, adult T-cell leukemia, chronic myeloid leukemia, lymphomas and multiple myeloma, as well as other tumors such as melanoma, mesothelioma, Wilms' tumor, glioblastomata, and astrocytomas (summarized in Liu et al. (2008) Expert Opin. Investig. Drugs 17(7):997-1011).
• the c-Met antibodies of the present invention can inhibit both HGF-dependent and HGF-independent tumors.
• the humanized IgG2 and IgG4 and murine IgG (also sometimes referred to as mlgGl) control antibodies are isotype control antibodies unrelated to the present c-Met antibodies.
• Antibodies C8, DI l, and optDl 1 are murine antibodies.
• human HGF is obtained from R&D Systems (#294).
• amino acid sequences of the light chain and heavy chain variable regions, the complete light and heavy chains, and the respective encoding nucleotide sequences of the foregoing, of the present human engineered antibodies are listed below in the section entitled "Amino Acid and Nucleotide Sequences.”
• the light chain and heavy chain CDR amino acid sequences are shown in Tables 1 and 2, respectively.
• Xi is Y or R, and X 2 is A or R;
• X 3 is S or R
• X 4 is I or Q
• X 5 is Q or V
• X 6 is S or R
• X 7 is N, I, or R, and X 8 is K or P;
• X 9 is Y or F;
• Xio is N or Y, Xn is G or R, and Xi 2 is G or S;
• Xi3 is T or A, and X 14 is W or I;
• Consensus sequences for Dl 1- and C8- antibody light and heavy chain variable regions are:
• Antibodies are transiently expressed in HEK293 EBNA cells (Edge BioSystems,
• Transfected cells are cultured in standard serum-free medium containing geneticin (G418) and tobramycin for 48 to 120 hours at 37°C after transfection.
• Antibodies are purified on a 60 mL rProtein A Sepharose column (Amersham Biosciences, #17-1279-04) by following the manufacturer's instructions, and further concentrated and purified by size exclusion chromatography (XK50/60 Superdex200, Pharmacia) with phosphate buffered saline (PBS), pH 7.4, as the mobile phase.
• Antibodies are then filtered using Millev-GV, PVDF membranes, 0.22 ⁇ m, 33mm, (Millipore, # SLGVO33RS), and stored at 4 to 8°C.
• Murine IgGl c-Met antibody 5D5 (U.S. Patent No. 5,686,292), discussed in many of the examples below, is isolated and purified from hybridoma HB-11895 obtained from the American Type Culture Collection, Manassas, VA, as described above.
• Example 2 Binding Kinetics of c-Met Antibodies to Various c-Met Extracellular Domains
• ECDs extracellular domains
• Fc fusion proteins with a flag- and His-tag (Flis-tag) at the C-terminus of the Fc (SEQ ID NOs:72-74).
• Fc-tag flag- and His-tag
• a Biacore® 2000 instrument is used to measure the binding kinetics of c-Met antibodies to human, cynomolgus monkey, and rat c-Met ECDs. Measurements are performed at 25°C. Samples are dissolved in HBS-EP buffer (150 mM sodium chloride, 3 mM EDTA, 0.005% (w/v) surfactant P-20, and 10 mM N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES) at pH 7.4; #BR-1001-88).
• HBS-EP buffer 150 mM sodium chloride, 3 mM EDTA, 0.005% (w/v) surfactant P-20, and 10 mM N-2-hydroxyethylpiperazine- N'-2-ethanesulfonic acid (HEPES) at pH 7.4; #BR-1001-88).
• F(ab') 2 fragment of goat anti human IgG, F(ab') 2 fragment specific is immobilized on flow cells 1 to 4 of a CM5 sensor chip at a level of 4000 response units (RUs) using amine coupling chemistry to capture anti-c-Met antibodies.
• Binding is evaluated using multiple cycles. Each cycle is performed at a flow rate of 50 ⁇ L/min., and consists of the following steps: injection of about 10 ⁇ L of a c-Met antibody at 10 ⁇ g/mL aiming at a capture of 40-100 RUs, injection of 250 ⁇ L of human, cynomolgus, or rat c-Met-Flis-Fc ECD (starting at 100 nM and using two-fold serial dilutions for each cycle) followed by 20 min. for dissociation, and regeneration using about 30 ⁇ L of 10 mM glycine hydrochloride, pH 1.5.
• association and dissociation rates for each cycle are evaluated using a "1 : 1 (Langmuir) binding" model in the BIAevaluation software, version 4.1.
• a heterogeneous ligand model is used to fit the data adequately; therefore, two binding affinities are obtained.
• the epitopes of the present c-Met antibodies are mapped by a combination of hydrogen-deuterium exchange mass spectrometry (HDXMS) (Yamada et al. (2002) Rapid Commun. Mass Spectrom. 16(4):293-299) and diethyl pyrocarbonate (DEPC) labeling (Mendoza et al. (2008) Analy. Chem. 80(8):2895-2904).
• Hydrogen-deuterium exchange reaction of human c-Met Sema domain is carried out in the presence or absence of c-Met antibodies. Sema domain regions that gain less deuterium in the presence of an antibody than in its absence are identified as the epitope(s) for the antibody.
• DEPC can react with amino groups of surface-exposed lysine or histidine residues in the Sema domain, forming ethyl carbamate lysine or histidine. If these amino acids are located in the epitope region, they will be protected, and will not react with DEPC upon antibody binding. This helps to further localize and/or confirm the epitope regions determined by HDXMS.
• the Sema domain of human c-Met is expressed with a FHs tag at the C-terminus (SEQ ID NO:76) in HEK293 EBNA cells and purified as described in Examples 1 and 2.
• the purified protein is then stored at 4 0 C in PBS at pH 7.4.
• This domain binds to the c-Met antibodies of the present invention with affinity similar to that of full length human c-Met ECD, indicating that the epitopes for these antibodies are located in this region of human c-Met.
• HDXMS Assays 4 ⁇ L of deglycosylated or desialylated c-Met Sema domain/antibody mixture are mixed with 16 ⁇ L of 100% D 2 O (Acros, Code 166310500; 80% D during exchange), and incubated at ambient temperature for 90 seconds. The exchange is then quenched with 50 ⁇ L of 0.5% (v/v) formic acid in water at O 0 C. The quenched solution is immediately treated with 2 ⁇ L of 5 mg/mL (v/v) pepsin solution (Sigma, Cat. # P6887) at O 0 C for 3.5 or 4 min.
• the digested solution is immediately manually injected onto an RP-HPLC column (Polymer Laboratories, Part #1912-1802; 2.1x50mm, 1000 A pore size, 8 ⁇ M particle size).
