EA045516B1 - MONOCLONAL ANTIBODIES TO C-MET - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to monoclonal antibodies against human c-Met, a hepatocyte growth factor receptor, and to uses of such antibodies, particularly to their use in the treatment of cancer.

Prior Art

The c-Met protein is a membrane-associated receptor tyrosine kinase. The predominantly single-chain precursor is posttranslationally cleaved to yield the mature form of the c-Met heterodimer, which consists of an extracellular α chain (50 kDa) and a longer transmembrane β chain (145 kDa) that are linked by a disulfide bond (Birchmeier et al. 2003. Nat Rev Mol Cell Biol 4:915). The extracellular portion of c-Met consists of three types of domains. The N-terminal domain of SEMA is formed from the entire α-subunit and part of the β-subunit and has homology with semaphorin proteins. The SEMA domain is followed by a cysteine-rich domain, and then 4 immunoglobulin (Ig)-like domains. The cytoplasmic part contains a juxtamembrane kinase domain and a C-terminal tail, which is essential for signal transduction into the cell. The only known high-affinity ligand for c-Met, hepatocyte growth factor (HGF), is expressed primarily by fibroblasts under normal conditions (Li and Tseng 1995. J Cell Physiol 163:61) and tumor cells (Ferracini et al. 1995. Oncogene 10:739). HGF (also called scattering factor: SF) is synthesized as a precursor that is proteolytically converted into the active a/p heterodimer. Based on the crystal structure of the receptor binding fragment, it is believed that HGF binds to c-Met as a dimer (Chirgadze et al. 1999. Nat Struct Biol 6:72). The HGF α-chain binds in c-Met with high affinity to the Ig-like domain, while the HGF β-chain binds in c-Met with low affinity to the SEMA domain (Basilico et al. 2008. J Biol Chem 283:21267). The latter interaction is responsible for dimerization of c-Met and activation of the receptor tyrosine kinase upon binding to the active HGF heterodimer. Autophosphorylation of the receptor results in a unique docking site for the recruitment of effectors, of which the binding of Gab1 (growth factor receptor-associated protein 2 [Grb2]-associated protein 1) binding is essential for subsequent steps of the major c-signaling pathways. Met (Comoglio et al. 2008. Nat Rev Drug Discov 7:504):

Ras-ERK1/2 pathway: proliferation;

Ras-Rac pathway: invasion, motility, epithelial-mesenchymal transition;

PI3K-Akt pathway: viability.

c-Met is expressed on the surface of epithelial and endothelial cells in many organs during embryogenesis and in adulthood, including the liver, pancreas, prostate, kidney, muscle, and bone marrow. Activation of c-Met plays an essential role in the so-called invasive growth program, which consists of a number of processes including proliferation, motility, angiogenesis and protection against apoptosis (Boccaccio and Comoglio 2006. Nat Rev Cancer 6:637). These c-Met-regulated processes occur under normal physiological conditions during embryonic development, during repair of liver and cardiac lesions, and in pathological cases during tumorigenesis (Eder et al. 2009. Clin Cancer Res 15:2207).

Inappropriate c-Met signaling occurs in virtually all types of solid tumors, such as bladder cancer, breast cancer, cervical cancer, colorectal cancer, stomach cancer, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer glands, kidney cancer and thyroid cancer, as well as in various sarcomas, hematopoietic malignancies and melanomas (Birchmeier et al. 2003. Nat Rev Mol Cell Biol 4:915; Comoglio et al. 2008. Nat Rev Drag Discov 7:504 ; Peruzzi and Bottaro 2006. Clin Cancer Res 12:3657). The underlying mechanisms of cMet oncogenicity typically occur through three different pathways:

autocrine HGF/c-Met loops, overexpression of c-Met or HGF, mutations in the c-Met receptor coding sequence, activating kinases.

Most significantly, c-Met activating mutations have been identified in patients with hereditary papillary renal cancer (Schmidt et al. 1997. Nat Genet 16:68). Constitutive activation of c-Met contributes to one or a combination of proliferative, invasive, life-sustaining, or angiogenic phenotypes of malignancies. Silencing of the endogenously expressed c-Met gene in tumor cells has been shown to result in cessation of proliferation and tumor growth and regression of established metastasis, as well as a reduction in the formation of new metastases (Corso et al. 2008. Oncogene 27:684).

Because c-Met contributes to many stages of malignancy development, from initial development to progression to metastasis, c-Met and its ligand HGF have emerged as leading candidates for targeted anticancer therapy (Comoglio et al. 2008. Nat Rev Drug Discov 7:504; Knudsen and Vande Woude 2008. Curr Opin Genet Dev 18:87). Several strategies are being explored to achieve this goal:

False receptors: HGF or c-Met subregions or molecular analogues may act ant

- 1 045516 gonistically as stoichiometric competitors by blocking ligand binding or receptor dimerization. One example of such an antagonistic HGF subregion is NK4 (Kringle Pharma).

Small molecule tyrosine kinase inhibitors (TKIs): Three c-Met-specific TKIs in various stages of clinical evaluation are ARQ197 (ArQule), JNJ 38877605 (Johnson & Johnson), and PF04217903 (Pfizer).

Monoclonal antibodies that bind HGF, such as AMG102, rilotumumab (Amgen), HuL2G7 (Takeda), and AV-299 (Schering).

Monoclonal antibodies that bind c-Met are described in WO 2005016382, WO 2006015371, WO 2007090807, WO 2007126799 WO 2009007427, WO 2009142738 and van der Horst et al. (van der Horst et al. 2009. Neoplasoa 11:355). MetMAb (Genentech) is a humanized monovalent (single-arm) OA-5D5 antibody that binds the extracellular domain of c-Met, thereby preventing HGF binding and subsequent receptor activation (Jin et al. 2008. Cancer Res 68:4360). In murine xenograft models, MetMAb treatment was found to inhibit tumor growth of HGF-driven orthotopic glioblastoma and subcutaneous pancreatic tumors (Jin et al. 2008. Cancer Res 68:4360; Martens et al. 2006. Clin Cancer Res 12:6144). Antibody h224G11 (Pierre Fabre) (Corvaia and Boute 2009. Abstract 835 AACR 100th Annual Meeting) is a humanized divalent IgG1 antibody against c-Met. The antitumor effect of this antibody was observed in mice (Goetsch et al. 2009. Abstract 2792 AACR 100th Annual Meeting). Antibody CE-355621 (Pfizer) is a human IgG2 that blocks ligand binding by binding to the extracellular domain of c-Met and inhibits HGF-dependent growth in tumor xenograft models (Tseng et al. 2008. J Nucl Med 49:129).

In conclusion, several anti-c-Met products have already been studied, but there is still no anti-c-Met product approved for therapeutic use. There is a need to provide effective and safe products for the treatment of severe c-Met-associated diseases such as cancer.

The essence of the invention

The purpose of the present invention is to provide new highly specific and effective monoclonal antibodies against c-Met for medical use. Antibodies of the invention exhibit c-Met binding characteristics that differ from those of antibodies described in the art. In preferred embodiments, the antibodies of the invention have high affinity for human c-Met, are antagonistic, and have a favorable pharmacokinetic profile for use in human patients.

Brief description of drawings

Fig. 1. Comparison of the sequences of the variable regions of the heavy chains of HuMab antibodies. Based on these sequences, consensus CDR sequences can be determined. These consensus sequences are presented in Table 4.

Fig. 2. Comparison of the sequences of the variable regions of the light chains of HuMab antibodies. Based on these sequences, consensus CDR sequences can be determined. These consensus sequences are presented in Table. 4.

Fig. 3. Binding curves of monovalent and divalent forms of antibodies against c-Met to A431 cells that express c-Met. Data presented are the MFI of one representative experiment. Since IgG1-024 and Uni-068 do not show strong binding to A431 cells, it is not possible to calculate an accurate EC ₅₀ value.

Fig. 4. Antibody binding to c-Met, which is expressed on rhesus epithelial cells. Data presented are the MFI of a single experiment.

Fig. 5. Inhibition of HGF binding to the extracellular domain of the c-Met receptor induced by anti-c-Met antibody. The data shown is one representative experiment.

Fig. 6. Inhibition curves of various anti-c-Met antibodies on HGF binding to cMetSEMA_567His8 obtained using TR-FRET. Data shown are the mean MFI ± standard deviation of three independent experiments.

Fig. 7. Percent inhibition of viable KP4 cells after treatment with anti-c-Met antibody compared to untreated cells (0%). Data shown are percentage inhibition of viable cells from two independent experiments ± standard deviation. IgG11016-022 is the only positive result in one experiment.

Fig. 8. Efficacy of anti-c-Met antibodies in inhibiting tumor growth in the KP4 xenograft model in SCID mice. Mice were treated with 400 cd antibody on day 9 after a weekly maintenance dose of 200 cd. Average tumor sizes per treatment group are presented.

Fig. 9. Efficacy of c-Met binding antibodies to inhibit tumor growth in the KP4 xenograft SCID mouse model. Mice were treated with 400 μg of antibody on day 9, followed by a maintenance dose of 200 μg once weekly. Processing effect on frequency

- 2 045516 holey, from time to time. The percentage of tumor-free mice (tumor size <500 mm ³ ) is presented. Tumor formation was slowed in mice treated with antagonistic antibodies compared to mice treated with control antibodies.

Fig. 10. Efficacy of anti-c-Met antibodies in inhibiting tumor growth in the MKN45 xenograft model in SCID mice. Mice were treated with 40 mg/kg antibody on day 7 and 20 mg/kg antibody on days 14, 21 and 28. Average tumor sizes until 50% of mice reached the cutoff of 700 mm ³ per treatment group are shown.

Fig. 11. Efficacy of anti-c-Met antibodies in inhibiting tumor growth in the MKN45 xenograft model in SCID mice. Mice were treated with 40 mg/kg antibody on day 7 and 20 mg/kg antibody on days 14, 21 and 28. The percentage of mice with tumor size less than 700 mm ³ is presented on the Kaplan-Meier plot. Tumor formation was slowed in mice treated with anti-c-Met antibodies compared with mice treated with an isotype control antibody.

Fig. 12. KP4 viability assay to determine the effect of antibody flexibility on its agonistic activity. The IgA2m(l) format does not induce proliferation, unlike the IgA1 and IgG1 formats of the same antibody. Variants of the anti-c-Met antibody 5D5 were used in this experiment (see US6468529 and Example 2).

Fig. 13. Analysis of flexible mutants in non-reducing SDS-PAGE (069). No aberrant multimers or degradation products were observed, whereas light chain pairing was visible as a 50 kDa band ((LC) ₂ ) in C220S, AC220 in IgG1-IgG3 hinge mutants.

Fig. 14. Antigen-binding ELISA to measure c-Met binding of anti-c-Met antibodies with hinge mutations. Using ELISA, all mutants were shown to bind c-Met with comparable affinities.

Fig. 15. Phosphorylation of c-Met as a recorded value of the agonistic activity of antibodies against c-Met. In fig. Figure 15 shows the results of Western blotting of A549 cell lysates; membranes are stained with antibodies against phosphorylated c-met, total c-Met, or β-actin.

Fig. 16. Proliferation assay using NCI-H441 cells. Cell mass was determined after 7 days of incubation in the presence of antibody or controls and was expressed as a percentage relative to untreated samples (which was set to 100%).

Fig. 17. KP4 viability assay. The effect of anti-cMet antibodies on overall KP4 cell viability was tested. The ability of IgG1-1016-069 to reduce KP4 viability was maintained and/or improved by introducing mutations that reduced antibody flexibility.

Fig. 18. Negative modulation measured by ELISA as total c-Met level in A549 lysates. All variants of antibody (069) retained the ability of negative modulation.

Fig. 19. ADCC assay to compare high and low fucose IgG1-1016-069 antibodies.

Fig. 20. Lack of c-Met antibody binding to cells in whole blood in FACS binding assay. Results are presented for B cells, monocytes and granulocytes.

Detailed Description of the Invention

Definitions

The term c-Met, as used herein, refers to hepatocyte growth factor receptor (Genbank accession number NM 000245) and includes any variants, isoforms, and interspecies homologues of human c-Met that are naturally expressed by cells or expressed on cells transfected with the c gene -Met.

The term immunoglobulin refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four linked by disulfide bonds. The structure of immunoglobulins is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically contains a heavy chain variable region (abbreviated herein as _VH OR VH) and a heavy chain constant region. The heavy chain constant region typically contains three domains, _CH1 , CH2 and CH3. Each light chain typically contains a light chain variable region (abbreviated herein as VL OR VL) and a light chain constant region. The light chain constant region typically contains one domain, CL. The VH and VL regions, which can be further subdivided into hypervariable regions (or hypervariable regions that may be hypervariable in sequence and/or shape of structurally defined loops), also called complementarity-determining regions (CDRs), are interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL typically contains three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J Mol Biol 196, 901-917 (1987). Typically, numbering of amino acid residues in a given region is done using the method described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, Na

- 3 045516 National Institutes of Health, Bethesda, MD. (1991) (phrases such as Kabat or Kabat variable domain residue numbering refer to the numbering system for heavy chain variable domains or light chain variable domains herein). Using this numbering system, the actual linear amino acid sequence of the peptide may contain fewer amino acids or additional amino acids, corresponding to shortening or insertion into the FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of _VH CDR2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82 of FR heavy chains. Kabat residue numbering can be determined for a given antibody by comparing regions of homology between the antibody sequence and a standard Kabat numbering sequence.

In the context of the present invention, the term antibody (AT) means an immunoglobulin molecule, a fragment of an immunoglobulin molecule or a derivative thereof, which has the ability to specifically bind to an antigen under normal physiological conditions, with a half-life of significant periods of time, for example, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 h or more, about 3, 4, 5, 6, 7 days or more, etc., or any other proportionate functionally defined period (such as a period of time sufficient to induce, stimulate, enhance and/or modulate a physiological response, associated with the antibody binding to the antigen, and/or a period of time sufficient for the antibody to induce effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. Antibody constant regions (ATs) can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system, such as C1q, the first component of the classical complement pathway. The c-Met binding antibody may also be a bispecific antibody, diabody, or the like (for a description of diabodies, see, for example, PNAS USA 90(14), 6444-8 (1993)). Indeed, bispecific antibodies, diabodies and the like provided by the present invention can bind to a suitable target in addition to the c-Met site. As stated above, the term antibody as used herein, unless otherwise indicated or otherwise obvious by context, includes fragments of an antibody that retains the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be achieved using fragments of a full-length antibody. Examples of binding fragments encompassed by the term antibody include (i) a Fab' fragment or a Fab fragment, a monovalent fragment consisting of V _L , V _H , C _L AND _CH 1 domains, OR a monovalent antibody described in WO2007059782 (Genmab); (ii) F(ab') ₂ fragments, divalent fragments comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment essentially consisting of VH and C _H 1 domains; (iv) an Fv fragment consisting essentially of the V _L AND VH domains of a SINGLE arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)) which essentially consists of a VH AND domain is called also a domain antibody (Holt et al; Trends Biotechnol. 2003 Nov;21(11):484-90); (vi) camel antibodies or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(1):111-24) and (vii) a dedicated hypervariable region (CDR). Moreover, although the two domains of the Fv fragment, V _L and VH, are encoded by separate genes, they can be combined, using recombinant methods, through a synthetic linker that allows them to create a separate protein chain in which the VL and VH regions are fused to form monovalent molecules (known as single chain antibodies or single chain fragment Fv (scFv), see for example Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)) . Such single chain antibodies are encompassed by the term antibody unless otherwise noted or clearly indicated in the context. Although such fragments are typically included in the antibody designation, they, collectively or individually, constitute unique features of the present invention, demonstrating distinct biological properties and utility. These and other useful antibody fragments in the context of the present invention are discussed later in this document. It should also be understood that the term antibody, unless otherwise noted, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies, and antibody fragments that retain the ability to specifically bind an antigen (antigen-binding fragments ), obtained using known methods such as enzymatic digestion, peptide synthesis and recombinant methods. When produced, an antibody can be of any isotype.

As used herein, the term isotype refers to any class of immunoglobulins (eg, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) that are encoded by heavy chain constant region genes.

In the context of the present invention, the term monovalent antibody means that the antibody molecule is capable of binding to a single antigen molecule, and thus is not capable of crosstalk.

- 4 045516 natural binding to antigens.

An antibody deficient in effector function or an antibody deficient in effector function refers to an antibody that has significantly less or no ability to activate one or more effector mechanisms, such as complement activation or Fc receptor binding. Thus, antibodies deficient in effector function have significantly less or no ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). An example of such an antibody is IgG4.

A c-Met binding antibody is an antibody described above that specifically binds to a c-Met antigen.

As used herein, the term human antibody is intended to include antibodies containing variable and constant regions derived from human immunoglobulin germline sequences. Human antibodies of the invention may include amino acid residues not encoded by human immunoglobulin germline sequences (eg, mutations introduced by nonspecific or site-specific mutagenesis in vitro or somatic mutations in vivo). However, as used herein, the term human antibody is not intended to include antibodies in which CDR sequences derived from the germ line of other mammalian species, such as mice, have been transplanted into human framework sequences.

As used herein, a human antibody is isolated from a germline sequence if the antibody is derived from a system using human immunoglobulin sequences, for example, by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a library of human immunoglobulin genes, and where the selected human antibody is identical in amino acid sequence with the amino acid sequence encoded by the germline immunoglobulin gene by at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98% , or for example at least 99%. Typically, in addition to the heavy chain CDR3, a human antibody derived from a particular human germline sequence will exhibit no more than 20 amino acid differences, such as no more than 10 amino acid differences, such as no more than 9, 8, 7, 6, or 5, for example, no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

In a preferred embodiment, the antibody of the invention is isolated. As used herein, isolated antibody is meant to mean an antibody that is substantially free from other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to c-Met is substantially free from antibodies that specifically bind to antigens other than c-Met). An isolated antibody that specifically binds to an epitope, isoform, or variant of human c-Met may, however, be cross-reactive with other related antigens, such as those from other species (eg, interspecies c-Met homologues). In addition, the isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment of the present invention, two or more isolated monoclonal antibodies having different antigen binding specificities are combined in a well characterized composition.

When used herein in the context of two or more antibodies, the term competes with or cross-competes with means that two or more antibodies compete for binding to c-Met, for example, compete for binding to c-Met in the assay described in the examples herein document. For some antibody pairs, competition in the example assays is observed only when the antibody is coated on a plate and the other antibody is used to compete, rather than vice versa. Also when used herein, the term is intended to compete with such combinations of antibodies.

The term epitope means a protein determinant capable of specifically binding to an antibody. Epitopes typically contain surface groupings of molecules such as amino acids or sugar side chains and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, but not the latter, is impaired in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding (also called immunodominant components of the epitope) and other amino acid residues that are involved in binding, such as amino acid residues that are effectively blocked by the specific antigen binding peptide (in other words, the amino acid residue is within the footprint of the specific antigen binding peptide). peptide).

When used herein, the term monoclonal antibody refers to the drug

- 5,045,516 antibody molecules of one molecular composition. The monoclonal antibody composition exhibits unique binding specificity and affinity for a particular epitope. Accordingly, the term human monoclonal antibody refers to antibodies that exhibit unique binding specificity and that contain variable and constant regions derived from human immunoglobulin germline sequences. Human monoclonal antibodies can be produced by a hybridoma that is a fused immortalized B cell derived from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a human light chain transgene.

As used herein, the term binding in the context of binding an antibody to a specific antigen with a binding affinity corresponding to a K _D of about 10 ^-7 M or less, such as about 10 ^-8 M or less, such as about 10 ^-9 M or less, about 10 ^-10 M or less, or about 10 ^-11 M or less, as determined by, for example, surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using an antigen as a ligand and an antibody as an analyte, which binds to a specific antigen with an affinity corresponding to a K _D that is at least ten times lower, for example 100 times lower, for example at least 1000 times lower, for example at least 10,000 times lower, for example, at least 100,000 times lower than the binding affinity for a nonspecific antigen (eg, BSA, casein) other than a specific antigen or closely related antigen. The amount by which affinity is reduced depends on the _KD of the antibody, so when the _KD of an antibody is very low (that is, the antibody is highly specific), the amount by which the affinity for the antigen is lower than the affinity for a nonspecific antigen may be less than at least tenfold.

The term k _d (c ^-1 ), as used herein, denotes the degree of dissociation constant for a particular antigen-antibody interaction. The specified value is also denoted as kf.

The term k _a (M ^-1 x c ^-1 ), as used herein, denotes the degree of association constant for a particular antigen-antibody interaction.

The term K _D (M), as used herein, denotes the equilibrium dissociation constant of a particular antigen-antibody interaction.

The term K _A (M ^-1 ), as used herein, refers to the equilibrium association constant of a particular antigen-antibody interaction, which is obtained by dividing k _a by kd.

As used herein, the term growth inhibitory (eg, in relation to cells such as tumor cells) is intended to include any measurable reduction in cell growth upon contact with a c-Met binding antibody compared to the growth of the same cells not exposed with a c-Met binding antibody, for example, inhibition of cell culture growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99 %, or 100%. This reduction in cell growth can occur through various mechanisms, such as effector cell phagocytosis, ADCC, CDC, and/or apoptosis.

The present invention also provides antibodies comprising functional variants of the V _L region, V H region, or one or more of the CDR antibodies of the examples. The functional variant V _L , V H , or CDR used in the context of an anti-c-Met antibody allows the antibody to retain at least a substantial portion (at least about 50%, 60%, 70%, 80%, 90%, 95% or greater) of the affinity/avidity and/or specificity/selectivity of the parent antibody and in some cases such anti-c-Met antibody may be associated with greater affinity, selectivity and/or specificity than the parent antibody.

Such functional variants typically retain substantial sequence identity to the parent antibody. The percentage identity between two sequences is a function of the number of identical positions in the sequences (i.e. % homology = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. The percentage of identity between two nucleotide or amino acid sequences can be determined, for example, using an algorithm by E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which is included in the ALIGN program (version 2.0) using the PAM120 residue mass table, with a gap extension penalty of 12 and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

The sequence of the CDR variants may differ from the CDR sequence of the parent antibody sequences by conservative substitutions; for example, at least 10, such as at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 of the substitutions in the variant are conservative substitutions of amino acid residues.

In the context of the present invention, conservative substitutions can be defined by substitutions within classes of amino acids reflected in the following table:

- 6 045516

Amino acid residue classes for conservative substitutions

Acidic residues Asp (D) and Glu (E) Main balances Lys (K), Arg (R), and His (H) Hydrophilic residues uncharged Ser (S), Thr (T), Asn (N), and Gin (Q) Aliphatic residues uncharged Gly (G), Ala (A), Vai (V), Leu (L), and Pe (I) Non-polar residues uncharged Cys (C), Met (M), and Pro (P) Aromatic residues Phe (F), Tyr (Y), and Trp (W)

As used herein, the term recombinant host cell (or simply host cell) is meant to mean a cell into which an expression vector, such as an expression vector encoding an antibody of the invention, is introduced. Recombinant host cells include, for example, transfectomes such as CHO cells, HEK293 cells, NS/0 cells and lymphocyte cells.

The term transgenic non-human animal means a non-human animal having a genome that includes one or more human heavy and/or light chain transgenes or that includes transchromosomes (whether integrated or not integrated into the natural genomic DNA of the animal), and which is capable of express fully human antibodies. For example, a transgenic mouse may have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome such that the mouse produces human anti-c-Met antibodies when immunized with c-Met antigen and/or cells expressing cMet. The human heavy chain transgene can be integrated into the mouse chromosomal DNA, as in the case of transgenic mice, such as HuMAb mice, such as HCo7 mice or HCo12 mice, or the human heavy chain transgene can be maintained extrachromosomal, as in the case of transchromosomal KM mice, as described in WO 02/43478. Similar mice having a large repertoire of human AT genes include HCo7 and HCo20 (see, for example, WO 2009097006). Such transgenic and transchromosomal mice (collectively referred to herein as transgenic mice) are capable of producing multiple isotypes of human monoclonal antibodies to a given antigen (such as IgG, IgA, IgM, IgD and/or IgE) through V-D-J recombination and isotype switching. A non-human transgenic animal can also be used to produce antibodies against a specific antigen by introducing genes encoding the specific antibody, for example by operably linking genes to a gene that is expressed in the animal's milk.

Treatment means the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of alleviating, ameliorating, reversing, or eliminating (cure) symptoms of disease states.

An effective amount means an amount effective at such dosage and for such periods of time as necessary to achieve the intended therapeutic result. The therapeutically effective amount of the anti-c-Met antibody may vary according to factors such as the disease state, age, sex and weight of the individual, as well as the ability of the anti-c-Met antibody to elicit a target response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects exceed any toxic or harmful effects of the antibody or antibody portion.

An idiotypic antibody is an antibody that recognizes unique determinants typically associated with the antigen binding site of the antibody.

Additional Aspects and Embodiments of the Invention

As described above in the first aspect, the invention relates to a monoclonal human antibody that binds human c-Met.

The monoclonal antibodies of the present invention can be produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or can be produced using recombinant DNA techniques. Monoclonal antibodies can also be isolated from a phage antibody library using methods described, for example, in Clackson et al., Nature 352: 624-628 (1991) and in Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, monoclonal antibodies can be obtained from hybridomas prepared from murine splenic B cells obtained from mice immunized with the antigen of interest, for example, in the form of cells expressing the antigen on their surface, or in the form of a nucleic acid encoding antigen of interest. Monoclonal antibodies can also be obtained from hybridomas isolated from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, primates, etc.

In one embodiment, the antibody of the invention is a human antibody. Human monoclonal antibodies directed against c-Met can be produced using trans

- 7 045516 gene or transchromosomal mice, carrying more parts of the human immune system rather than the murine one. Such transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as transgenic mice.

The HuMAb mouse contains the human immunoglobulin gene minilocus, which encodes the immunoglobulin heavy chain (μ and γ) and light chain sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856859 (1994)). Accordingly, mice exhibit reduced expression of murine IgM or K and, in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutations to produce high-affinity human IgG,K monoclonal antibodies (Lonberg, N. et al. (1994) , above; reviewed in Lonberg, N. Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. N. Y. Acad. Sci 764 536-546 (1995)). The production of HuMAb mice is described in detail in Taylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J. et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5545806, US 5569825, US 5625126, US 5633425, US 5789650, US 5877397, US 5661016, US 5814318, US 5874299, US 5770429, US 5545807, WO 98/24 884, WO 94/25585, WO 93/ 1227, WO 92/22645, WO 92/03918 and WO 01/09187.

HCo7 mice contain a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), and a CMD disruption in their endogenous heavy chain genes (as described in Example 1 WO 01/14424), the human kappa light chain KCo5 transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and the human heavy chain HCo7 transgene (as described in US 5,770,429).

HCo12 mice contain a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), and a CMD disruption in their endogenous heavy chain genes (as described in Example 1 WO 01/14424), the human kappa light chain KCo5 transgene (as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996)), and the human heavy chain HCo12 transgene (as described in Example 2 of WO 01/14424 ).

In the KM mouse strain, the endogenous mouse kappa light chain gene is homozygously disrupted, as described in Chen et al., EMBO J. 12, 811-820 (1993), and the endogenous mouse heavy chain gene is homozygously disrupted, as described in Example 1 of WO 01 /09187. This mouse strain carries the human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome consisting of the hCF fragment (SC20) of chromosome 14, as described in WO 02/43478.

Splenocytes from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies according to known methods.

In addition, human antibodies of the present invention or antibodies of the present invention from other species can be identified by display methods, including, but not limited to, phage display, retroviral display, ribosomal display, and other methods well known in the art, and the resulting molecules may be subjected to additional maturation, such as affinity maturation, as such a method is well known in the art (see, for example, Hoogenboom et al., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al. al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Pluckthau, PNAS USA 94, 4937-4942 (1997) (ribosome display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990), Russell et al., Nucl. Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol Reviews 130, 43-68 (1992), CH1swell and McCafferty TIBTECH 10, 80-84 (1992), and US 5733743). If display technologies are used to produce antibodies that are not human, then such antibodies can be humanized.

In one embodiment, the antibody of the invention is of the IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM isotype.

In the first basic embodiment of the antibody of the invention, the antibody competes for binding to soluble cMetECDHis with an immobilized antibody that contains a VH region comprising the sequence SEQ ID NO:33 and a VL region comprising the sequence SEQ ID NO:37 (024), preferably where the antibody competes for more than 50%, such as more than 75% with the specified immobilized antibody, as defined in Example 17.

In a further embodiment, the antibody does not compete for binding to soluble cMetECDHis with an antibody selected from the group consisting of:

a) an immobilized antibody comprising a VH region comprising the sequence SEQ ID NO: 1 and a VL region comprising the sequence SEQ ID NO: 5 (005);

b) an immobilized antibody containing a VH region including the sequence SEQ

- 8 045516

ID NO: 17, and containing the VL region including the sequence SEQ ID NO: 21 (008);

c) an immobilized antibody containing the VH region and the VL region of the 5D5 antibody, AND

d) an immobilized antibody containing a VH region comprising the sequence of SEQ ID NO: 49, and containing a VL region comprising the sequence of SEQ ID NO: 53 (045), preferably where the antibody competes by less than 25%, such as by less than 20% with the indicated immobilized antibody, according to the definitions given in Example 17.

In a further embodiment, the antibody binds to the same epitope as the antibody selected from the group consisting of:

a) an antibody containing a VH region comprising the sequence of SEQ ID NO: 33 and containing a VL region comprising the sequence of SEQ ID NO: 37 (024);

b) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 65 and a VL region comprising the sequence of SEQ ID NO: 69 (061);

c) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 73 and a VL region comprising the sequence of SEQ ID NO: 77 (062);

d) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 81 and a VL region comprising the sequence of SEQ ID NO: 85 (064);

e) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 89 and a VL region comprising the sequence of SEQ ID NO: 93 (068);

f) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 97 and a VL region comprising the sequence of SEQ ID NO: 101 (069);

g) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 113 and a VL region comprising the sequence of SEQ ID NO: 117 (098);

h) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 121 and a VL region comprising the sequence of SEQ ID NO: 125 (101), and

i) an antibody comprising a VH region comprising the sequence of SEQ ID NO: 129 and a VL region comprising the sequence of SEQ ID NO: 133 (181).

In a further embodiment, the antibody comprises the CDR3 region of VH having the sequence shown in:

a) SEQ ID NO: 36 (024);

b) SEQ ID NO: 193, such as the CDR3 region of VH represented in SEQ ID NO: 68, 76, 84 or 92 (061, 062, 064, 068);

c) SEQ ID NO: 196, such as the CDR3 region of VH represented in SEQ ID NO: 100 or 132 (069, 181)

d) SEQ ID NO: 116 (098) or

e) SEQ ID NO: 201, such as the CDR3 region of VH shown in SEQ ID NO: 124 (101).