• the HPLC buffer stream from the HPLC pump (Waters, 2795 HPLC) passes through a metal tube (approximately 1 mL) to a manual injector.
• the column eluate is then passed to a Micromass LCT Premier or SYNAPT mass spectrometer.
• the metal tube, injector loop, and column are submerged in an ice water bath.
• the column is equilibrated with 99% A (0.05% (v/v) aqueous TFA (trifluoroacetic acid) and 1% B (0.04% (v/v) TFA in acetonitrile) at a flow rate of 0.2 mL/min.
• An isocratic gradient elution is performed for 1 min., from 1 to 10% B over 1 min., to 40% B over 12 min., to 90% B over 4 min. with a 3 min. hold, and then rapidly returned to the initial conditions.
• the mass spectrometry is performed on a Micromass LCT Premier
• Mass Spectrometer with a positive spray, W mode, and the following settings: a capillary voltage of 1.5 kV, a cone voltage of 100 V, Aperture 1 of 25 V, a mass range of 200 to 2000, a desolvation temperature of 150 0 C, and a desolvation gas flow of 500 L/h.
• the mass spectrum of each peptic peptide of c-Met is obtained after D/H exchange with or without c-Met antibody. The average mass of each peptide is calculated according to the isotopic distribution of the most intense ion peak.
• Each digested solution is acidified with 2 ⁇ L of 10% (v/v) acetic acid solution. 2 ⁇ L of 100 mg/mL TCEP (tris(2-carboxyethyl) phosphine hydrochloride (Sigma, Cat. # C4702-2G) solution are added to the unreduced digest without added dithiothreitol.
• TCEP tris(2-carboxyethyl) phosphine hydrochloride (Sigma, Cat. # C4702-2G) solution are added to the unreduced digest without added dithiothreitol.
• TCEP tris(2-carboxyethyl) phosphine hydrochloride (Sigma, Cat. # C4702-2G) solution are added to the unreduced digest without added dithiothreitol.
• Each of the solutions is subjected to LC/MS analysis using a Waters Acquity UPLC and Waters SYNAPT Mass spectrometer.
• a gradient elution is performed in isocratic state for 2 min., from 1 to 25% B over 25 min., to 45% B over 10 min., to 90% B over 1 min., with 1.5 min. hold (at the same period of time, flow rate of 0.3 mL/min.), and then rapidly returned to 1% B.
• Mab C8-H241 appears to bind a conformational epitope comprising four regions of the c-Met Sema domain, located in the ⁇ -chain of the human c-Met extracellular domain (amino acid residues 25-307 of SEQ ID NO:75): 12 iVVDTYYDDQL 13 o (SEQ ID NO:77),
• Mab C8-H241 binds c-Met by interacting with one or more amino acid residues within:
• I23 DTYYDDi 28 (SEQ ID NO:81) inclusive, I 44 HVFPHNHTADIQSI 56 (SEQ ID NO: 82) inclusive, I 92 FINFi 95 (SEQ ID NO:83) inclusive, and 2 20 KETKDGFM 227 (SEQ ID NO:84) inclusive.
• Binding of Mab C8-H241 to the region I 44 HVFPHNHTADIQS I 56 renders it capable of binding both human (SEQ ID NO:75) and cynomolgus monkey (amino acids 25 to 932 of SEQ ID NO:73) c-Met extracellular domain with comparable affinity, but not rat or mouse c-Met, up to 100 nM of antibody in binding assays.
• Mab Dl 1-8B8 binding to the ⁇ -chain of the c-Met Sema domain appears to be localized to one region, i.e., amino acid residues within a linear epitope within the amino acid sequence S 4 YKTGPVLEHPDCFPCQDCSSKANLIO 7 (SEQ ID NO:85) inclusive, more particularly the region 95 CFPCQDCSSKAi 05 (SEQ ID NO:86) inclusive.
• the epitope for Mab Dl 1-8B8 is further confirmed by epitope extraction experiments (Dhungana et al. (2009) Methods MoI Biol. 524:87-101).
• c-Met Sema domain is digested with porcine trypsin (Promega) and the digest is then mixed with biotinylated Mab DI l- 8B8, using the EZ-LinkTM Sulfo-NHS-LC-Biotin kit (Pierce, Prod. #1754210), to bind the c-Met peptides.
• Biotinylated Dl 1-8B8 with or without bound c-Met peptides is captured by high capacity Streptavidin agarose resin (Thermo Scientific, Prod. # 20359). The bound peptides are released by 0.15% formic acid (v/v) in H 2 O, and then identified by LC/MS.
• Example 4 Preferential Binding of Antibodies to c-Met vs. RON and PlexinA2 ECDs
• the human proteins with the greatest sequence identity to c-Met are RON and Plexin A2. This experiment compares the specificity of c-Met antibody binding to c-Met, RON, and PlexinA2 ECDs.
• Wells of 96-well EIA/RIA high binding plates (Costar, #2592) are coated with 100 ⁇ L of 1 ⁇ g/mL human c-Met extracellular domain (ECD)-Fc-Flis fusion (SEQ ID NO:72), RON-ECD-Fc (R&D Systems, #1947-MS), or PlexinA2 -ECD-Fc (Abnova, Taipei, Taiwan #H00005362-P01) in coating buffer (BioFX Labs, # COAT-1000-01) overnight at 4°C.
• ECD c-Met extracellular domain
• SEQ ID NO:72 human c-Met extracellular domain
• RON-ECD-Fc R&D Systems, #1947-MS
• PlexinA2 -ECD-Fc (Abnova, Taipei, Taiwan #H00005362-P01) in coating buffer (BioFX Labs, # COAT-1000-01) overnight at 4°C.
• the wells are aspirated and non-specific binding sites are blocked by adding 200 ⁇ L of blocking buffer (Tris-buffered saline, pH 8.0, with 0.05% (v/v) polysorbate 20 (Tween-20) (TBS-T) (BioFX Labs, # WSHW-1000-01) plus 1% (w/v) bovine serum albumin (BSA) (Jackson Immuno, #001-000-162) and incubating for 1 hour at room temperature. After the plates are washed three times with wash buffer (TBS-T), 100 ⁇ L/well of 1 :6 serial dilutions of c-Met antibodies in blocking buffer (starting from 100 ⁇ g/mL) are added and incubated at room temperature for 2 hours.