In a further embodiment, the antibody contains:

a) a VH region including the sequences CDR1, 2 and 3, SEQ ID NO: 34, 185 and 36, and a VL region including the sequences CDR1, 2 and 3, SEQ ID NO: 38, 39 and 206, such as an antibody, which contains a VH region comprising the sequences SEQ ID NOs: 34, 35 and 36, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 38, 39 and 40, (024);

b) a VH region including the sequences CDR1, 2 and 3, SEQ ID NO: 191, 192 and 193, and a VL region including the sequences CDR1, 2 and 3, SEQ ID NO: 78, 79 and 208, such as an antibody, including:

a) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 66, 67 and 68, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 70, 71 and 72, (061);

b) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 74, 75 and 76, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 78, 79 and 80, (062);

c) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 82, 83 and 84, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 86, 87 and 88, (064);

d. the VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 90, 91 and 92, and the VL region including the sequences CDR1, 2 and 3, SEQ ID NOs: 94, 95 and 96, (068);

c) a VH region including the sequences CDR1, 2 and 3, SEQ ID NO: 194, 195 and 196, and a VL region including the sequences CDR1, 2 and 3, SEQ ID NO: 209, 210 and 104, such as an antibody, including:

a) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 98, 99 and 100, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 102, 103 and 104, (069), or

b) the VH region, including the sequences CDR1, 2 and 3, SEQ ID NO: 130, 131 and 132, and the VL region, including the sequences CDR1, 2 and 3, SEQ ID NO: 134, 135 and 136, (181),

d) a VH region including the sequences CDR1, 2 and 3, SEQ ID NO: 197, 198 and 116, and a VL region including the sequences CDR1, 2 and 3, SEQ ID NO: 118, 119 and 211, such as an antibody, which contains the VH region, including the sequences CDR1, 2 and 3, SEQ ID NO: 114, 115 and 116, and the VL region, including the sequences CDR1, 2 and 3, SEQ ID NO: 117, 118 and 120,

- 9 045516 (098) or

e) a VH region comprising the sequences CDR1, 2 and 3 of SEQ ID NOs: 199, 200 and 201 and a VL region comprising the sequences CDR1, 2 and 3 of SEQ ID NOs: 126, 212 and 128, such as an antibody that contains the region VH, including the sequences CDR1, 2 and 3, SEQ ID NO: 122, 123 and 124 and the VL region, including the sequences CDR1, 2 and 3, SEQ ID NO: 126, 127 and 128 (101).

In a further embodiment, the antibody comprises:

a) a VH region comprising the sequence SEQ ID NO: 33, and preferably a VL region comprising the sequence SEQ ID NO: 37 (024);

b) a VH region comprising the sequence SEQ ID NO: 61, and preferably a VL region comprising the sequence SEQ ID NO: 69 (061);

c) a VH region comprising the sequence SEQ ID NO: 73, and preferably a VL region comprising the sequence SEQ ID NO: 77 (062);

d) a VH region comprising the sequence SEQ ID NO: 81, and preferably a VL region comprising the sequence SEQ ID NO: 85 (064);

e) a VH region comprising the sequence SEQ ID NO: 89, and preferably a VL region comprising the sequence SEQ ID NO: 93 (068);

f) a VH region comprising the sequence SEQ ID NO: 97, and preferably a VL region comprising the sequence SEQ ID NO: 101 (069);

g) a VH region comprising the sequence SEQ ID NO: 113, and preferably a VL region comprising the sequence SEQ ID NO: 117 (098);

h) a VH region comprising the sequence SEQ ID NO: 121, and preferably a VL region comprising the sequence SEQ ID NO: 125 (101);

i) a VH region comprising the sequence SEQ ID NO: 129, and preferably a VL region comprising the sequence SEQ ID NO: 133 (181);

j) a VH region comprising the sequence SEQ ID NO: 159 and preferably a VL region comprising the sequence SEQ ID NO: 160 (078);

k) a VH region comprising the sequence SEQ ID NO: 161, and preferably a VL region comprising the sequence SEQ ID NO: 162 (084);

i) a VH region comprising the sequence SEQ ID NO: 163, and preferably a VL region comprising the sequence SEQ ID NO: 164 (063);

m) a VH region comprising the sequence SEQ ID NO: 165, and preferably a VL region comprising the sequence SEQ ID NO: 166 (087);

n) a VH region comprising the sequence SEQ ID NO: 137, and preferably a VL region comprising the sequence SEQ ID NO: 138 (066);

o) a VH region comprising the sequence SEQ ID NO: 139, and preferably a VL region comprising the sequence SEQ ID NO: 140 (065);

p) a VH region comprising the sequence SEQ ID NO: 141, and preferably a VL region comprising the sequence SEQ ID NO: 142 (082);

q) a VH region comprising the sequence SEQ ID NO: 143, and preferably a VL region comprising the sequence SEQ ID NO: 144 (089), or

r) a variant of any of said antibodies, wherein said variant preferably contains at least 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, for example conservative amino acid substitutions in said sequences.

In one embodiment, the antibody comprises a VH region comprising the CDR3 sequence of SEQ ID NO: 100 and a VL region comprising the CDR3 sequence of SEQ ID NO: 104 (069).

In one embodiment, the antibody comprises a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 98, 99 and 100, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 102, 103 and 104, (069 ).

In one embodiment, the antibody contains a VH region comprising the sequence SEQ ID NO:97 and a VL region comprising the sequence SEQ ID NO:101 (069).

In another basic embodiment, the antibody of the invention:

competes for binding to soluble cMetECDHis with an immobilized antibody, wherein said immobilized antibody comprises a VH region comprising the sequence SEQ ID NO: 9 and a VL region comprising the sequence SEQ ID NO: 13 (006), preferably where the antibody competes by more than 50 %, such as, for example, more than 75% with the specified immobilized antibody, as defined in example 17, and does not compete for binding with soluble cMetECDHis with the immobilized antibody containing the VH region comprising the sequence SEQ ID NO:49, and the region VL comprising the sequence SEQ ID NO: 53 (045), preferably where the antibody competes less than 50%, for example less than 25%, for example less than 20% with the specified immobilized antibody, as defined in the example 17, and binds the SEMA domain of -Met, preferably where the antibody is capable of inhibiting binding

- 10 045516

SEMA domain HGF with an IC50 of less than 10 μg/ml, for example less than 2 μg/ml, as described in Example 9.

In a further embodiment, the antibody does not compete for binding to soluble cMetECDHis with an immobilized antibody comprising a VH region comprising the sequence SEQ ID NO: 33 and a VL region comprising the sequence SEQ ID NO: 37 (024), preferably where the antibody competes by less than 25%, for example, less than 20% with the specified immobilized antibody, as defined in Example 17.

In a further embodiment, the antibody binds the same epitope as the antibody selected from the group consisting of:

a) an antibody containing a VH region comprising the sequence SEQ ID NO: 1, and a VL region comprising the sequence SEQ ID NO: 5 (005);

b) an antibody comprising a VH region comprising the sequence SEQ ID NO: 9 and a VL region comprising the sequence SEQ ID NO: 13 (006);

c) an antibody comprising a VH region comprising the sequence SEQ ID NO: 25 and a VL region comprising the sequence SEQ ID NO: 29 (022), and

d) an antibody comprising a VH region comprising the sequence SEQ ID NO: 57 and a VL region comprising the sequence SEQ ID NO: 61 (058).

In a further embodiment, the antibody comprises the CDR3 region of VH having the sequence shown in:

a) SEQ ID NO: 181, such as the CDR3 region of VH represented in SEQ ID NO: 4 or 12 (005, 006);

b) SEQ ID NO: 28 (022) or

c) SEQ ID NO: 60 (058).

In a further embodiment, the antibody comprises:

a) a VH region including the sequences CDR1, 2 and 3, SEQ ID NO: 179, 180 and 181, and a VL region including the sequences CDR1, 2 and 3, SEQ ID NO: 6, 7 and 202, such as an antibody, containing:

a) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 3, 4 and 3, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 6, 7 and 8, (005), or

b) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 10, 11 and 12, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NO: 14, 15 and 16, (006) b' ) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 26, 184 and 28, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 30, 31 and 205, such as an antibody containing the VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 26, 27 and 28, and the VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 30, 31 and 32 (62), or

c) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 189, 190 and 60, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 62, 63 and 207, for example, an antibody containing the VH region, including the sequences CDR1, 2 and 3, SEQ ID NO: 58, 59 and 60, and the VL region, including the sequences CDR1, 2 and 3, SEQ ID NO: 62, 63 and 64 (058).

In a further embodiment, the antibody comprises:

a) a VH region comprising the sequence SEQ ID NO: 1, and preferably a VL region comprising the sequence SEQ ID NO: 5 (005);

b) a VH region comprising the sequence SEQ ID NO: 9, and preferably a VL region comprising the sequence SEQ ID NO: 13 (006);

c) a VH region comprising the sequence SEQ ID NO: 25, and preferably a VL region comprising the sequence SEQ ID NO: 29 (022);

d) a VH region comprising the sequence SEQ ID NO: 57, and preferably a VL region comprising the sequence SEQ ID NO: 61 (058);

e) a VH region comprising the sequence SEQ ID NO: 145, and preferably a VL region comprising the sequence SEQ ID NO: 146 (031);

f) a VH region comprising the sequence SEQ ID NO: 147, and preferably a VL region comprising the sequence SEQ ID NO: 148 (007);

g) a VH region comprising the sequence SEQ ID NO: 149, and preferably a VL region comprising the sequence SEQ ID NO: 150 (011);

h) a VH region comprising the sequence SEQ ID NO: 151, and preferably a VL region comprising the sequence SEQ ID NO: 152 (017);

i) a VH region comprising the sequence SEQ ID NO: 153, and preferably a VL region comprising the sequence SEQ ID NO: 154 (025), or

j) a variant of any of said antibodies, wherein said variant contains at least 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, for example conservative amino acid substitutions in said sequences.

In another basic embodiment, the antibody of the invention

- 11 045516 competes for binding to soluble cMetECDHis with an immobilized antibody, wherein said immobilized antibody comprises a VH region comprising the sequence SEQ ID NO: 49 and a VL region comprising the sequence SEQ ID NO: 53 (045), preferably where the antibody competes more less than 50%, such as greater than 75%, with said captive antibody as defined in Example 17, and the antibody does not compete for binding to soluble cMetECDHis with the captive antibody, wherein said captive antibody comprises a VH region comprising the sequence SEQ ID NO:9, and the VL region comprising the sequence SEQ ID NO: 13 (006), preferably where the antibody competes less than 25%, such as less than 20% with the specified immobilized antibody, as defined in example 17 .

In a further embodiment, the antibody does not compete for binding to soluble cMetECDHis with an antibody selected from the group consisting of:

a) an immobilized antibody comprising a VH region comprising the sequence SEQ ID NO: 17 and a VL region comprising the sequence SEQ ID NO: 21(008), and

b) an immobilized antibody comprising a VH region comprising the sequence SEQ ID NO: 33 and a VL region comprising the sequence SEQ ID NO: 37 (024), preferably where the antibody competes by less than 25%, for example, by less than 20% with said immobilized antibody as defined in Example 17.

In a further embodiment, the antibody binds to the same epitope as an antibody comprising a VH region comprising the sequence SEQ ID NO: 49 and comprising a VL region comprising the sequence SEQ ID NO: 53 (045).

In another embodiment, the antibody comprises a VH CDR3 region having the sequence set forth in SEQ ID NO: 188, for example, the VH CDR3 region set forth in SEQ ID NO: 52 (045).

In a further embodiment, the antibody comprises a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 186, 187 and 188, and a VL region comprising the sequences CDR1, 2 and 3 SEQ ID NO: 54, 55 and 56, for example an antibody, containing a VH region comprising the sequences CDR1, 2 and 3 of SEQ ID NOs: 50, 51 and 52, and a VL region comprising the sequences CDR1, 2 and 3 of SEQ ID NOs: 54, 55 and 56, (045).

In a further embodiment, the antibody comprises:

a) a VH region comprising the sequence SEQ ID NO: 49, and preferably a VL region comprising the sequence SEQ ID NO: 53 (045);

b) a VH region comprising the sequence SEQ ID NO:155, and preferably a VL region comprising the sequence SEQ ID NO:156 (040);

c) a VH region comprising the sequence SEQ ID NO: 157, and preferably a VL region comprising the sequence SEQ ID NO: 158 (039), or

d) a variant of any of said antibodies, wherein said variant preferably contains at least 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in said sequences.

In a further embodiment, the antibody binds to the SEMA domain of c-Met, preferably the antibody is capable of inhibiting the binding of HGF to the SEMA domain with an IC50 of less than 10 μg/ml, for example, less than 2 μg/ml, as described in Example 9.

In another basic embodiment of an antibody of the invention, the antibody binds the same epitope as an antibody comprising a VH region comprising the sequence SEQ ID NO: 17 and a VL region comprising the sequence SEQ ID NO: 21 (008), or binds the same epitope the same as an antibody containing a VH region comprising the sequence SEQ ID NO: 41 and a VL region comprising the sequence SEQ ID NO: 45 (035), or binds the same epitope as an antibody containing a VH region comprising the sequence SEQ ID NO: 105, and the VL region including the sequence SEQ ID NO: 109 (096).

In another embodiment, the antibody comprises a VH CDR3 region having the sequence set forth in SEQ ID NO: 183, for example, a VH CDR3 region set forth in SEQ ID NO: 20, 44, or 108 (008, 035, 096).

In a further embodiment, the antibody comprises a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 18, 182 and 183, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 22, 203 and 204, e.g. containing:

a) the VH region, including the sequences CDR1, 2 and 3, SEQ ID NO: 18, 19 and 20, and the VL region, including the sequences CDR1, 2 and 3, SEQ ID NO: 22, 23 and 24, (008),

b) the VH region, including the sequences CDR1, 2 and 3, SEQ ID NO: 42,43 and 44, and the VL region, including the sequences CDR1, 2 and 3, SEQ ID NO: 46, 47 and 48, (035),

c) a VH region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 106, 107 and 108, and a VL region comprising the sequences CDR1, 2 and 3, SEQ ID NOs: 110, 111 and 112, (096).

In another embodiment, the antibody contains:

- 12 045516

a) a VH region comprising the sequence SEQ ID NO: 17, and preferably a VL region comprising the sequence SEQ ID NO: 21 (008);

b) a VH region comprising the sequence SEQ ID NO: 41, and preferably a VL region comprising the sequence SEQ ID NO: 45 (035);

c) a VH region comprising the sequence SEQ ID NO: 105, and preferably a VL region comprising the sequence SEQ ID NO: 109 (096); or

d) a variant of any of said antibodies, wherein said variant preferably contains at least 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions in said sequences.

In a further embodiment, the antibody binds to A431 cells with an EC50 of 10 nM or less, such as an EC50 of 2 nM or less, preferably as determined according to Example ^13.

In a further embodiment, the antibody binds to c-Met with an affinity constant (KD) of 20 nM or less, e.g., 5 nM or less, preferably as determined according to Example 14.

In a further embodiment, the antibody binds to rhesus c-Met, preferably if the antibody binding signal to rhesus c-Met is at least 5 times greater than the signal for the negative control antibody as determined according to Example 15.

In a further embodiment, the antibody inhibits the binding of HGF to the extracellular domain of c-Met, preferably the antibody inhibits binding by more than 40%, such as by more than 50%, for example by more than 60%, for example by more than 70 %, for example more than 80%, for example more than 90%, as determined according to example 16.

In yet another embodiment, the antibody is capable of inhibiting the viability of KP4 cells, preferably the antibody is capable of inhibiting viability by more than 10%, e.g., more than 25%, e.g., more than 40%, preferably as described in Example 19.

Antibody formats

The present invention provides antagonistic and non-antagonistic c-Met binding antibodies. While some antibodies act antagonistically on target cells regardless of whether they are monovalent or bivalent, for other antibodies the functional effect depends on valency. As presented in Example 19 herein, antibodies 024, 062, 064, 068, 069, 098, 101, 181 (which are all in the same cross-blocking group, see Example 17), for example, exhibit antagonistic properties in analysis of the viability of KP4 cells, regardless of their format. Antibodies 022 and 058, on the other hand, behave antagonistically in this assay in a monovalent format, but are agonistic (or at least not antagonistic) in a bivalent format. Thus, depending on the desired functional properties for a particular use, specific antibodies can be selected from the set of antibodies proposed in the present invention and/or their format can be adapted by changing the valency.

Moreover, the antibody of the invention can be of any isotype. Isotype selection will typically be guided by the effector functions sought, such as ADCC induction. Examples of isotypes are IgG1, IgG2, IgG3 and IgG4. One of two human light chain constant regions, kappa or lambda, may be used. If necessary, the class of the anti-c-Met antibody of the present invention can be changed by known methods. For example, the antibody of the present invention, which was originally IgM, can be changed to the IgG antibody class of the present invention. Additionally, class change techniques can be used to convert one IgG subclass to another, for example from IgG1 to IgG2. Thus, the effector functions of the antibodies of the present invention can be changed by changing the isotype, for example, to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibody for various therapeutic applications. In one embodiment, the antibody of the present invention is an IgG1 antibody, for example, IgG1,K.

Negative modulation induced by antagonistic antibodies is the mechanism of action of therapeutic antibodies against c-Met. Accordingly, in one aspect of the invention, antibodies are sought that have reduced agonistic properties but retain the ability to induce negative modulation of c-Met.

It was found that with reduced antibody conformational flexibility, the potential residual agonist activity of bivalent IgG1 antibodies was minimized.

Accordingly, in one embodiment, the antibody of the invention has been modified to make it less flexible, for example, through mutations in the hinge region.

The greatest conformational changes result from the flexibility of the hinge, which allows a wide range of Fab-Fc angles to be accessible (Ollmann Saphire, E., R.L. Stanfield, M.D.M. Crispin, P.W.H.I. Parren, P.M. Rudd, R.A. Dwek, D.R. Burton and L.A. Wilson. 2002. Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 319: 9-18). One way to reduce the flexibility of the Fab arm in immunoglobulins is to prevent the formation of disulfide bonds between the light and heavy chains through genetic modification. In the natural IgG1 antibody, the light chain is

- 13 045516 is united to the heavy chain covalently through a disulfide bond connecting the C-terminal cysteine of the light chain to the cysteine at position 220 (C220 EU numbering) in the Fc hinge of the heavy chain. Either by mutating the amino acid C220 to serine or any other naturally occurring amino acid, or by removing C220, or by removing the hinge entirely, or by replacing the IgG1 hinge with an IgG3 hinge, a molecule is formed in which the light chains are connected through their C-terminal cysteines, similar to the situation found in the human isotype IgA2m(1). This results in a decrease in the flexibility of the Fab relative to the Fc and therefore reduces the ability to cross-link, as shown in the examples.

Another strategy to reduce the flexibility of the IgG1 molecule is to replace the IgG1 hinge with an IgG2 hinge or an IgG2-like hinge (Dangl et al. EMBO J. 1988;7:1989-94). This hinge region has two properties that are different from those of the IgG1 hinge, which are thought to make the molecule less flexible. First, compared to the IgG1 hinge, the IgG2 hinge is 3 amino acids shorter. Second, the IgG2 hinge contains an additional cysteine, thus forming three, instead of two, disulfide bridges within the heavy chain. Alternatively, a variant IgG1 hinge may be introduced that is similar to the IgG2 hinge. This mutant (TH7A6-9) (WO2010063746) contains the T223C mutation and two deletions (K222 and T225) to produce a shorter hinge with an additional cysteine.

In a further embodiment, the antibody of the invention is an IgG1 subtype in which the hinge region has been modified by:

(i) removing the hinge region with the sequence EPKSCDKTHTCPPCP and replacing it with an IgG2 hinge region with the sequence: ERKCCVECPPCP (IgG1 Hinge-IgG2);

(ii) deletion at position 220 such that the modified hinge region contains the sequence EPKSDKTHTCPPCP (IgG1 AC220);

(iii) replacing the cysteine at position 220 with any other naturally occurring amino acid (X) such that the modified hinge region contains the sequence EPKSXDKTHTCPPCP (IgG1 C220X);

(iv) deleting the hinge region with the sequence EPKSCDKTHTCPPCP (UniBody IgG1);

(v) removing the hinge region with the sequence EPKSCDKTHTCPPCP and replacing it with an IgG3 hinge region with the sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPP CPRCP (IgG1 Hinge-IgG3); or (vi) replacing the threonine at position 223 with a cysteine and removing the lysine at position 222 and the threonine at position 225 such that the modified hinge region contains the sequence EPKSCDCHCPPCP (IgG1 TH7A6-9).

In one embodiment of the invention, the antibody of the invention has an IgG1 subtype in which the hinge region has been modified by deletion at position 220 such that the modified hinge region has the sequence EPKSDKTHTCPPCP (IgG1 AC220) or by replacing the cysteine at position 220 with any other naturally occurring amino acid (X), such that that the modified hinge region has the sequence EPKSXDKTHTCPPCP (IgG1 C220X);

In a further embodiment, the antibody of the invention is of the IgG1 subtype in which the hinge region has been modified by replacing the cysteine at position 220 with a serine such that the modified hinge region has the sequence EPKSSDKTHTCPPCP (IgG1 C220S).

In a further embodiment, the antibody of the invention is of the IgG2 subtype.

In a further embodiment, the antibody of the invention has sugars modified to reduce the amount of fucose and thus enhance ADCC, for example by adding compounds to the culture medium during antibody production, as described in US 2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350 or using FUT8 knockout cells, for example, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, ADCC can be optimized using the method described by Umana et al. (1999) Nature Biotech 17:176.

In one embodiment, the antibody comprises a VH region comprising the CDR3 sequence, SEQ ID NO: 100, and a VL region comprising the CDR3 sequence, SEQ ID NO: 104 (069), of the IgG1 subtype in which the hinge region has been modified by replacing a cysteine at the position 220 to serine, so that the modified hinge region has the sequence EPKSSDKTHTCPPCP (IgG1 C220S).

In one embodiment, the antibody comprises a VH region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 98, 99 and 100 and a VL region comprising the CDR1, 2 and 3 sequences of SEQ ID NOs: 102, 103 and 104 (069) of the IgG1 subtype, in which the hinge region was modified by replacing the cysteine at position 220 with a serine, such that the modified hinge region had the sequence EPKSSDKTHTCPPCP (IgG1 C220S).

In one embodiment, the antibody comprises a VH region comprising the sequence SEQ ID NO:97 and a VL region comprising the sequence SEQ ID NO:101 (069) of the IgG1 subtype, in which the hinge region has been modified by replacing the cysteine at position 220 with a serine such that the modified hinge the region had the sequence EPKSSDKTHTCPPCP (IgG1 C220S).

- 14 045516

Various publications have demonstrated a correlation between reduced core fucosylation and increased ADCC activity in vitro (Shields RL. 2002 JBC; 277:26733-26740, Shinkawa T. 2003 JBC; 278(5):3466-3473, Umana P. Nat Biotechnol. 1999 Feb;17(2):176-80).

In a further embodiment, the antibody of the invention has been modified to reduce core fucosylation below 10%, eg below 5%, as determined by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). This test can be carried out by methods well known in the prior art, for example, treatment with kifunensin or obtaining cells negative for FUT8.

In another embodiment, the antibody of the invention was designed to enhance complement activation, for example, as described in Natsume et al. (2009) Cancer Sci. 100:2411.

In one embodiment, the antibody of the invention is a full length, preferably an IgG1 antibody, in particular an IgG1 antibody, k. In another embodiment, the antibody of the invention is an antibody fragment or a single chain antibody.

Antibody fragments can be prepared, for example, by fragmentation using conventional methods, and the fragments are screened in the same manner as described herein for whole antibodies. For example, F(ab') ₂ fragments can be obtained by treating the antibody with pepsin. The resulting F(ab') ₂ fragment can be processed to reduce disulfide bridges to produce Fab' fragments. Fab fragments can be produced by treating an IgG antibody with papain; Fab' fragments can be obtained by pepsin digestion of an IgG antibody. The F(ab') fragment can also be obtained by linking Fab', described below via a thioether bond or a disulfide bond. A Fab' fragment is an antibody fragment produced by cleavage of the disulfide bond of the F(ab')2 hinge region. The Fab' fragment can be prepared by treating the F(ab')2 fragment with a reducing agent such as dithiothreitol. An antibody fragment can also be produced by expressing nucleic acids encoding such fragments in recombinant cells (see, for example, Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimeric gene encoding part of an F(ab') ₂ fragment may include DNA sequences encoding a CH1 domain and an H-chain hinge region followed by a translation stop codon, thereby producing a truncated antibody fragment molecule.

As stated above, in one embodiment, the anti-c-Met antibody of the invention is a bivalent antibody.

In one embodiment, the anti-c-Met antibody of the invention is a monovalent antibody.

In one embodiment, the antibody of the invention is a Fab fragment or single-arm antibody such as described in US 20080063641 (Genentech) or another monovalent antibody, for example, such as described in WO 2007048037 (Amgen).

In a preferred embodiment, the monovalent antibody has the structure described in WO 2007059782 (Genmab) (which is incorporated herein by reference) having a deletion in the hinge region. Accordingly, in one embodiment, the antibody is a monovalent antibody, wherein said anti-c-Met antibody is constructed by a method comprising:

i) providing a nucleotide construct encoding a light chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VL region of a selected antigen-specific anti-c-Met antibody, and a nucleotide sequence encoding a constant region (CL) of an Ig, wherein said nucleotide sequence encoding the region The VL of the selected antigen-specific antibody and said nucleotide sequence encoding the CL region of the Ig are operably linked to each other, and wherein in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified so that the CL region does not contain amino acids capable of forming disulfide bonds or covalent bonds with other peptides containing an identical amino acid sequence to the CL region in the presence of polyclonal human IgG or when administered to an animal or human;

ii) providing a nucleotide construct encoding the heavy chain of said monovalent antibody, said construct comprising a nucleotide sequence encoding the VH region of the selected antigen-specific antibody and a nucleotide sequence encoding the CH constant region of a human Ig, wherein the nucleotide sequence encoding the CH region is modified such that the region corresponding to the hinge region and, as required by the Ig subtype, other CH regions, such as the CH3 region, did not contain any amino acid residues that are involved in the formation of disulfide bonds or covalent or stable non-covalent heavy chain interchain bonds with other peptides containing an amino acid sequence identical to the CH constant region of a human Ig, in the presence of a polyclonal human IgG or when administered to a human being, wherein said nucleotide sequence encoding the VH region of the selected antigen-specific antibody and said nucleotide sequence encoding the CH region of the specified IIIg are operably linked to each other;

iii) providing a cellular expression system to produce said monovalent

- 15 045516 antibodies;

iv) obtaining said monovalent antibody by co-expression of nucleotide constructs (i) and (ii) in cells of the cellular expression system (iii).

Similarly, in one embodiment, an anti-c-Met antibody is a monovalent antibody that contains (i) a variable region of an antibody of the invention as described herein or an antigen binding portion of said region, and (ii) an immunoglobulin CH region or fragment thereof including CH2 regions and CH3, wherein the CH region or fragment thereof is modified so that the region corresponding to the hinge region, and, if the immunoglobulin is not of the IgG4 isotype, other CH regions, such as CH3, do not carry any amino acid residues that would not be capable of forming disulfide bonds to an identical C _H region or are unable to form other covalent or stable non-covalent heavy chain bonds to an identical C _H region in the presence of polyclonal human IgG.

In a further embodiment, the monovalent anti-c-Met antibody heavy chain was modified such that the entire hinge was removed.

In another further embodiment, said monovalent antibody is an IgG4 subtype, but the CH3 region has been modified so that one or more of the following amino acid substitutions are made:

Numbering of SNZ mutations

KAVAT* EU index G4* Mutations E378 E357 E357A or E357T or E357V or E357I S387 S3 64 S364R or S364K T389 T366 Т366А or T366R or Т366К or T366N L391 L368 L368A or L368V or L368E or L368G or L368S or L368T D427 D399 D399A or D399T or D399S F405A or F405L or F405T or F405D or F405R or F405Q F436 F405 or F405K or F405Y Y438 Y407 Y407A or Y407E or Y407Q or Y407K or Y407F F436 and Y438 F405 and Y407 (F405T and Y407E) or (F405D and Y407E) (D399S and Y407Q) or (D399S and Y407K) or (D399S D427 and Y438 D399 and Y407 and Y407E)

*KABAT indicates amino acid numbering according to Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). EU index indicates amino acid numbering according to the EU index as outlined in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

In a further embodiment, the sequence of said monovalent antibody has been modified so that it does not contain any acceptor sites for N-linked glycosylation.

The anti-c-Met antibodies of the invention also include single chain antibodies. Single chain antibodies are peptides in which the Fv regions of the heavy and light chains are joined. In one embodiment, the present invention provides a single chain Fv (scFv) in which the heavy and light chains in the Fv of the anti-c-Met antibody of the present invention are combined with a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues) in a single chain peptide . Methods for producing such antibodies are described, for example, in US 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269315 (1994), Bird et al., Science 242, 423-426 (1988), Huston et al., PNAS USA 5879, 5883-1988 (85) and McCafferty et al., Nature 348, 552-554 (1990) . A single chain antibody may be monovalent if one type of VH and VL is used, bivalent if two types of VH and VL are used, or multivalent if more than two types of _VH and _VL are used.

In one embodiment, the anti-c-Met antibody of the invention is an effector function-deficient antibody. In one embodiment, the effector function-deficient anti-c-Met antibody is a stabilized IgG4 antibody that has been modified to prevent Fab arm replacement (van der Neut Kolfschoten et al. (2007) Science 317(5844): 1554-7). Examples of suitable stabilized IgG4 antibodies are those in which the arginine at position 409 in the heavy chain constant region of human IgG4, which is indicated by the EU index as in Kabat et al., is replaced by lysine, threonine, methionine or leucine, preferably lysine (described in WO2006033386 (Kirin)) and/or where the hinge region has been modified to contain the sequence Cys-ProPro-Cys.

In another embodiment, the stabilized anti-c-Met IgG4 antibody is an antibody comprising a heavy and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met and Leu at the position corresponding to 409 and/or the residue is selected from the group consisting of: Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and wherein said antibody optionally includes one or more additional substitutions, deletions and/or insertions, and wherein said antibody comprises a Cys-ProPro-Cys sequence in the hinge region. Preferably, the antibody contains a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody is replaced by the CH3 region of human IgG1, or human IgG2, or human IgG3. See also WO 2008145142 (Genmab).

In yet another embodiment, the stabilized anti-c-Met IgG4 antibody is an antibody comprising a heavy and a light chain, wherein said heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met and Leu at position corresponding to 409 and/or the residue is selected from the group consisting of Ala, Val, Gly, Ile and Leu at the position corresponding to 405, and wherein said antibody optionally includes one or more additional substitutions, deletions and/or insertions and wherein said antibody contains sequence CysPro-Pro-Cys in the hinge region. Preferably, the antibody contains a Lys or Ala residue at the position corresponding to 409 or the CH3 region of the antibody is replaced by the CH3 region of human IgG1, or human IgG2, or human IgG3.

In a further embodiment, the effector function-deficient anti-c-Met antibody is a non-IgG4 antibody, such as IgG1, IgG2, or IgG3, that has been mutated such that the ability to mediate effector functions such as ADCC is reduced or even eliminated. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(21:1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168(2001).