• blocking buffer Tris-buffered saline, pH 8.0, with 0.05% (v/v) polysorbate 20 (Tween-20) (TBS-T) (BioFX Labs, # WSHW-1000-01) plus 1% (w
• the plates are washed and incubated with 100 ⁇ L/well of HRP-conjugated goat anti-human F(ab) 2 IgG (Jackson ImmunoResearch Labs #109-036-097) in blocking buffer for 90 min. After the plates are washed, 100 ⁇ L/well of substrate solution (BioFx, #TMBW- 1000-01) are added and the plates are incubated for 10 min. at room temperature. 100 ⁇ L/well of stop solution (BioFx, # LSTP-1000-01) are added to stop the reaction. The colorimetric signals are developed and read at 450 nm using a SpectraMax 190 plate reader (Molecular Devices, Sunnyvale, CA). c-Met antibody binding to c-Met, RON and PlexinA2 ECDs is proportional to color signal production.
• In vitro binding assays are used to determine inhibition of HGF binding to c-Met by the present c-Met antibodies.
• Wells of 96-well EIA/RIA high binding plates (Costar, #2592) are coated with 100 ⁇ L of human c-Met ECD-Fc-Flis (SEQ ID NO:72) (2 ⁇ g/mL) in Dulbecco's phosphate buffered saline (DPBS) overnight at room temperature; washed four times with wash buffer (Tris-buffered saline, pH 8.0, with 0.05% (v/v) polysorbate 20 (TWEEN ® - 20) (TBS-T) (BioFX Labs, # WSHW-1000-01) in a plate washer; blocked by adding 300 ⁇ L of blocking buffer (TBS-T plus 1% (w/v) bovine serum albumin (BSA) (Jackson Immuno, #001-000-162); and incubating for 60 min.
• wash buffer Tris-buffered saline, pH 8.0, with 0.05% (v/v) polysorbate 20 (TWEEN ®
• Blocking buffer is then removed from the wells and 50 ⁇ L of antibodies in blocking buffer at final concentrations as indicated in Table 7 are added into each well, respectively. Blocking buffer is added to the HGF-only control wells. The plates with c-Met antibodies are incubated for 90 min. at 37°C. 50 ⁇ L of human hepatocyte growth factor (HGF) (R&D Systems, #294) in blocking buffer at a final concentration of 10 ng/mL are then added to each well except the antibody-only control wells. The plates containing the c-Met antibody/HGF mixture are incubated on a plate shaker for two hours at room temperature. The wells are then washed four times with TBS-T in a plate washer.
• HGF human hepatocyte growth factor
• AVG average
• STDErr Standard Error
• a phosphorylated c-Met (p-Met) ELISA assay is used to measure the agonistic activity of various c-Met antibodies in the absence of HGF, as well as of Mab 5D5 and HGF itself.
• A549 cells (ATCC, #CCL-185) are cultured in Ham's F12K + 2 mM glutamine (Invitrogen, # 21127-022) + 10% FBS medium (Invitrogen, #10082). Cells are plated at 6 x 10 4 cells/well in the full serum medium as above in 96-well plates and incubated overnight at 37°C under 5% (v/v) CO 2 . The medium is removed from the wells and the cells are starved in 100 ⁇ L of Ham's F12K + 2 mM glutamine medium + 0.5% FBS for six hours at 37°C under 5% (v/v) CO 2 .
• Antibodies or HGF are diluted in the starvation culture medium, added at final concentrations as indicated in Table 8, and incubated for 15 min. at 37°C.
• the medium is aspirated and cells are lysed in 50 ⁇ L lysis buffer (10 mM Tris (Invitrogen, #15567-027), 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) (Invitrogen, #15575-038), 50 mM NaF (Sigma, #S1504), 1% (v/v) octylphenoxy polyethoxy ethanol (TRITON ® -X 100; Sigma, #T8787), 2 mM sodium orthovanadate (EMD Chemicals, Gibbstown, NJ, #567540), protease inhibitor (Sigma, St.
• phosphatase inhibitor cocktail I Sigma #P2850
• phosphatase inhibitor cocktail II Sigma #P5726
• HGF binding protein to c-Met results in tyrosine phosphorylation of c-Met molecules and activation of the c-Met signaling pathway.
• human colon cancer cell line HCTl 16 which is responsive to HGF, is used to assess the inhibition of HGF- induced phosphorylation at tyrosine residues 1230, 1234, and 1235 of c-Met by the present c-Met antibodies.
• HCTl 16 cells (ATCC, Manassas, VA, #CCL-247) are resuspended to 150,000 cells/mL in McCoy's 5A medium (Invitrogen, Carlsbad, CA, #16600-082) plus penicillin/streptomycin (Invitrogen, #15140-122) with 10% (v/v) fetal bovine serum (FBS) (Invitrogen, #10082-147).
• 0.2 mL of the resuspended HCTl 16 cells are added to 96-well microtiter plates (Corning, Lowell, MA, #3596) at 30,000 cells/well, and the cells are then incubated for 48 hours at 37°C under 5% (v/v) CO 2 .
• the culture medium is then aspirated and the cells are starved in 100 ⁇ L of McCoy's 5A medium plus penicillin/streptomycin with 0.1% (w/v) BSA for 24 hours at 37°C under 5% (v/v) CO 2 .
• 25 ⁇ L of sodium azide (final concentration of 0.01% (w/v)) (Sigma, #S2002) are added, followed immediately by adding 25 ⁇ L of 8X c-Met antibody dilutions to McCoy's 5 A medium plus penicillin/streptomycin with 0.1% (w/v) BSA, and the cells are incubated for 30 min. at 37°C under 5% (v/v) CO 2 .
• HGF HGF
• cell lysis buffer 10 mM Tris (Invitrogen, #15567-027), 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) (Invitrogen, #15575-038), 50 mM NaF (Sigma, #S1504), 1% (v/v) octylphenoxy polyethoxy ethanol (TRITON ® -X 100; Sigma, #T8787), 2 mM sodium orthovanadate (EMD Chemicals, Gibbstown, NJ, #567540), and complete protease inhibitor, EDTA free (Roche, Basel, Switzerland, #11836170001).
• c-Met capture antibody is diluted in coating buffer (BioFX, Glendora, CA,
• the plates are then washed four times with TBS-T. 100 ⁇ L of 0.5 ⁇ g/mL of pYpYpY1230/1234/1235 c-Met polyclonal antibody (Invitrogen, #44-888G) in blocking buffer are then added to each well, and the plates are incubated for 2 hours at room temperature. Next, the plates are washed four times with TBS-T. 100 ⁇ L of 1/12,000 diluted peroxidase-conjugated goat anti-rabbit IgG polyclonal antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, #111-035-144) in blocking buffer are then added, and the mixture is incubated for 1 hour at room temperature.