Conjugates

In a further embodiment, the present invention provides an anti-c-Met antibody conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Such conjugates are referred to herein as immunoconjugates. Immunoconjugates that include one or more cytotoxins are called immunotoxins.

A cytotoxin or cytotoxic agent includes any agent that has an adverse effect on cells (eg, kills them). Suitable therapeutic agents for forming the immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine-dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, clad ribin), alkylating agents (such as mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin ), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (such as diphtheria chain A and their active fragments and hybrid molecules), ricin toxin (for example, ricin or deglycosylated ricin A chain), cholera toxin, Shiga toxin-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Baumann-Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modescine, gelanin, abrin A chain, moddecin chain, alpha-sarcin, Aleurites fordii proteins, diantine proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica Charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin and enomycin. Other suitable conjugated molecules include ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, sweet potato antiviral protein, diphtheria toxin and pseudomonas endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that can be administered in combination with the anti-c-Met antibody of the present invention, as described elsewhere herein, may also be candidates for therapeutic moieties useful in conjugating the antibody of the present invention.

In another embodiment, the anti-c-Met antibody of the invention includes a conjugated nucleic acid or a molecule associated with a nucleic acid. In one such aspect of the present invention, the conjugated nucleic acid is a cytotoxic ribonuclease, an antisense nucleic acid, an inhibitory RNA molecule (eg, an siRNA molecule), or an immunostimulatory nucleic acid (eg, a DNA molecule containing an immunostimulatory CpG motif). In another embodiment, the anti-c-Met antibody of the invention is conjugated to an aptamer or ribozyme.

In one embodiment, anti-c-Met antibodies are provided containing one or more radiolabeled amino acids. Radiolabeled anti-c-Met antibody can be used for both diagnostic and therapeutic purposes (conjugation with radiolabeled molecules

- 17 045516 lami is another possible feature). Non-limiting examples of tags for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc and 1251, 1311 and 186Re.

Antibodies against c-Met can also be chemically modified by covalent conjugation to a polymer, for example, to increase their half-life in the bloodstream. Exemplary polymers and methods for attaching them to polypeptides are illustrated, for example, in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Additional polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (for example, PEG with a molecular weight of about 1000 to about 40,000, for example, from about 2000 to about 20,000).

To conjugate an anti-c-Met antibody to conjugated molecule(s), such as those described above, any method known in the art can be used, including those described by Hunter et al., Nature 144, 945 (1962), David et al., Biochemistry 13, 1014 (1974), Pain et al., J. Immunol. Meth. 40, 219 (1981) and Nygren, J. Histochem. and Cytochem. 30, 407 (1982). Such antibodies can be prepared by chemically conjugating another moiety to the N-terminal side or C-terminal side of an anti-c-Met antibody or fragment thereof (e.g., the H or L chain of an anti-c-Met antibody) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated antibody derivatives can be prepared by conjugation of internal moieties or sugars, where necessary. The agents can be paired either directly or indirectly with the anti-c-Met antibody of the present invention. One example of indirect pairing with a second agent is pairing via a spacer. In one embodiment, the anti-C-Met antibody of the present invention is attached to a chelator linker, such as thiuxitan, which allows the antibody to be conjugated to a radioisotope.

Bispecific antibodies

In a further aspect, the invention provides a bispecific molecule comprising a first antigen binding region from an anti-c-Met antibody of the invention as described hereinabove, and a second antigen binding region, with different binding specificities, such as binding specificity to a human effector cell, human Fc -receptor, T-cell receptor, B-cell receptor, or binding specificity to a non-overlapping c-Met epitope, i.e. a bispecific antibody in which the first and second antigen-binding regions do not compete for binding to c-Met, for example, a bispecific antibody in which the first and second antigen-binding regions do not compete for binding to c-Met, for example, in the test described in Example 17.

Exemplary bispecific antibody molecules of the invention include (i) two antibodies, one with specificity for c-Met and one with specificity for a second target, that are conjugated together, (ii) a single antibody that has a single chain or arm specific for c-Met , and a second chain or arm specific for the second molecule, and (iii) a single chain antibody that has specificity for c-Met and the second molecule. In one embodiment, the second molecule is a cancer antigen/tumor antigen, such as oncoembryonic antigen (CEA), prostate specific antigen (PSA), RAGE (renal antigen), α-fetoprotein, CAMEL (CTL melanoma antigen), CT antigens (such as MAGE -B5, -B6, -C2, -C3 and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE and SAGE), mucin antigens (for example, MUC1, mucin-CA125 and etc.), ganglioside antigens, tyrosinases, gp75, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM or cancer associated integrin such as α5β3 integrin. In another embodiment, the second molecule is an angiogenic factor or other cancer-associated growth factor, such as vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, angiogenin, or a receptor of any of these, especially receptors associated with cancer progression (eg , one of the HER1-HER4 receptors). In one embodiment, the bispecific antibody of the present invention is a diabody.

Nucleotide sequences, vectors and host cells

In a further aspect, the invention relates to nucleotide sequences, such as DNA sequences, encoding the heavy and light chains of an antibody of the invention.

In one embodiment, the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 1 67, 168, 169, 170, 171,172, 173, 174, 175, 176, 177 and 178.

In another specific embodiment, the nucleic acid sequence encodes a VH amino acid sequence selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105 113, 121, 129, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175 and 177.

In another specific embodiment, the nucleic acid sequence encodes a VL amino acid sequence selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53,

- 18 045516

61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176 and 178.

In a further aspect, the invention relates to an expression vector, or set of expression vectors, encoding an antibody of the invention. The antibody heavy or light chain may be encoded by the same vector or by a different vector.

Such expression vectors can be used to recombinantly produce antibodies of the invention.

In one embodiment, the expression vector of the invention contains a nucleotide sequence encoding one or more amino acid sequences selected from the group consisting of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45 , 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 138, 139 , 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 , 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 and 178.

In another specific embodiment, the expression vector of the invention contains a nucleotide sequence encoding one or more VH amino acid sequences selected from the group consisting of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81 , 89, 97, 105, 113, 121, 129, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 1 73 , 175 and 177.

In another specific embodiment, the expression vector of the invention contains a nucleotide sequence encoding one or more VL amino acid sequences selected from the group consisting of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85 , 93, 101, 109, 117, 125, 133, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174 , 176 and 178.

In another embodiment, the expression vector further includes a nucleotide sequence encoding a constant region of a light chain, a heavy chain, or both a light and heavy chain of an antibody, such as a human antibody.

An expression vector in the context of the present invention can be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleotide vectors (a nucleotide sequence containing a set of expression control elements). Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculoviruses, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, a nucleic acid encoding an antibody against c-Met, contains a naked DNA or RNA vector, including, for example, a linear expression element (as described, for example, by Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a packaged nucleotide vector (as described, for example, in US 6077835 and/or WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119, a midge nucleotide vector (as described, for example, in Schakowski et al., Mol Ther 3 , 793-800 (2001)), or a precipitated nucleotide vector construct, for example a CaPO4-precipitated construct (as described, for example, in WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleotide vectors and their uses are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

In one embodiment, the vector is suitable for expressing an anti-c-Myc antibody in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989), pET vectors (Novagen, Madison, Wis.), etc.).

The expression vector may also be, or otherwise is, a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, but are not limited to, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

The expression vector may also be or otherwise be a vector suitable for expression in mammalian cells, for example a vector containing glutamine synthetase as a selectable marker, such as the vectors described in (Bebbington (1992) Biotechnology (NY) 10:169-175 ).

The nucleic acid and/or vector may also contain a nucleotide sequence encoding a secretion/localization sequence that can target a polypeptide, such as a growing polypeptide chain, into the periplasmic space or cell culture medium. Such sequences are known in the art and include secretion leader or signal peptides.

In the expression vector of the invention, the nucleic acids encoding the anti-cMet antibody may contain or be associated with any suitable promoter, enhancer and other expression facilitating elements. Examples of such elements include promoters for strong expression (e.g., human CMV IE promoter/enhancer and RSV promoters,

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SV40, SL3-3, MMTV and HIV LTR), effective polyA termination sequences, and origins of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and/or a common site for cloning (e.g., polylinker ). The nucleic acids may also contain an inducible promoter, as opposed to a constitutive promoter such as CMV IE.

In one embodiment, an expression vector encoding an anti-c-Met antibody may be located in and/or delivered to a host cell or host animal via a viral vector.

In a further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectome, that produces an antibody of the invention as defined herein. Examples of host cells include yeast, bacterial and mammalian cells such as CHO or HEK cells. For example, in one embodiment, the present invention provides a cell containing a nucleic acid stably integrated into the cell genome that contains a coding sequence expressing an anti-c-Met antibody of the present invention. In another embodiment, the present invention provides a cell containing a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid or linear expression element, which contains a coding sequence that expresses an anti-c-Met antibody of the invention.

In a further aspect, the invention relates to a hybridoma that produces an antibody of the invention as defined herein. In yet another additional aspect, the invention relates to a transgenic non-human animal or plant containing nucleic acids encoding a human heavy and light chain, wherein the animal or plant produces an antibody of the invention.

In a further aspect, the invention relates to a method for producing an anti-c-Met antibody of the invention, which includes the steps of:

a) culturing the hybridoma or host cell of the invention as described above herein, and

b) purifying the antibody of the invention from the culture medium.

Compositions

In a further main aspect, the invention provides a pharmaceutical composition comprising an anti-c-Met antibody as defined herein and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may contain one antibody of the present invention or a combination of different antibodies of the present invention.

Pharmaceutical compositions can be formulated in accordance with conventional methods, such as those described in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutical composition of the present invention may, for example, include diluents, excipients, salts, buffers, detergents (eg, nonionic detergent such as Tween-20 or Tween-80), stabilizers (eg, sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/ or other materials suitable for inclusion in a pharmaceutical composition.

Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption retarding agents, and the like, which are physiologically compatible with the compound of the present invention. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerin, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, colloidal solutions of carboxymethylcellulose, gum tragacanth, and injectable organic esters such as ethyl oleate, and/or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate administration. Correct flow can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, for example, (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants such as ascorbyl palmitate, butyloxyanisole, butyloxytoluene, lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The pharmaceutical compositions of the present invention may also contain isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, glycerin or sodium chloride in the composition

- 20 045516 tions.

The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the chosen route of administration, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which can enhance the shelf life or effectiveness of the pharmaceutical composition. The compounds may be formulated with carriers that will prevent the compound from being rapidly released, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid alone or with waxes, or other materials well known in the art. Methods for preparing such compositions are patented or well known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount into an appropriate diluent with one or a combination of the ingredients listed above, as required, followed by microfiltration sterilization. In general, dispersions are prepared by introducing the active compound into a sterile vehicle that contains a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional required ingredient from a pre-sterile filtered solution thereof.

Actual dosage levels of active ingredients in pharmaceutical compositions may vary so that the amount of active ingredient is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxic effects to the patient. The dosage level selected will depend on a number of pharmacokinetic factors, including the potency of the particular compositions used in the present invention or their amide, route of administration, time of administration, elimination rate of the particular compound used, duration of treatment, other drugs, compounds and/or materials used in combination with the particular compositions used, the age, sex, weight, condition, general health level and previous medical history of the patient being treated, and the like. factors well known in medicine.

The pharmaceutical composition may be administered by any suitable route or method. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. The phrase parenterally administered as used herein means modes of administration other than enteral or local administration, usually by injection, and includes, but is not limited to, epidermal, intravenous, intramuscular, intraarterial, intratracheal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and epigastric injection or infusion.

In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

Applications

In a further main aspect, the invention relates to an anti-c-Met antibody of the invention for use as a drug.

The anti-c-Met antibodies of the invention can be used for several purposes. Specifically, the antibodies of the invention can be used to treat various forms of cancer, including metastatic cancers and treatment-resistant cancers. Such malignancies may be HGF-dependent or HGF-independent.

In one embodiment, the anti-c-Met antibodies of the invention are used in the treatment of a form of cancer selected from the group consisting of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head cancer and neck, kidney cancer, liver cancer, lung cancer (such as non-small cell lung cancer (NSCLC)), nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, thyroid cancer.

In another embodiment, the anti-c-Met antibodies of the invention are used in the treatment of a form of malignancy selected from the group consisting of osteosarcoma, rhabdomyosarcoma and synovial sarcoma.

In another embodiment, the anti-c-Met antibodies of the invention are used in the treatment of a form of malignancy selected from the group consisting of Kaposi's sarcoma, leiomyosarcoma, malignant fibrous histiocytoma and fibrosarcoma.

In another embodiment, the anti-c-Met antibodies of the invention are used in the treatment of hematopoietic

- 21 045516 Chinese malignant tumors, for example malignant tumors selected from the group consisting of acute myelogenous leukemia, adult T-cell leukemia, chronic myeloid leukemia, lymphoma and multiple myeloma.

In a further embodiment, the anti-c-Met antibodies of the invention are used in the treatment of neoplasias selected from the group consisting of glioblastoma, astrocytoma, melanoma, mesothelioma and Wilms tumor.

In a further embodiment, the anti-c-Met antibodies of the invention are used in the treatment of MiT tumors including clear cell sarcoma (CCS), alveolar soft tissue sarcoma (ASPS) and translocation-associated renal cell carcinoma.

In another embodiment, the anti-c-Met agonistic antibodies of the invention are used to regulate cytokine production and induce endothelial progenitor cell mobilization, for example in patients with coronary artery disease (Yang et al. (2009) Clin Exp Pharmacol Physiol. 36:790).

In another embodiment, the anti-c-Met agonistic antibodies of the invention are used to inhibit or improve chronic renal failure (Mizuno et al. (2008) Front Biosci. 13:7072).

Likewise, the invention relates to a method of inhibiting the growth and/or proliferation of a tumor cell expressing c-Met, the method comprising administering to an individual in need thereof an effective amount of an antibody of the invention.

In one embodiment, said tumor cell is involved as a malignancy selected from the group consisting of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer , liver cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, Kaposi's sarcoma, leiomyosarcoma, malignant fibrous histiocytoxanthoma, fibrosarcoma, acute myelogenous leukemia , T-cell leukemia of adults, chronic myeloid leukemia, lymphoma, multiple myeloma, glioblastoma, astrocytoma, melanoma, mesothelioma, Wilms tumor.

The invention also relates to the use of a monoclonal antibody that binds human c-Met for the preparation of a medicament for the treatment of cancer, such as one of the specific manifestations of cancer mentioned above.

In one embodiment, the selection of patients for treatment using an anti-c-Met antibody is based on the level of (over)expression of c-Met and/or HGF on the respective tumor cells of said patients.

In a further embodiment of the treatment methods of the present invention, the effectiveness of treatment is monitored during therapy, for example at certain time points by determining the level of c-Met expression on the corresponding tumor cells.

Dosage regimens in the above treatments and applications are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple doses may be administered over a period of time, or doses may be proportionally decreased or increased depending on the needs of the therapeutic situation. Parenteral compositions can be formulated in unit dosage form for ease of administration and uniformity of dosage.

Effective dosages and dosage regimens for anti-c-Met antibodies depend on the disease or condition being treated, or can be determined by one skilled in the art. A typical non-limiting range for a therapeutically effective amount of a compound of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, such as about 0.1-20 mg/kg, such as about 0. 1-10 mg/kg, for example about 0.5, about 0.3, about 1, about 3, about 5, or about 8 mg/kg.

A physician or veterinarian skilled in the art can readily determine and prescribe the required effective amount of the therapeutic composition. For example, a physician or veterinarian may begin with a dosage of the anti-c-Met antibody in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dosage until the target effect is achieved. In general, a suitable daily dosage of a composition of the present invention will be that amount of compound that is the lowest dose effective to produce a therapeutic effect. Administration may be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, anti-c-Met antibodies can be administered by infusion at a weekly dosage of 10 to 500 mg/m ² , such as 200 to 400 mg/m ² . Such administration may be repeated, for example, 1-8 times, for example, 3-5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, such as 2 to 12 hours. In one embodiment, anti-c-Met antibodies may be administered by slow continuous infusion over a long period, such as over 24 hours, with to reduce toxic side effects.

- 22 045516

In one embodiment, anti-c-Met antibodies may be administered at a weekly dosage of 250 to 2000 mg, such as 500 mg, 700 mg, 1000 mg, 1500 mg, 2000 mg, or 300 mg, for up to 8 times, e.g. from 4 to 6 times. This regimen can be repeated one or more times as necessary, for example after 6 or 12 months. The dosage can be determined or adjusted by measuring the amount of a compound of the present invention in the blood upon administration by, for example, collecting a biological sample and using anti-idiotypic antibodies that target the antigen binding region of the anti-c-Met antibodies of the invention.

In one embodiment, anti-c-Met antibodies can be administered maintenance therapy, for example, once a week for 6 months or more.

Antibodies against c-Met may also be administered prophylactically to reduce the risk of developing a cancer, delay the onset of an event in the progression of a cancer, and/or reduce the risk of relapse when the cancer is in remission.

Antibodies against c-Met can also be administered in combinatorial therapy, i.e. combinations with other therapeutic agents relevant to the disease or condition being treated. Accordingly, in one embodiment, the antibody-containing drug is in combination with one or more additional therapeutic agents, for example, a cytotoxic, chemotherapeutic or antiangiogenic agent.

Such combined administration may be simultaneous, separate or sequential. For simultaneous administration, the agents may be administered as a single composition or as separate compositions, as appropriate. The present invention therefore provides methods for treating a disease involving c-Met expressing cells as described above, wherein the methods comprise administering an anti-c-Met antibody of the present invention combined with one or more additional therapeutic agents as described below.

In one embodiment, the present invention provides a method of treating a disorder that involves c-Met expressing cells in a subject matter, wherein the method includes administering a therapeutically effective amount of an anti-c-Met antibody of the present invention, and at least one additional therapeutic agent to a patient in need of this object.

In one embodiment, the present invention provides a method of treating or preventing cancer, wherein the method comprises administering a therapeutically effective amount of an anti-c-Met antibody of the present invention, and at least one additional therapeutic agent, to a subject in need thereof.

In one embodiment, such additional therapeutic agent may be selected from an antimetabolite such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine.

In another embodiment, such additional therapeutic agent may be selected from an alkylating agent such as mechloroamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, decarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin.

In another embodiment, such additional therapeutic agent may be selected from an antimitotic agent such as taxanes, eg doxetacel and paclitaxel, and vinca alkaloids, eg vindesine, vicristine, vinblastine and vinorelbine.

In another embodiment, such additional therapeutic agent may be selected from a topoisomerase inhibitor such as topotecan or irinotecan, or a cytostatic drug such as etoposide and teniposide.

In another embodiment, such additional therapeutic agent may be selected from a growth factor inhibitor, such as an ErbB1 (EGFR) inhibitor (such as an anti-EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors such as gefitinib or erlotinib), an ErbB2 (Her2/neu) inhibitor (such as an anti-HER2 antibody such as trastuzumab, trastuzumab DMI or pertuzumab) or an inhibitor of both EGFR and HER2 such as lapatinib).

In another embodiment, such additional therapeutic agent may be selected from a tyrosine kinase inhibitor such as imatinib (Glivec, Gleevec STI571) or lapatinib, PTK787/ZK222584.

In one embodiment, the present invention provides a method of treating a disorder involving c-Met expressing cells, wherein the method comprises administering a therapeutically effective amount of an anti-c-Met antibody of the present invention, and at least one angiogenesis inhibitor, neovascularization and/or other vascularization in need of this object.

Examples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloprotease inhibitors (such as marimastat, Neovastat, BAY 12-9566, A.G. 3340, BMS-275291, etc.), inhibitors of endothelial cell migration and proliferation (such as TNP -470, squalamine, 2methoxyestradiol, combretastatins, endostatin, angiostatin, penicillamine, SCH66336 (ScheringPlough Corp, Meedison, NJ), R115777 (Janssen Pharmaceuticals, Inc, Titusville, NJ) and

- 23 045516 similar agents), antagonists of angiogenic growth factors (for example, such as ZD6474, SU6668, antibodies against angiogenic agents and/or their receptors (such as VEGF (for example, bevacizumab), bFGF, and angiopoietin-1), thalidomide, analogues thalidomide (e.g. CC-5013), 5416 Sugen, SU5402, antiangiogenic ribozyme (e.g. angiozyme), interferon (e.g. interferon a2a), suramin, etc.), inhibitors of VEGF-R kinase and other antiangiogenic tyrosine kinases (e.g. SU 011248), endothelium-specific integrin/survival signaling pathway inhibitors (such as Vitaxin and similar agents), copper antagonists/chelators (such as tetrathiomolybdate, captopril, etc.), carboxyamidotriazole (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E, and nucleotide molecules that inhibit angiogenesis (eg, antisense-VEGF cDNA, angiostatin cDNA, p53 cDNA, and defective VEGF receptor-2 cDNA).

Other examples of such inhibitors of angiogenesis, neovascularization, and/or other vascularization include antiangiogenic heparin derivatives (eg, heparinase III), temozolomide, NK4, macrophage migration inhibitory factor, cyclooxygenase-2 inhibitors, hypoxia-inducible factor 1 inhibitors, antiangiogenic soy isoflavones, oltipraz, fumagillin and its analogs, somatostatin analogs, pentosan polysulfate, sodium tecohalane, dalteparin, tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, antibodies against other targets such as anti-alphaV/beta-3 integrin antibodies and anti-kinitostatin antibodies.

In one embodiment, the therapeutic agent for use in combination with an anti-cMet antibody in the treatment of the disorders described above may be an anti-cancer immunogen, such as a cancer antigen/tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM/TACSTDl), mucin 1 (MUC1), oncoembryonic antigen (OAA), tumor associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, vaccines against viruses associated with malignancies (e.g. human papillomavirus vaccines) or heat shock tumor proteins.

In one embodiment, the therapeutic agent for use in combination with an anti-cMet antibody in the treatment of the disorders described above may be an antitumor cytokine, a chemokine, or a combination thereof. Examples of suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (eg, INFa2b), IFNe, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim and TNFa. Suitable chemokines may include Glu-Leu-Arg (ELR)-negative chemokines such as IP-10, MCP-3, MIG and SDF-1a from the CXC and C-C families of human chemokines. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine-based chimeric proteins.

In one embodiment, the therapeutic agent for use in combination with an anti-cMet antibody in the treatment of the disorders described above may be a cell cycle control/apoptosis regulator (or regulatory agent). The cell cycle control/apoptosis regulator may include molecules that target and modulate cell cycle control/apoptosis regulators such as (i) cdc-25 (e.g., NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (e.g., flavopiridol ( L868275, HMR1275), 7-hydroxycytosporine (UCN-01, KW-2401) and roscovitine (R-roscovitine, CYC202)) and (iii) telomerase modulators (e.g. BIBR1532, SOT-095, GRN163 and compositions described in e.g. US 6440735 and US 6713055). Non-limiting examples of molecules that interfere with apoptotic signaling pathways include TNF-related apoptosis-inducing ligand (TRAIL)/apoptosis ligand 2 (Apo-2L), antibodies that activate TRAIL receptors, IFN, and antisense Bcl-2.

In one embodiment, the therapeutic agent for use in combination with an anti-cMet antibody in the treatment of the disorders described above may be a hormonal regulatory agent, such as agents useful in anti-androgen and anti-estrogen therapy. Examples of such hormonal regulatory agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, antiandrogen (such as flutamind/eulexin), progestin (eg, such as hydroxyprogesterone caproate, medroxyprogesterone/provera, megestrol acepate/ Megace), adrenocorticosteroid (eg, hydrocortisone, prednisolone), luteinizing hormone releasing factor (and its analogs and other LHRH agonists such as buserelin and goserelin), aromatase inhibitor (eg, anastrazole/arimidex, aminoglutethimide/citraden, exemestane) or hormonal inhibitor (eg octreotide/sandostatin).

In one embodiment, the therapeutic agent for use in combination with an anti-cMet antibody in the treatment of the disorders described above may be any anti-anergic agent, for example, compounds that are molecules that block the activity of CTLA-4, for example, may be ipilimumab.

In one embodiment, the therapeutic agent for use in combination with an anti-cMet antibody in the treatment of the disorders described above may be an antitumor nucleic acid

- 24 045516 or antitumor inhibitory RNA molecule.

Examples of other antineoplastic agents that may be useful as therapeutic agents for use in combination with an anti-c-Met antibody in the treatment of the disorders described above include differentiation-inducing agents, retinoic acid analogues (such as all of trans-retinoic acid, 13-cis-retinoic acid and similar agents), vitamin D analogues (such as seocalcitol and similar agents), ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk/2, Flt4, FGFR1, FGFR2, FGFR3 inhibitors , FGFR4, TRKA, TRKC, RON (eg anti-RON antibody), Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7 and similar agents.

Examples of other antineoplastic agents that can be used as therapeutic agents in combination with an anti-c-Met antibody in the treatment of the disorders described above are estramustine and epirubicin.

Examples of other antitumor agents that can be used as therapeutic agents for use in combination with an anti-c-Met antibody in the treatment of the disorders described above include an HSP90 inhibitor such as 17-allylaminogeldamycin, antibodies against a tumor antigen such as PSA, CA125, KSA, integrin such as beta1 integrin, or VCAM inhibitors.

Examples of other antineoplastic agents that may be used as therapeutic agents for use in combination with an anti-c-Met antibody in the treatment of the disorders described above include calcineurin inhibitors (such as valspodar, PSC 833 and other MDR-1 or p-glycoprotein inhibitors) , TOR inhibitors (such as sirolimus, everlimus and rapamycin), and lymphocyte homing mechanism inhibitors (such as FTY720), and agents that affect cell signaling pathways, such as adhesion molecule inhibitors (for example, anti-LFA).

In one embodiment, the anti-c-Met antibody of the invention is for use in combination with one or more other therapeutic antibodies, such as ofatumumab, zanolimumab, daratumumab, ranibizumab, Zenapax, Simulect, Remicade, Himura, Tusabri, Xolair, Raptiva and/or rituximab.

Other therapeutic antibodies that can be used in combination with an antibody of the present invention are anti-c-Met antibodies that bind other c-Met regions, such as those described in WO 2005016382, WO 2006015371, WO 2007090807, WO 2007126799 or WO 2009007427 ( all are incorporated herein by reference).

In another embodiment, a combination of two or more different antibodies of the invention described herein is used in the treatment of a disease. Particularly interesting combinations include two or more non-competing antibodies. Such combination therapy may result in the binding of increased numbers of antibody molecules per cell, which may increase efficacy through activation of complement-mediated lysis.

In addition to the above, other embodiments of the combination therapies of the invention include the following:

In the treatment of non-small cell lung cancer, an anti-c-Met antibody is used in combination with EGFR inhibitors, such as an anti-EGFR antibody such as zalutumumab, cetuximab, panitumumab or nimotuzumab, or other EGFR inhibitors such as gefitinib or erlotinib) or in combination with an ErbB2 inhibitor (Her2/neu) (such as an anti-HER2 antibody such as trastuzumab, trastuzumab-DMI or pertuzmumab) or in combination with an inhibitor of both EGFR and HER2, such as lapatinib, or in combination with a HER3 inhibitor.

Glioma is treated with an anti-c-Met antibody in combination with temozolomide or an angiogenesis inhibitor such as bevacizumab.

In the treatment of colorectal cancer, an anti-c-Met antibody is used in combination with one or more compounds selected from: gemcitabine, bevacizumab, Folfox, Folfiri, Xelox, IFL, oxaliplatin, irinotecan, 5-FU/LV, capecitabine, UFT, targeting EGFR agents such as cetuximab, panitumumab, zalutumumab; VEGF inhibitors or tyrosine kinase inhibitors such as sunitinib.

In the treatment of prostate cancer, an anti-c-Met antibody is used in combination with one or more compounds selected from: hormonal/antihormonal therapy; for example, antiandrogens, luteinizing hormone releasing hormone (LHRH) agonists, and chemotherapeutic agents such as taxanes, mitoxantrone, estramustine, 5FU, vinblastine, ixabepilone.

Radiation therapy - surgery

In one embodiment, the present invention provides a method of treating a disorder involving c-Met expressing cells in a subject matter, wherein the method comprises administering a therapeutically effective amount of an anti-c-Met antibody of the present invention and at least one additional therapeutic agent to a subject in need of this object.

In one embodiment, the present invention provides a method of treating or preventing cancer, the method comprising administering a therapeutically effective amount of an anti-c-Met antibody of the present invention, and administering radiation therapy to the desired

- 25 045516 given in this object.

In one embodiment, the present invention provides the use of an anti-c-Met antibody of the present invention for the preparation of a pharmaceutical composition for the treatment of cancer by administering the antibody in combination with radiation therapy.

Radiation therapy may involve irradiation or associated administration of radiopharmaceuticals to the patient. The radiation source may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or in the form of brachotherapy (BT)). Radioactive elements that may be used in the practical implementation of such methods include, for example, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodide-123, iodide-131, and indium-111.

In a further embodiment, the present invention provides a method of treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-c-Met antibody, such as the anti-cMet antibody of the present invention, in combination with surgery.

Diagnostic applications

Antibodies of the invention against c-Met can also be used for diagnostic purposes. Thus, in a further aspect, the invention relates to a diagnostic composition comprising an anti-c-Met antibody as defined above.

In one embodiment, the anti-c-Met antibodies of the present invention can be used in vivo or in vitro to diagnose diseases in which activated cMet-expressing cells play an active role in pathogenesis, by determining the level of c-Met, or the level of cells that contain c-Met on its membrane surface. This can be achieved, for example, by contacting the test sample, optionally together with a control sample, with an anti-c-Met antibody under conditions that allow the formation of a complex between the antibody and c-Met.

Thus, in a further aspect, the invention relates to a method for detecting the presence of c-Met antigen or a cell expressing c-Met in a sample, comprising contacting the sample with an antibody under conditions that allow formation of a complex between the antibody and c-Met; and analysis of complex formation.

In one embodiment, the method is carried out in vitro.

More specifically, the present invention provides methods for identifying and diagnosing invasive cells and tissues and other cells targeted by the anti-c-Met antibodies of the present invention, and methods for monitoring the progress of therapeutic treatment, post-treatment status, risk of cancer, progression of cancer and so on.

Suitable labels for the anti-c-Met antibody and/or secondary antibodies used in such methods are well known in the art.

In a further aspect, the invention relates to a reagent kit for detecting the presence of c-Met antigen or a cell expressing c-Met in a sample that contains an anti-c-Met antibody of the invention or a bispecific molecule of the invention and instructions for use of the reagent kit.

In one embodiment, the present invention provides a kit for the diagnosis of cancer comprising a container containing an anti-c-Met antibody and one or more reagents for detecting binding of the anti-c-Met antibody to c-Met. Reagents include, for example, fluorescent tags, enzyme tags, or other detectable tags. Reagents may also include secondary or tertiary antibodies or reagents for enzymatic reactions, where the enzymatic reactions produce a product that can be visualized.

Anti-idiotypic antibodies

In a further aspect, the invention provides an anti-idiotypic antibody that binds the anti-c-Met antibody of the invention described herein.