• the percentage of inhibition of c-Met tyrosine phosphorylation is set at the average of HGF treatment without c-Met antibody addition (0% inhibition), and 100% inhibition is set at the average of sodium azide treatment (no HGF or c-Met antibody treatment).
• Table 9 shows the average of triplicate treatments per experiment for three experiments with standard errors. The data demonstrate that five of six c-Met antibodies of the present invention significantly inhibit HGF-induced tyrosine phosphorylation of c-Met compared to that of the human IgG2 and IgG4 isotype controls. Table 9
• the experiments described in this example employ fluorescence-activated cell sorting (FACS) analysis to demonstrate that the present c-Met antibodies bind to cell surface c-Met molecules and induce c-Met internalization.
• FACS fluorescence-activated cell sorting
• the MKN45 cells employed constitutively express high levels of total c-Met, and exhibit HGF-independent phosphorylation of c-Met as a result of MET gene amplification.
• c-Met internalization appears to induce c-Met degradation (see Examples 10 and 19), resulting in inhibition of the c-Met signaling pathway.
• Wells of 6-well tissue culture plates (Costar, #3598) are seeded with 1.5 x 10 5 human gastric tumor MKN45 cells (Japan Health Sciences Foundation, Health Science Research Resource Bank, #JCRB0254) in 2 mL of culture medium (RPMI- 1640 (Invitrogen, #11835); 10% (v/v) FBS (Invitrogen, #10082); 2 mM L-glutamine (Invitrogen, #25030); 100 U/500mL penicillin G, and 100 ⁇ g/500 mL streptomycin (Invitrogen, #15140)).
• the plates are incubated for 24 hours at 37°C under 95% relative humidity and 5% (v/v) CO 2 .
• Antibodies are then added to the wells at a final concentration of 5 ⁇ g/mL. After overnight treatment, the culture medium is removed from the wells and replaced with 1 mL of enzyme-free cell dissociation solution (Chemicon, #S-014-B). The cells are collected into centrifuge tubes after being incubated for 5 min. at room temperature, and washed once in culture medium followed by one more wash in binding buffer (DPBS with 1% (w/v) BSA and 0.01% (w/v) sodium azide).
• DPBS with 1% (w/v) BSA and 0.01% (w/v) sodium azide
• a c-Met antibody that recognizes a different epitope from the present c-Met antibodies is labeled by using an Alexa Fluor 488 Monoclonal Antibody Labeling Kit (Molecular Probes, Eugene, OR, #A-20181) according to the supplier's instructions.
• 100 ⁇ L of binding buffer containing 2 ⁇ g/mL of the Alexa Fluor 488- labeled antibody are added to the cells, which are then incubated for 60 min. on ice.
• the cells are then washed once with binding buffer and resuspended in DPBS containing 2 ⁇ g/mL propidium iodide (to stain the dead cells).
• the amount of c-Met molecules remaining on the cell surface is analyzed by FACS analysis, and 10,000 events are acquired for each sample.
• the mean fluorescence intensity on the cell surface reflects the quantity of c-Met molecules that remain on the cell surface after treatment with c-Met antibodies.
• the data from one representative experiment are shown in Table 10.
• % internalization [1- (mean fluorescence of test antibody divided by mean fluorescence of isotype antibody control)] multiplied by 100].
• Complementary internalization results are obtained by confocal microscopy using fluorescently labeled murine C8 antibody in MKN45 and Caki-1 cells.
• C8-H241, C8-6, C8-CO-16, and optDl l antibodies also induce internalization of c-Met in NIH3T3 cells transfected with nucleic acid encoding human c-Met kinase domain mutation Ml 149T.
• Antibodies C8-H241, C8-6, C8-CO-16, and murine Dl 1 also induce internalization of c-Met in non-small cell lung cancer H 1437 cells containing c- Met juxtamembrane domain mutation R988C. Both mutations cause gain-of-function constitutive activation of the c-Met.
• Example 9 Antibody Inhibition of Ligand-Independent Tumor Cell Proliferation In Vitro
• c-Met and isotype control antibodies are diluted with culture medium (RPMI- 1640 (Invitrogen, #11835), 10% (v/v) FBS, 2 mM L-glutamine (Invitrogen, #25030), 100 U/500mL penicillin G, and 100 ⁇ g/500 mL streptomycin (Invitrogen, #15140)) to achieve 2X the final concentrations indicated in Table 11, and 50 ⁇ L of the 2X antibody solutions are added to each well of 96-well tissue culture plates (PerkinElmer# 1450-517).
• culture medium RPMI- 1640 (Invitrogen, #11835), 10% (v/v) FBS, 2 mM L-glutamine (Invitrogen, #25030), 100 U/500mL penicillin G, and 100 ⁇ g/500 mL streptomycin (Invitrogen, #15140)
• MKN45 cells (Japan Health Sciences Foundation, Health Science Research Resource Bank, #JCRB0254) are maintained in the medium indicated above, and are resuspended to 1x10 5 cells/mL in the same medium. 50 ⁇ L of this MKN45 resuspension are added to each well to achieve 5xlO 3 cells/well. The plates are then incubated for 48 hours at 37°C under 95% relative humidity and 5% (v/v) CO 2 . For the last six hours of the culture, the cells are pulsed with 3 H-thymidine (MP Biomedicals, Solon, OH #24066) at 1 ⁇ ci/well at 37°C, 95% relative humidity, 5% (v/v) CO 2 .
• 3 H-thymidine MP Biomedicals, Solon, OH #24066
• the medium is then removed and the cells are washed once with DPBS. After this, 200 ⁇ L of Optiphase Supermix (PerkinElmer, #1200-439) are added to each well. The plates are sealed and incubated for at least one hour at room temperature. 3 H-thymidine incorporated in the cells is counted for one min. using a scintillation counter.
• AVG average
• CPM count per minute
• STDErr Standard Error
• This example investigates whether treatment of MKN45 cells with c-Met antibodies of the present invention results in reduction of phosphorylated c-Met (p-Met) and total c-Met.
• this assay is used to determine if c-Met antibody treatment induces the shedding of c-Met ECD into MKN45 -conditioned medium.
• c-Met and isotype control antibodies are diluted with culture medium (RPMI- 1640 (Invitrogen, #11835), 10% (v/v) FBS (Invitrogen, #10082), 2 mM L-glutamine (Invitrogen, #25030), 100 U/500mL penicillin G, and 100 ⁇ g/500 mL streptomycin (Invitrogen, #15140)) to achieve 2X the final concentrations indicated in Table 12, and 50 ⁇ L of the 2X antibody solutions are added to each well of 96-well tissue culture plates (Costar, #3596).