An anti-idiotypic (Id) antibody is an antibody that recognizes unique determinants typically associated with the antigen-binding site of the antibody. As a source of the anti-c-Met mAb with the Id mAb, the Id antibody can be produced by immunizing an animal of the same species and genetic type for which the anti-Id antibody is produced. The immunized animal typically recognizes and responds to the idiotypic determinants of the immunized antibody by producing an antibody to the idiotypic determinants (anti-Id antibody).

Anti-Id antibodies can also be used as an immunogen to induce an immune response in another animal, producing a so-called anti-Id antibody. The anti-Id antibody may be epitopically identical to the parent mAb that induces the anti-Id antibody. Thus, by using antibodies to idiotypic mAb determinants, other clones expressing antibodies of identical specificity can be identified.

The present invention is further illustrated by the following examples, which are not

- 26 045516 should be construed as limiting.

Examples

Example 1: c-Met Expression Constructs

Codon-optimized constructs were obtained for the expression of c-Met, the extracellular domain (ECD) (aa 1-932 and the C-terminal His6 tag or the SEMA domain of c-Met (aa 1-567 and the C-terminal His9 tag) in HEK cells and CHO The proteins encoded by these constructs are identical to Genbank account NM 000245 for c-Met. The constructs contain suitable restriction sites for cloning and the optimal Kozak sequence (Kozak et al. (1999) Gene 234: 187-208). The constructs are cloned into a vector for mammalian expression of pEE13.4 (Lonza Biologies) (Bebbington (1992) Biotechnology (NY) 10:169-175), yielding pEE13.4cMet, pEE13.4cMetECDHis and pEE13.4cMetSEMA-567His8.

Example 2: Expression constructs for 5D5v1, 5D5 and G11-HZ

Codon-optimized constructs were obtained for the expression of heavy (HC) and light chains (LC) of IgG1 antibodies 5D5vl, 5D5 and G11-HZ in HEK cells. The proteins encoded by these constructs are identical to those described in Pat. US 6468529 (sequences 3 and 4) for the heavy and light chains 5D5vl, WO 2006/015371 A2 (Fig. 13) for the heavy and light chains 5D5, and WO 2009/007427 A2 (sequence was isolated from multiple figures) for the heavy and light chains 224G11. 224G11 is also referred to in this document as G11-HZ.

Example 3 Transient Expression in HEK-293F Cells

Freestyle™ 293-F cells (a subclone of HEK-293 adapted for suspension growth and chemically specified Freestyle media (HEK-293F)) were obtained from Invitrogen and transfected with the appropriate plasmid DNA using 293fectin (Invitrogen) according to the manufacturer's instructions. c-Met expression was checked by FACS analysis as described below. In the case of antibody expression, the corresponding expression vectors for the heavy and light chains were co-expressed.

Example 4 Transient Expression in CHO Cells pEE13.4cMet was transiently transfected into the Freestyle™ CHO-S cell line (Invitrogen) using Freestyle MAX transfection reagent (Invitrogen). c-Met expression was checked by FACS analysis as described below.

Example 5: Cloning and expression of monovalent antibodies (UniBody® molecules)

To express monovalent antibodies in mammalian cells, the HC constant region from IgG4, which had lost the hinge (Ch) region (amino acids E99-P110) and contained two mutations F405T and Y407E in the CH3 region, was synthesized as a codon-optimized construct in the mammalian-expressing vector pcDNA3. 3 (Invitrogen) and named pUniTE. By inserting a codon optimized constant region into the human kappa light chain region in pcDNA3.3, a separate vector was constructed.

The relevant VH and VL regions were inserted into pUniTE and pKappa, respectively, to obtain vectors for the expression of the heavy and light chains of specific antibodies. Cotransfection of vectors with heavy and light chains of specific antibodies in HEK-293F cells (Invitrogen) led to the transient production of monovalent antibodies with the desired specificities. Antibody purification was carried out using Protein A affinity column chromatography (as described in Example 11).

Example 6 Purification of His-tagged c-Met cMetECDHis and cMetSEMAHis were expressed in HEK-293F cells. The His tag in cMetECDHis and cMetSEMAHis allows them to be purified by immobilized metal affinity chromatography. In this method, the chelator fixed in the chromatography resin is loaded with Co ²⁺ . Supernatants containing cMetECDHis and cMetSEMAHis were incubated with the resin in batch mode (i.e., in solution). The His-tagged protein binds strongly to the resin beads, whereas other proteins present in the culture supernatant do not bind strongly. After incubation, the beads were removed from the supernatant and placed on a column. The column was washed to remove loosely bound proteins. The highly bound proteins cMetECDHis and cMetSEMAHis were then eluted with a buffer containing imidazole, which competes with His for binding to Co ²⁺ . The eluent was removed from the protein by exchanging the buffer on the desalting column.

Example 7. Procedure for immunization of transgenic mice

Antibodies 005, 006, 007, 008, 011, 012, 016, 017, 022, 024, 025, 028, 031, 035, 039, 040, 045, 093, 095, 096, 101 and 104 were allocated from the following immunizations: one one HCo20 mouse (1 female, strain GG2713), one HCo17 mouse (female, strain GG2714) and two HCol2-Balb/C mice (2 females, strain GG2811) (Medarex, San Jose, CA, USA; for reference see above paragraph on HuMab mice, WO2009097006 and US2005191293) were immunized every two weeks in turn with 5x106 NCI-H441 tumor cells intraperitoneally (i.p.) and subcutaneously (s.c.) with 20 μg of cMetECDHis protein coupled to the keyhole limpet hemocyanin hapten (KLH) .

Antibodies 058, 061, 062, 063, 064, 065, 066, 068, 069, 078, 082, 084, 087, 089, 098 and 181 were isolated from the following immunizations: two HCo20 mice (1 male and 1 female, strain GG2713) , one HCol2Balb/C mouse (1 male, strain GG2811) (Medarex, San Jose, CA, USA; for reference, see above paragraph on

- 27 045516 HuMab mice) were immunized every two weeks in turn with 5x10 ⁶ CHO-K1SV cells transiently transfected with cMetECD, intraperitoneally (i.p.) and subcutaneously (s.c.) with 20 μg of cMetECDHis protein associated with the hemocyanin hapten of the snail lymph ( KLH).

A maximum of eight immunizations per mouse were performed, four i.p. and four p.c. immunization in the sacrum. The first immunization was performed with cells in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). For all other immunizations, cells were injected i.p. in PBS, and cMetECD coupled to KLH was injected s.c. using incomplete Freund's adjuvant (IFA; Difco Laboratories, Detroit, MI, USA). Mice with at least two consecutive c-Met-specific antibody titers of 200 (200/1 serum dilution) or greater, as determined by the FMAT antigen-specific screening assay, as described in Example 8, were pooled.

Example 8 Homogeneous antigen-specific screening assay

The presence of anti-c-Met antibodies in the sera of immunized mice or in HuMab hybridomas (human monoclonal antibodies) or in transfectome culture supernatants was determined in a homogeneous antigen-specific screening assay (fourth quadrant) using Fluorometric Micro volume Assay Technology (FMAT; Applied Biosystems, Foster City,

California, USA). For this purpose, a combination of 3 cell-based assays and one bead-based assay was used. In cell-based assays, binding to TH1016-cMet (HEK-293F cells transiently expressing the extracellular domain of the c-Met receptor; prepared as described above) and HT29 (which express c-Met on the cell surface), as well as HEK293 cells, was determined. wild type (negative control which does not express c-Met). For the bead-based assay, binding to SB1016-cMet (cMetECDHis produced by transient expression in HEK-293F cells as described above, biotinylated and coupled to streptavidin-coated beads) was determined. Samples were added to cells/beads to bind to c-Met. HuMab binding was then detected using a fluorescent conjugate (goat anti-human IgG-Cy5; Jackson ImmunoResearch). The c-Met-specific chimeric antibody 5D5vl (produced in HEK-293F cells) was used as a positive control, and HuMab and HuMab-KLH mixed sera were used as negative controls. Samples were scanned using an Applied Biosystems 8200 Cellular Detection System (8200 CDS) and the counts x fluorescence values were used as output data. Samples were determined to be positive when counts were greater than 50 and the 'counts x fluorescence' was at least three times higher than the HuMab-KLH negative control.

Example 9. Preparation of HuMab hybridoma

HuMab mice with sufficient development of an antigen-specific titer (defined above) were sacrificed and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Fusion of splenocytes and lymph node cells with a murine myeloma cell line was performed by electrofusion using the CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), essentially according to the manufacturer's instructions. Fusion plates were screened using antigen-specific binding assays as described above, and cells positive for this assay were tested in the Alphascreen® SureFire® ERK phosphorylation assay and the Octet affinity assay as described below. Antibodies 031, 035, 087, and 089 were raised and cultured using standard protocols (e.g., as described in Coligan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M. and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).

In parallel, antibodies 005, 006, 007, 008, 011, 012, 016, 017, 022, 024, 025, 028, 035, 039, 040, 045, 058, 061, 062, 063, 064, 065, 06 were cloned 6,068 , 069, 078, 082, 084, 093, 095, 096, 098, 101, 104 and 181 using the ClonePix system (Genetix, Hampshire, UK). Specific primary well hybridomas were plated on semisolid medium prepared with 40% CloneMedia (Genetix, Hampshire, UK) and 60% HyQ 2x complete medium (Hyclone, Waltham, USA), and approximately 100 subclones of each primary well were punctured. Subclones were tested in an antigen-specific binding assay as previously described, and IgG levels were measured using Octet to select the most specific and productive clone from the primary well for subsequent expansion. Subsequent expansion and cultivation of the resulting HuMab hybridomas was carried out using standard protocols (e.g., as described in Coligan, J.E., Bierer, B.E., Margulies, D.H., Shevach, E.M. and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons , Inc., 2006).

Example 10: Mass spectrometry of purified antibodies

A small 0.8 ml aliquot of antibody containing hybridoma supernatant from the 6-well or Hyperflask stage was purified using PhyTip columns containing protein G resin (PhyNexus Inc., San Jose, USA) on a Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton, USA) PhyTip columns were used according to the manufacturer's instructions, but the buffers were replaced with: PBS Binding Buffer (B. Braun, Medical B.V., Occ, The Netherlands) and Elution Buffer 0.1 M Glycine-HCl pH 2.7 (Fluka Riedel -de Haen, Buchs, Germany), after cleaning the samples were neutralized

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M 2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zeindrecht, The Netherlands). Alternatively, in some cases large volumes of culture supernatant were purified by protein A affinity column chromatography.

After purification, samples were placed in a 384-well plate (Waters, 100 µl square well plate, part* 186002631). Samples were deglycosylated overnight at 37°C with Nglycosidase F. DTT (15 mg/ml) was added (1 μl/well) and incubated for 1 hour at 37°C. Samples (5 or 6 μl) were desalted on an Acquity UPLC™ (Waters, Milford, USA) using a BEN300 C18, 1.7 ct, 2.1 x 50 mm column at 60°C. MQ water and LC-MS grade acetonitrile (Biosolve, cat no 01204101, Valkenswaard, The Netherlands), both with 0.1% formic acid (Fluka, cat no 56302, Buchs, Germany), were used as Eluents A and B, respectively. Electrospray ionization time-of-flight mass spectra were recorded in real time on a micrOTOF™ mass spectrometer (Bruker, Bremen, Germany) operating in positive ion mode. Prior to analysis, the 900–3000 m/z scale was calibrated using ES Setup Mix (Agilent Technologies, Santa Clara, USA). Mass spectra were deconvoluted using DataAnalysis™ software v. 3.4 (Bruker) using the maximum entropy algorithm to find molecular masses between 5 and 80 kDa.

After deconvolution, the resulting heavy and light chain masses for all samples were compared to detect duplicate antibodies. In heavy chain comparisons, the possible presence of C-terminal lysine variants was taken into account. A list of unique antibodies was obtained, in which unique is defined as a unique combination of heavy and light chains. In the event that duplicate antibodies were found, results from other tests were used to decide which antibody was the best material to proceed with the experiments.

Example 11. Analysis and cloning of anti-c-Met antibody variable domain sequences into expression vectors

HuMab anti-c-Met total RNA was prepared from 5x106 hybridoma cells, and 5'-RACE complementary DNA (cDNA) was prepared from 100 ng of total RNA using the SMART RACE cDNA Amplification kit (Clontech), according to the manufacturer's instructions. The VH (heavy chain variable region) and VL (light chain variable region) coding regions were amplified by PCR and cloned in frame into constant region vectors pGlf (containing a codon optimized, fully synthetic, heavy chain constant region of human IgG1 (allotype f) in the mammalian expression vector pEE6.4 (Lonza Biologies, Slough, UK (Bebbington et al. (1992) Biotechnology 10:169-175)) and pKappa (containing a codon optimized, fully synthetic, human kappa light chain constant region (allotype Km3 ) in the mammalian expression vector pEE12.4 (Lonza Biologies, Slough, UK (Bebbington et al. (1992) Biotechnology 10:169-175)) using a ligation-independent cloning strategy (Aslanidis et al. Nucleic Acids Res. 18: 6069-6074) For each HuMab, 12 VL clones and 8 VH clones were sequenced and their theoretical masses were calculated and compared with available antibody mass spectrometry data. The sequences are given in the sequence list and in table. 1 below in this document. CDR sequences were determined according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). In table 2 and 3 provide an overview of antibody sequence information and most homologous germline sequences.