• culture medium RPMI- 1640 (Invitrogen, #11835), 10% (v/v) FBS (Invitrogen, #10082), 2 mM L-glutamine (Invitrogen, #25030), 100 U/500mL penicillin G, and 100 ⁇ g/500 mL streptomycin (Invitrogen, #15140)
• MKN45 cells (Japan Health Sciences Foundation, Health Science Research Resource Bank, #JCRB0254) are maintained in the medium indicated above and are resuspended to 1x10 5 cells/mL in the same medium. 50 ⁇ L of this MKN45 resuspension are added to each well to achieve 5xlO 3 cells/well. The plates are then incubated for 24 hours at 37°C under 95% relative humidity and 5% (v/v) CO 2 , and cell lysates are prepared as described in Example 7. In addition, the conditioned medium from each treatment is collected for c-Met-ECD quantitation. p-Met and total c-Met levels in the cell lysates are determined by ELISA, and normalized to lysate protein concentration (as determined by BCA, Pierce #23225).
• Phosphorylation of c-Met at tyrosine residues 1230, 1234, and 1235 is determined as described in Example 7.
• a c-Met capture antibody is diluted in coating buffer (BioFX, Glendora, CA, COAT-1000-01) to 2 ⁇ g/mL. 110 ⁇ L of the diluted antibody are added per well to ELISA plates (Greiner Bio-One, Monroe, NC, #655081) and the plates are incubated overnight at 4°C. The wells are aspirated, washed twice with TBS-T, and then blocked with 200 ⁇ L of blocking buffer (TBS-T plus 2%
• the plates are washed four times with TBS-T.
• 100 ⁇ L of 1/12,000 diluted peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA, #016-030-084) in blocking buffer are then added, and the mixture is incubated for 1 hour at room temperature.
• the plates are then washed six times with TBS-T.
• 100 ⁇ L of 3,3 ',5,5 '- tetramethylbenzidine solution (BioFX, ⁇ MBW-1000-01) are added to each well, followed by the addition of 100 ⁇ L stop solution (BioFX, #LSTP- 1000-01).
• the plates are read at 450 nm with a 570 nm correction using a SpectraMax 190 plate reader (Molecular Devices, Sunnyvale, CA).
• the standard curve is established using 4-para- meter analysis, and sample values are determined using SOFTmax Pro 3.1.2 software (Molecular Devices).
• Caki-1 renal carcinoma cells proliferate in response to HGF.
• the activation of c-Met in Caki-1 cells by c-Met antibodies in the absence of HGF is examined in this example to assess the agonist activity of c-Met antibodies of the present invention.
• Wells of 96-well tissue culture plates are seeded with 5,000 human kidney clear cell carcinoma Caki-1 cells (ATCC, #HTB-46) in McCoy's 5A culture medium (Invitrogen, #16600) supplemented with 10% (v/v) FBS, 2 mM L-glutamine (Invitrogen, #25030), 100 U/500mL penicillin G and 100 ⁇ g/500 mL streptomycin (Invitrogen, #15140).
• cells are starved in low serum medium (0.5% (v/v) FBS) for another 24 hours.
• the cells are then cultured in the presence of anti-c-Met and control antibodies in low serum medium at final concentrations as indicated in Table 13 for 24 hours at 37°C, 95% relative humidity, 5% (v/v) CO 2 .
• the cells are pulsed with 3 H-thymidine (MP Biomedicals, Solon, OH #24066) at 1 ⁇ ci/well at 37°C, 95% relative humidity, 5% (v/v) CO 2 .
• the medium is removed and the cells are washed once with DPBS.
• AVG average
• CPM count per minute
• STDErr Standard Error
• the agonist activity of the present c-Met antibodies is further assessed in primary human hepatocytes (PHH), which are HGF -responsive, in the absence of HGF.
• PHL primary human hepatocytes
• Cryopreserved, plateable PHH cells (KQG Celsis, Chicago, IL, RD#00002) are thawed at 37°C and resuspended to 175,000 cells/mL in InVitroGRO CP medium (Celsis, #Z99029) with torpedo antibiotic mix (Celsis, #Z99000).
• 0.2 mL of resuspended PHH cells are added per well to collagen I coated 96-well microtiter plates (BD, Franklin Lakes, NJ, #354407) at 35,000 cells/well, and the cells are incubated for 24 hours at 37°C, 5% (v/v) CO 2 .
• the culture medium is then aspirated and 150 ⁇ L of InVitroGRO HI medium (Celsis, #Z99009) with torpedo antibiotic mix plus 0.1% (w/v) BSA are added per well, plus 50 ⁇ L of c-Met and control antibodies in a final concentration range of 10 ⁇ g/mL to 0.0032 ⁇ g/mL, or HGF at a final concentration of 200 ng/mL, in InVitroGRO HI medium with torpedo antibiotic mix plus 0.1% (w/v) BSA.
• the agonist activity of the present c-Met antibodies is significantly lower than that of the 5D5 control c-Met antibody.
• Agonist antibody 5D5 stimulates PHH proliferation in a dose-dependent manner, with a 5-fold increase at a concentration of 3 ⁇ g/mL.
• HGF stimulates a 5-fold increase in H-thymidine uptake.
• Mab C8-H241 does not induce proliferation even when used at 10 ⁇ g/mL.
• HGF induces tubular morphologenic changes in HepG2 cells grown in Matrigel TM (Becton-Dickinson, #354234), an extracellular matrix material containing components of the basement membrane.
• Matrigel TM Becton-Dickinson, #3542314
• the HGF-like agonist activity of antibodies of the present invention in inducing tubular morphologenic changes in HepG2 cells is assessed.
• HepG2 cells ATCC, #HB-8065
• DMEM fetal bovine serum
• a MatrigelTM solution (MatrigelTM, Becton-Dickinson) diluted in Opi- MEMI (Invitrogen, #31985) supplemented with 10% (v/v) FBS, 2 mM L-glutamine (Invitrogen, #25030), 100 U/500mL penicillin G and 100 ⁇ g/500 mL streptomycin (Invitrogen, #15140) are plated in wells of 96-well tissue culture plates (Costar, #3596). After the MatigelTM solution solidifies, 2000 HepG2 cells in 50 ⁇ L of culture medium supplemented with 10% serum are added.
• c-Met and control antibodies at a final concentration of 50 ⁇ g/mL, or HGF at a final concentration of 50 ng/mL, are added to the cells.
• the cells are grown for 4 days at 37°C in a humidified atmosphere containing 5% (v/v) CO 2 . After 4 days, the top medium is removed and replaced with 50 ⁇ L of 1 mg/mL p-Iodonitro-tetrazolium violet (Sigma, #18377) in PBS, and the cells are incubated for another 48 hours under the same conditions. Photographs are taken of the stained 32 mm area, and analyzed using Image-Pro Plus 6 (Media Cybernetics, Inc., MD).
• HGF and control agonist antibody 5D5 induce approximately 5-fold tubular morphologenic changes in HepG2 cells as compared to the isotype control.
• present c-Met antibodies C8-H241 and C8-C0I6 do not induce significant tubular morphologenic changes in HepG2 cells under the same conditions, while c-Met antibody C8-6 induces only a low level of stimulation.
• H441 Effect of Antibodies on Cell Motility; DU145 Scatter Assay and H441 Cell Scratch Assay
• DU 145 prostate cancer cells dissociate from each other and H441 cells fill in a scratch made in a confluent cell layer.
• H441 cells exhibit a high level of c-Met expression and constitutive phosphorylation of this receptor, but are still HGF-responsive.
• the following experiments assess the agonist effect of antibodies of the present invention on cell motility in a scatter assay and a scratch assay.
• DU145 cells (ATCC, # HTB-81) grown in MEM medium (Invitrogen, #11095) + 10% FBS (Invitrogen, #10082) at 37°C under 5% (v/v) CO 2 are plated at 2 x 10 3 cells/well in 70 ⁇ L volume in black ViewPlate 96-well plates (Perkin Elmer, Waltham, MA, #6005182) and incubated overnight at 37°C under 5% (v/v) CO 2 .
• c-Met and control antibodies are diluted in the cell culture medium and added at a final concentration of 20 ⁇ g/mL, and HGF is added at a final concentration of 20 ng/mL, each in 30 ⁇ L volume with twelve replicates, and incubated for 48 hours at 37°C under 5% (v/v) CO 2 .
• the medium is then aspirated and cells are fixed in 2% formaldehyde for 15 min. at room temperature.
• Wells are washed three times with PBS, and 50 ⁇ L of 5 U/mL Alexa Fluor 488 phalloidin (Invitrogen, #A12379) are added for 30 min. at room temperature.
• H441 Cell Scratch Assay For the H441 Scratch assay, H441 cells (ATCC, # HTB-174) are grown in RPMI-
• the confluent cell layers on the bottom of the wells are scratched with 5 mL pipette tips in the middle of each well, and floating cells are aspirated. The remaining cells are washed IX with low serum medium. Low serum medium is added, and the scratched areas are imaged using a bright field microscope with a 4X objective. These gaps are defined as Gaps at 0 hours.
• the testing antibodies are added to the cells at a final concentration of 10 ⁇ g/mL, followed by incubation at 37°C under 5% CO 2 (v/v) for 16 hours.
• HGF is tested at a final concentration of 200 ng/mL.
• Each treatment group is tested at least in duplicate wells.
• the scratched areas are imaged again using a bright field microscope at 16 hours. These gaps are defined as Gaps at 16 hours.
• the effect of c-Met antibodies or HGF on the movement of H441 cells to fill the gaps are calculated as follows:
• Treatment group (Gap at 0 hour - Gap at 16 hours) x 100
• HGF and agonist c-Met antibodies stimulate invasion of c-Met bearing cells.
• This example examines the agonist activity of the present c-Met antibodies in a cell invasion assay employing HepG2 cells, which are HGF-responsive in an invasion assay.
• HepG2 cells (ATCC, #HB-8065) are starved overnight in serum-free MEM medium (Invitrogen, #11095) and then 5 x 10 4 cells in a total volume of 500 ⁇ L are added to each well of the top chamber of a matrigel invasion chamber (BD, Franklin Lakes, NJ, #354483), with the bottom chamber containing antibodies in a total volume of 750 ⁇ L of serum- free medium at a concentration of 10 ⁇ g/mL, or HGF at 50 ng/mL in serum- free medium, followed by incubation for forty-eight hours at 37 0 C under 5% (v/v) CO 2 .
• serum-free MEM medium Invitrogen, #11095
• 5 x 10 4 cells in a total volume of 500 ⁇ L are added to each well of the top chamber of a matrigel invasion chamber (BD, Franklin Lakes, NJ, #354483), with the bottom chamber containing antibodies in a total volume of 750 ⁇ L of serum- free medium at
• Non- invading cells are removed from the top chamber with a swab, followed by membrane fixation with 95% ethanol and staining with 0.2% (w/v) crystal violet. After washing and drying, the number of invading cells is counted using Image-Pro Plus 6 Manual Tag (Media Cybernetics, Inc., MD) software analysis of photographs taken of stained cells with a 2.5X objective.
• Image-Pro Plus 6 Manual Tag Media Cybernetics, Inc., MD
• HGF and agonist c-Met antibodies protect cells from staurosporine-induced cell death.
• This example examines the agonist activity of c-Met antibodies of the present invention in a staurosporine-induced apoptosis assay employing HGF -responsive Caki-1 cells.
• Caki- 1 cells (ATCC, #HTB-46) are grown as described in Example 11 , seeded at 1 x 10 4 cells/well in 96 well plates (Costar, #3596) in culture medium, and pre-treated with antibodies (diluted from 30000 ng/mL to 3 ng/mL in the cell culture medium), or HGF (diluted from 225 ng/mL to 0.02 ng/mL), for one hour followed by treatment with 0.1 ⁇ M staurosporine (final concentration) for forty-eight hours at 37° C. The medium is aspirated and the cells are lysed for 30 min. with 0.2 mL of the lysis buffer component of the Cell Death Detection ELISA kit (Roche Applied Science, Indianapolis, IN, #11774425001).
• This kit utilizes 20 ⁇ L of each lysate to measure cell death by detection of cytoplasmic histone-associated DNA fragments as determined by the absorption at 450 nm. A higher optical density at 450 nm indicates greater apoptosis.
• HGF and agonist c-Met antibodies stimulate angiogenesis.
• c-Met antibodies of the present invention are evaluated for this functional agonist property in the
• ADSC Adipose-derived stem cells
• ECFC endothelial colony forming cells
• ADCS Longza, Allendale, NJ, # PT-5006
• basal medium MCDB- 131 medium (Sigma, St. Louis, MO #M8537) + 30 ⁇ g/mL L-ascorbic
• ECFC EndGenitor Technologies, Inc., Indianapolis, IN #EGT-ECFC 100506
• HGF and antibodies are diluted in the cell culture medium and added to separate wells at the following final concentrations: HGF: 100 ng/mL; antibodies: 10 ⁇ g/mL.
• the HGF antibody (R&D Systems #AB-294-NA) is also added at 10 ⁇ g/mL final concentration.
• the cells are incubated for an additional 4 days at 37 0 C.
• the wells are aspirated, and 100 ⁇ L/well of 1% paraformaldehyde are added, followed by incubation for 20-30 min.
• Cells are washed three times with PBS-BSA (0.1% BSA, Invitrogen #15260-037) and treated with 50 ⁇ L of 1 ⁇ g/mL anti-human CD31 antibody (R&D Systems, #AF806) for 1 hour at 37°C or overnight at 4°C.
• Cells are washed twice with PBS-BSA and treated with 50 ⁇ L of 4 ⁇ g/mL anti-sheep IgG AlexaFluor488 conjugate (Invitrogen, #A11015) for 1 hour at room temperature. Cells are washed twice with PBS-BSA and stained with 100 ⁇ L of Hoechst3342 dye and read on a Cellomics ArrayScan (Thermo Fisher Scientific, Waltham, MA). vHCS View Version 1.4.6 software is used to determine total tube areas, which are used to evaluate the effect of the various antibodies and HGF on stimulation of angiogenesis.
• MKN45 cell and U87MG human glioblastoma cell mouse xenograft models, respectively.
• MKN45 cells constitutively express high levels of c-Met and c-Met phosphorylation in the absence of HGF.
• U87MG cells secrete HGF in an autocrine manner, and are HGF -responsive.
• MKN45 cells (Japan Health Sciences Foundation, Health Science Research Resource, #JCRB0254) are expanded in culture as described in Example 8, trypsinized to single cells, harvested, and resuspended in PBS.
• Two million MKN45 cells in PBS are injected subcutaneously into the rear flank of athymic nude mice (Harlan, Indianapolis, IN).
• c-Met antibodies and corresponding IgG2 and IgG4 antibodies are diluted in PBS, pH 7.2, and administered on a weekly basis by intravenous injection starting from 3 or 7 days after tumor cell implantation at 1, 5, or 20 mg/mL. Inhibition of tumor cell growth is determined by three dimensional caliper measurement of tumor volumes twice weekly during the course of treatment. Body weight is measured as a general measurement of toxicity.
• U87MG cells (ATCC, #HTB-14) are grown in MEM (Invitrogen, #11095) at 37 C, expanded in culture, trypsinized to single cells, harvested, and resuspended in PBS (Invitrogen, # 14190). Five million cells are injected subcutaneously into the rear flank of athymic nude mice (Harlan, Indianapolis, IN).
• c-Met antibodies are diluted in PBS, and administered on a weekly basis by intravenous injection at the doses indicated in Table 22 starting 7 days after tumor cell implantation.
• Control IgG4 antibody is administered at 10 mg/kg. Inhibition of tumor cell growth is determined by three dimensional caliper measurement of tumor volumes twice weekly during the course of treatment. Body weight is measured as a general measurement of toxicity.
• a dose-dependent inhibition of tumor cell growth by C8-H241 is also observed in the HGF-dependent U87MG cell xenograft model, as summarized in Table 22.
• Maximum % Inhibition represents the percent inhibition of tumor growth compared to treatment with corresponding IgG4 control antibody (0% inhibition).
• C8-H241 antibody also inhibits H441 non-small cell lung cancer xenograft tumor growth 58% and 60%, respectively.
• H441 cells exhibit a high level of c-Met expression and constitutive phosphorylation of c-Met, but are still responsive to HGF.
• MKN45 cells are expanded in culture as described in Example 8, trypsinized, and harvested. Two million MKN45 cells in PBS are injected subcutaneous Iy into the rear flank of athymic nude mice (Harlan, Indianapolis, IN). c-Met antibody C8-H241 is diluted in PBS, pH 7.2, and administered by intravenous injection eight days after tumor cell implantation at 2.5, 5, 10, 20, and 40 mg/kg. Control antibody hIgG4 is administered at 40 mg/kg. After 24 hours of treatment, tumors are removed, flash frozen, stored temporarily at -80°C, and lysed in lysis buffer (5 mM ethylenediaminetetraacetic acid
• EDTA 5 mM ethyleneglycol-bis(b-aminoethyl)-N,N,N',N'-tetracetic acid
• HEPES 20 mM sodium pyrophosphate
• 150 mM NaCl 20 mM NaF
• 1% (v/v) octylphenoxy polyethoxy ethanol TRITON ® -X 100
• complete protease inhibitor EDTA free (Roche, Basel, Switzerland, # 1836153) phosphatase inhibitor cocktail I (Sigma #P2850), and phosphatase inhibitor cocktail II (Sigma #P5726)).
• a c-Met capture antibody is diluted in Bup H coating buffer (Thermo Fisher Scientific, Waltham, MA, #28382) to 2 ⁇ g/mL. 100 ⁇ L of the diluted antibody is added per well to ELISA plates (ThermoFisherScientific, Waltham, MA #439454), and the plates are incubated overnight at 4°C. The wells are aspirated, washed twice with TBS-T, and then blocked with 200 ⁇ L of blocking buffer (TBS-T plus 2% (w/v) BSA) for 1 hour at room temperature. The plates are washed twice with TBS- T.
• Bup H coating buffer Thermo Fisher Scientific, Waltham, MA, #28382
• 100 ⁇ L of the diluted antibody is added per well to ELISA plates (ThermoFisherScientific, Waltham, MA #439454), and the plates are incubated overnight at 4°C.
• the wells are aspirated, washed twice with T
• dilutions of tumor lysates or c-Met extracellular domain are added, and the plates are incubated overnight at 4°C.
• the plates are then washed three times with TBS-T.
• 100 ⁇ L of 0.5 ⁇ g/mL of biotinylated Mab 5D5 (as second c-Met antibody that binds a different c-Met epitope from the capture antibody) diluted in blocking buffer are then added to each well, and the plates are incubated for 2 hours at room temperature. Next, the plates are washed three times with TBS-T.
• a c-Met capture antibody is diluted in Bup H coating buffer (Thermo Fisher Scientific, Waltham, MA, #28382) to 2 ⁇ g/mL. 100 ⁇ L of the diluted antibody are added per well to ELISA plates (ThermoFisherScientific, Waltham, MA #439454), and the plates are incubated overnight at 4° C. The wells are aspirated, washed twice with TBS-T, and then blocked with 200 ⁇ L of blocking buffer (TBS-T plus 2% (w/v) BSA) for 1 hour at room temperature. The plates are washed twice with TBS-T.
• MKN45 cell lysates are added, and the plates are incubated overnight at room temperature.
• the plates are then washed three times with TBS-T.
• 100 ⁇ L of 0.5 ⁇ g/mL anti-pY1349 c-Met antibody (Cell Signaling Technology, Danvers, Massachusetts, #3121) diluted in blocking buffer are then added to each well, and the plates are incubated for 2 hours at room temperature.
• the plates are washed three times with TBS-T.
• 100 ⁇ L of 1/10,000 diluted peroxidase conjugated anti-rabbit IgG Jackson ImmunoResearch Laboratories, West Grove, PA, #111-035-144) in blocking buffer are then added, and the mixture is incubated for 1 hour at room temperature.
• the plates are then washed three times with TBS-T. 100 ⁇ L of 3,3 ,5,5 '-tetramethylbenzidine solution (BioFX, #TMBW- 1000-01) are added to each well, followed by the addition of 100 ⁇ L stop solution (BioFX, ⁇ STP-IOOO-Ol). The plates are read at 450 nm using a 5 SpectraMax 250 plate reader (Molecular Devices, Sunnyvale, CA) with SOFTmax Pro 3.1.2 software (Molecular Devices).
• D11-C27G3 E ⁇ VLTQSPGTLSLSPGERATLSCSVSSSISSTNLHWYQQKPGQAPRLL ⁇ YGTSRLRS GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWSSYPYSFGQGTKLEIK
• D11-C27G3 (SEQ ID NO:21) CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTC TCCTGCAAGGCTTCTGGCTACACCTTCACAAGTAGGTATATACACTGGGTGCGACAGGCC CCTGGACAAGGGCTTGAGTGGATGGGATGGATTTATCCTGTAACTGGTGATACTTACTAC AGAGAGCCTTTCAAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTAC ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGCTAT GGGGCTTTTTACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCC
• Rat cMet-ECD-Fc-Flis (SEQ ID NO:74)
• DIl- Antibody Light Chain CDR2 GTSXiLX 2 S (SEQ ID NO:87), wherein Xi is Y or R, and X 2 is A or R;
• SVSSSVX 3 SIYLH (SEQ ID NO:88), wherein X 3 is S or R;
• X 4 X 5 YX 6 GYPLT (SEQ ID NO:89), wherein X 4 is I or Q, X 5 is Q or V, and Xe is S or R;
• DIl- Heavy Chain Antibody CDR2 WIYPVTGDTYYX 7 EX 8 FKG (SEQ ID NO: 90), wherein X 7 is N, I, or R, and Xs is K or P;
• GYGAFX 9 Y (SEQ ID NO:91), wherein X 9 is Y or F;
• RVNPXioRXiiXi 2 TTYNQKFEG (SEQ ID NO:92), wherein X i0 is N or Y, Xn is G or R, and X 12 is G or S;
• Xi 3 NXi 4 LDY (SEQ ID NO:93), wherein X i3 is T or A, and X 14 is W or I;

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Immunology (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Pharmacology & Pharmacy (AREA)
• Molecular Biology (AREA)
• Animal Behavior & Ethology (AREA)
• Biochemistry (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Oncology (AREA)
• Hematology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Epidemiology (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)

Priority Applications (19)

Applications Claiming Priority (4)

Publications (1)

Family

ID=41435259

Family Applications (1)

Country Status (28)

Cited By (31)

Families Citing this family (41)

Citations (4)

Family Cites Families (20)

• 2009
  • 2009-11-13 PA PA20098849001A patent/PA8849001A1/es unknown
  • 2009-11-15 JO JOP/2009/0424A patent/JO3097B1/ar active
  • 2009-11-16 AR ARP090104438A patent/AR074360A1/es unknown
  • 2009-11-17 TW TW098139025A patent/TWI477284B/zh not_active IP Right Cessation
  • 2009-11-18 EA EA201170716A patent/EA020398B1/ru not_active IP Right Cessation
  • 2009-11-18 EP EP09752708.9A patent/EP2358755B1/en active Active
  • 2009-11-18 PT PT97527089T patent/PT2358755E/pt unknown
  • 2009-11-18 DK DK09752708.9T patent/DK2358755T3/en active
  • 2009-11-18 PL PL09752708T patent/PL2358755T3/pl unknown
  • 2009-11-18 ES ES15175051.0T patent/ES2663825T3/es active Active
  • 2009-11-18 TR TR2018/02841T patent/TR201802841T4/tr unknown
  • 2009-11-18 MX MX2011005400A patent/MX2011005400A/es active IP Right Grant
  • 2009-11-18 HU HUE09752708A patent/HUE026058T2/en unknown
  • 2009-11-18 KR KR1020117011522A patent/KR101334450B1/ko not_active IP Right Cessation
  • 2009-11-18 BR BRPI0922800A patent/BRPI0922800A2/pt not_active IP Right Cessation
  • 2009-11-18 JP JP2011537557A patent/JP5688027B2/ja active Active
  • 2009-11-18 AU AU2009316742A patent/AU2009316742B2/en not_active Ceased
  • 2009-11-18 CA CA2743508A patent/CA2743508C/en not_active Expired - Fee Related
  • 2009-11-18 NZ NZ592215A patent/NZ592215A/xx not_active IP Right Cessation
  • 2009-11-18 SI SI200931262T patent/SI2358755T1/sl unknown
  • 2009-11-18 CN CN200980146173.1A patent/CN102216333B/zh active Active
  • 2009-11-18 WO PCT/US2009/064881 patent/WO2010059654A1/en active Application Filing
  • 2009-11-18 EP EP15175051.0A patent/EP2963058B1/en active Active
  • 2009-11-18 US US12/620,617 patent/US8217148B2/en active Active
  • 2009-11-18 ES ES09752708.9T patent/ES2549760T3/es active Active
• 2011
  • 2011-05-02 IL IL212633A patent/IL212633A/en not_active IP Right Cessation
  • 2011-05-16 ZA ZA2011/03586A patent/ZA201103586B/en unknown
  • 2011-11-24 HK HK11112740.5A patent/HK1158229A1/xx not_active IP Right Cessation
• 2012
  • 2012-06-25 US US13/531,858 patent/US8398974B2/en active Active
• 2015
  • 2015-09-28 HR HRP20151019TT patent/HRP20151019T1/hr unknown
  • 2015-10-02 CY CY20151100880T patent/CY1116886T1/el unknown

Patent Citations (4)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events