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Table 1. Variable heavy chain (VH), variable light chain (VL) and CDR sequences from HuMab

SEQ ID No: 1 VH 005 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYGFGWVRQAPGQGLEWMGRISPILGIANY AQMFQGRVTITADKSTSTAYMELSSLRSED TAVYYCARDVGYDWPDTFDIWGQGTMVIV ss SEQ ID No: 2 VH 005, CDR1 SYGFG SEQ ID No: 3 VH 005, CDR2 RISPILGIANYAQMFQG SEQ ID No: 4 VH 005, CDR3 DVGYDWPDTFDI SEQ ID No: 5 VL 005 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGGGSGTDFTLTISSLQPEDFATYYCQQYN SFPPTFGQGTKVEIK SEQ ID No: 6 VL 005, CDR1 RASQGISSWLA SEQ ID No: 7 VL 005, CDR2 AASSLQS SEQ ID No: 8 VL 005, CDR3 QQYNSFPPT SEQ ID No: 9 VH006 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SFGIGWVRQAPGQGLEWMGRIFPILGTANY AQMFQGRVTITADKSTSTAYMELTSLRSED TAVYYCARDVGYDSADAFDIWGQGTMVTV SS SEQ ID No: 10 VH 006, CDR1 S.F.G.I.G. SEQ ID No: 11 VH 006, CDR2 RIFPILGTANYAQMFQG SEQ ID No: 12 VH 006, CDR3 DVGYDSADAFDI SEQ ID No: 13 VL006 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPPTFGQGTKVEIK SEQ ID No: 14 VL 006, CDR1 RASQGISSWLA SEQ ID No: 15 VL 006, CDR2 AASSLQS SEQ ID No: 16 VL 006, CDR3 QQYNSYPPT SEQ ID No: 17 VH 008 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS Y WIG WVRQMPGKGLEWMGIIYPGDS ETRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEITGEFDYW GQGTLVTVSS SEQ ID No: 18 VH 005, CDR1 SYWIG SEQ ID No: 19 VH 008, CDR2 IIYPGDSETRYSPSFQG SEQ ID No: 20 VH 008, CDR3 QEITGEFDY SEQ ID No: 21 VL008 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PRTFGQGTKVEIK SEQ ID No: 22 VL 008, CDR1 RASQGISSALA SEQ ID No: 23 VL 008, CDR2 DASSLES SEQ ID No: 24 VL 008, CDR3 QQFNSYPRT

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SEQ ID No: 25 VH022 QVQLVESGGGWQPGRSLRLSCAASGTFFS SYAMHWVRQAPGKGLEWVAVISYDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARELLWFGELW GYFDLWGRGTLVTVSS SEQ ID No: 26 VH 022, CDR1 SYAMH SEQ ID No: 27 VH 022, CDR2 VISYDGSNKYYADSVKG SEQ ID No: 28 VH 022, CDR3 ELLWFGELWGYFDL SEQ ID No: 29 VL022 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQEASS FTWTFGQGTKVEIK SEQ ID No: 30 VL 022, CDR1 RASQGISSWLA SEQ ID No: 31 VL 022, CDR2 AASSLQS SEQ ID No: 32 VL 022, CDR3 QEASSFTWT SEQ ID No: 33 VH024 EVQLLESGGGLVQPGGSLRLSCVASGFTFSS YAMSWVRQAPGKGLEWVSAISGSSGGSTY YVDSVKGRFTISRANSKNTLYLQMNSLRAE DTAVYYCAKDLDRGWMG YFGYWGQGTLVTVSS SEQ ID No: 34 VH 024, CDR1 SYAMS SEQ ID No: 35 VH 024, CDR2 AISGSSGGSTYYVDSVKG SEQ ID No: 36 VH 024, CDR3 DLDRGWMGYFGY SEQ ID No: 28 VL024 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPTFGQGTRLEIK SEQ ID No: 38 VL 024, CDR1 RASQGISSWLA SEQ ID No: 39 VL 024, CDR2 AASSLQS SEQ ID No: 40 VL 024, CDR3 QQANSFPT SEQ ID No: 41 VH035 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS YWIGWVRQMPGKGLEWMGIIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWNSLKASDT AMYYCARQEITGEFDYW GQGTLVTVSS SEQ ID No: 42 VH 035, CDR1 SYWIG SEQ ID No: 43 VH 035, CDR2 IIYPGDSDTRYSPSFQG SEQ ID No: 44 VH 035, CDR3 QEITGEFDY SEQ ID No: 45 VL035 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PMYTFGQGTKLEIK SEQ ID No: 46 VL 035, CDR1 RASQGISSALA SEQ ID No: 47 VL 035, CDR2 DASSLES SEQ ID No: 48 VL 035, CDR3 QQFNSYPMYT SEQ ID No: 49 VH 045 EVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMSWVRQAPGKGLEWVSVISGSGGITYY ADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARDRGWGSDYW GQGTLVTVSS SEQ ID No: 50 VH 045, CDR1 SYAMS SEQ ID No: 51 VH 045, CDR2 VISGSGGITYYADSVKG SEQ ID No: 52 VH 045, CDR3 DRGWGSDY

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SEQ ID No: 53 VL045 EIVLTQSPATLSLSPGERATLSCRASQSVSSY LAWYQQKPGQAPRLLIYDASNRATGIPARF SSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN WPFTFGPGTKVDIK SEQ ID No: 54 VL 045, CDR1 RASQSVSSYLA SEQ ID No: 55 VL 045, CDR2 DASNRAT SEQ ID No: 56 VL 045, CDR3 QQRSNWPFT SEQ ID No: 57 VH058 EVQLVESGGGLVKPGGSLKLSCAASGTFFS DYYMYWVRQTPEKRLEWVATISDDGSYTY YPDSVKGRFTISRDNAKNNLYLQMSSLKSE DTAMYYCAREGLYYYGSGSYYNQDYWGQ GTLVTVSS SEQ ID No: 58 VH 058, CDR1 DYYMY SEQ ID No: 59 VH 058, CDR2 TISDDGSYTYYPDSVKG SEQ ID No: 60 VH 058, CDR3 EGLYYYGSSGSYYNQDY SEQ ID No: 61 VL 058 AIQLTQSPSSLSASVGDRVTITCRASQGLSSA LAWYRQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFTSYP QITFGQGTRLEIK SEQ ID No: 28 VL 058, CDR1 RASQGLSSALA SEQ ID No: 63 VL 058, CDR2 DASSLES SEQ ID No: 64 VL 058, CDR3 QQFTSYPQIT SEQ ID No: 65 VH061 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWS WIRQPPGKGLEWIGX1IYHSGNTY DNPSLKSRVTIAVDRSKNQLSLKLSFLTAAD TAVYYCARSSYDFLTDWG QGTLVTVSS where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 66 VHO61,CDR1 SGGHSWS SEQ ID No: 67 VH061,CDR2 X1IYHSGNTYDNPSLKS, where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 68 VH061,CDR3 SSYDFLTD SEQ ID No: 69 VL061 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN GFPITFGQGTRLEIK SEQ ID No: 70 VLO61,CDR1 RASQGISSWLA SEQ ID No: 71 VL061,CDR2 AASSLQS SEQ ID No: 72 VL061,CDR3 QQANGFPIT SEQ ID No: 73 VH062 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWS WIRQPPGKGLEWIGX 1 IYHSGNTY DNPSLKSRVTIAVDRSKNQLSLKLSFVTAAD TAVYYCARSSYDILTDWGQGTLVTVSS where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 74 VH 062, CDR1 SGGHSWS SEQ ID No: 75 VH 062, CDR2 X1IYHSGNTYDNPSLKS, where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 76 VH 062, CDR3 SSYDILTD

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SEQ ID No: 77 VL062 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN GFPITFGQGTRLEIK SEQ ID No: 78 VL 062, CDR1 RASQGISSWLA SEQ ID No: 79 VL 062, CDR2 AASSLQS SEQ ID No: 80 VL 062, CDR3 QQANGFPIT SEQ ID No: 81 VH 064 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHS WS WIRQPPGKGLEWIGX11YHSGNTY DNPSLKSRVTISVDRSKNQVSLKLSSVTAAD TAVYYCARSSYDILTDWGQGTLVTVSS, where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 82 VH 064, CDR1 SGGHSWS SEQ ID No: 83 VH 064, CDR2 X1IYHSGNTYDNPSLKS, where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 84 VH 064, CDR3 SSYDILTD SEQ ID No: 85 VL064 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN GFPITFGQGTRLEIK SEQ ID No: 86 VL 064, CDR1 RASQGISSWLA SEQ ID No: 87 VL 064, CDR2 AASSLQS SEQ ID No: 88 VL 064, CDR3 QQANGFPIT SEQ ID No: 89 VH 068 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGYSWSWIRQPPGKGLEWIGXHYHSGSTYY NPSLKSRVTISVDRSKNQFSLKLSSVTAADT AVYYCARSSYDILTDW GQGTLVTVSS where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 90 VH 068, CDR1 SGGYSWS SEQ ID No: 91 VH 068, CDR2 X1IYHSGSTYYNPSLKS, where XI is any amino acid, preferably C, S, Y or A SEQ ID No: 92 VH 068, CDR3 SSYDILTD SEQ ID No: 93 VL068 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQ ID No: 94 VL 068, CDR1 RASQGISSWLA SEQ ID No: 95 VL 068, CDR2 AASSLQS SEQ ID No: 96 VL 068, CDR3 QQANSFPIT SEQ ID No: 97 VH069 QVQLVQSGAEVKKPGASVKVSCETSGYTFT SYGISWVRQAPGHGLEWMGWISAYNGYTN YAQKLQGRVTMTTDTDTSTSTAYMELRSLRSD DTAVYYCARDLRGTNYFDYWGQGTLVTVS S SEQ ID No: 98 VH 069, CDR1 SYGIS SEQ ID No: 99 VH 069, CDR2 WISAYNGYTNYAQKLQG SEQ ID No: 100 VH 069, CDR3 DLRGTNYFDY

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SEQIDNo: 101 VL069 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWFQHKPGKAPKLLIYAASSLLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQIDNo: 102 VL 069, CDR1 RASQGISNWLA SEQ ID No: 103 VL 069, CDR2 AASSLLS SEQIDNo: 104 VL 069, CDR3 QQANSFPIT SEQIDNo: 105 VH096 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS YWIGWVRQMPGKGLEWMGIIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEITGDFDYWGQGTLVTVSS SEQ ID No: 106 VH 096, CDR1 SYWIG SEQ ID No: 107 VH 096, CDR2 IIYPGDSDTRYSPSFQG SEQIDNo: 108 VH 096, CDR3 QEITGDFDY SEQ ID No: 109 VL096 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPNLLIYAASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PLTFGGGTKVEIK SEQ ID No: 110 VL 096, CDR1 RASQGISSALA SEQ ID No: 111 VL 096, CDR2 AASSLES SEQ ID No: 112 VL 096, CDR3 QQFNSYPLT SEQ ID No: 113 VH098 QVQLVQSGAEVKKPGASVKVSCKASGYTF TNFGISWVRQAPGQGLEWMGWISAFNGHT DYSQKVQGRVTMTTDTSTSTAYMELRSLRS DDTAVFYCARSHYYGSGSPFDYWGQGTLV TVSS SEQIDNo: 114 VH 098, CDR1 NFGIS SEQIDNo: 115 VH 098, CDR2 WISAFNGHTDYSQKVQG SEQIDNo: 116 VH 098, CDR3 SHYYGSSGSPFDY SEQ ID No: 117 VL098 DIQMTQSPSSLSASVGDRVTITCRASQGISN WLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCHQYK SYPWTFGQGTKVEIK SEQ ID No: 118 VL 098, CDR1 RASQGISNWLA SEQIDNo: 119 VL 098, CDR2 AASSLQS SEQ ID No: 120 VL 098, CDR3 HQYKSYPWT SEQIDNo: 121 VH 101 QVQLVQSGGEVKKPGASVKVSCKASGYTF TRHGITWVRQAPGQGLEWMGWISADNGNT NYAQKFQDRVTMTTDTSTSTAYMELRSLRS DDTAVYFCARVFRYFDWLLPYFDYWGQGT LVTVST SEQ ID No: 122 VH 101,CDRl RHGIT SEQ ID No: 123 VH 101,CDR2 WISADNGNTNYAQKFQD SEQ ID No: 124 VH 101,CDR3 VFRYFDWLLPYFDY SEQ ID No: 125 VL 101 EIVLTQSPGTLLSLSPGERATLSCRASQSVSSS YLAWYQQKPGQAPRLLIYGVFSRATGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYG SSPYTFGQGTKLEIK SEQIDNo: 126 VL 101,CDRl RASQSVSSSYLA SEQ ID No: 127 VL 101,CDR2 GVFSRAT SEQ ID No: 128 VL 101, CDR3 QQYGSSPYT

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SEQ ID No: 129 VH 181 QVQLVQSGAEVKKPGASVKVSCKASGYTF TSYGISWVRQAPGQGLEWMGWISTYNGYT NYAQKLQGRVTMTTDTSTSTAYMELRSLRS DDTAVYYCARDLRGTAYFDYWGQGTLVTV ss SEQ ID No: 130 VH 181,CDR1 SYGIS SEQ ID No: 131 VH 181,CDR2 WISTYNGYTNYAQKLQG SEQ ID No: 132 VH 181,CDR3 DLRGTAYFDY SEQ ID No: 133 VL 181 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQ ID No: 134 VL 181,CDR1 RASQGISNWLA SEQ ID No: 135 VL 181,CDR2 AASSLLS SEQ ID No: 136 VL 181,CDR3 QQANSFPIT SEQ ID No: 137 VH066 QVQLVQSGAEVKKPGASVKVSCEASGYTFT SYGISWVRQAPGHGLEWMGWISAYNGYTN YAQKLQGRVTMTADTSTSTAYMELRSLRS DDTAVYYCARDLRGTNYFDYWGQGTLVTV SS SEQ ID No: 138 VL066 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQ ID No: 139 VH 065 QVQLVQSGAEVKKPGASVKVSCEASGYTFT NYGISWVRQAPGHGLEWMGWISAYNGYTN YAQKLQGRVTMTTDTSTTTAYMELRSLRSD DTAVYYCARDLRGTNYFDYWGQGTLVTVS S SEQ ID No: 140 VL065 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQ ID No: 141 VH 082 QVQLVQSGAEVKKPGASVKVSCETSGYTFT SYGISWVRQAPGHGLEWMGWISAYNGYTN YAQKLQGRVTMTTDTDTSTSTAYMELRSLRSD DTAVYYCARDLRGTNYFDYWGQGTLVTVS S SEQ ID No: 142 VL082 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWYQHKPGKAPKLLIYAASSLLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQ ID No: 143 VH 089 QVQLVQSGAEVKKPGASVKVSCETSGYTFT SYGISWVRQAPGHGLEWMGWISAYNGYTN YAQKLQGRVTMTTDTDTSTSTAYMELRSLRSD DTAVYYCARDLRGTNYFDYWGQGTLVTVS S SEQ ID No: 144 VL 089 DIQMTQSPSSVSASVGDRVTITCRASQGISN WLAWFQHKPGKAPKLLIYAASSLLSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK

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SEQ ID No: 145 VH031 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYGFGWVRQAPGQGLEWMGRISPILGITNY AQMFQGRVTITADKSTSTAYMELSSLRSED TAVYYCARDVGYDWPDTFDIWGQGTMVIV ss SEQ ID No: 146 VL031 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGGGSGTDFTLTISSLQPEDFATYYCQQYN SFPPTFGQGTKVEIK SEQ ID No: 147 VH 007 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYGIGWVRQAPGQGLEWMGRIFPILGTANY AQMFQGRVTITADKSTSTAYIELTSLRSEDT AVYYCARDVGYDSADAFDIWGQGTMVTVS S SEQ ID No: 148 VL 007 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPPTFGQGTKVEIK SEQ ID No: 149 VH011 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYGIGWVRQAPGQGLEWMGRVFPILGTAN YAQMFQGRVTITADKSTSTAYMELTSLRSE DTAVYYCARDVGYDSADAFDIWGQGTMVT VSS SEQ ID No: 150 VL011 DIQMTQSPSSLSASVGDRVTITCRASQGISS WLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYN SYPPTFGQGTKVEIK SEQ ID No: 151 VH017 QVQLVESGGGWQPGRSLRLSCAASGTFFS SYAMHWVRQAPGKGLEWVAFISYDGSNKY FADSVKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCARELLWFGELWGYFDLWGRGTL VTVSS SEQ ID No: 152 VL017 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQEANS FTWTFGQGTKVEIK SEQ ID No: 153 VH 025 QVQLVESGGGWQPGRSLRLSCAASGTFFS SYAMHWVRQAPGKGLEWVAFISYDGSSKD YADSVKGRFTIFRDNSKNTLYLQMSSLRAA DTAVYYCARELLWFGELWGYFDLWGRGTL VTVSS SEQ ID No: 154 VL 025 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQTNS FTWTFGQGTKVEIK SEQ ID No: 155 VH040 EVQLLESGGGLVQPGGSLRLSCAASGFTFSS YAMTWVRQAPGKGLEWVSVISGSGGITYY ADSVKGRFTISRDNSKNTLYLQMNSLRAED TAVYYCARDRGWGSDYWGQGTL VTVSS

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SEQIDNo: 156 VL040 EIVLTQSPATLSLSPGERATLSCRASQSVSSY LAWYQQKPGQAPRLLIYDASNRATGIPARF SSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN WPFTFGPGTKVDIK SEQ ID No: 157 VH 039 EVQLLESGGGLVQPGGSLRLSCAASGFTFN NYAMSWVRQAPGKGLEWVSAISGSGGITY YADSEKGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCAKDRGWGSDCWGQGTLVTVSS SEQIDNo: 158 VL039 EIVLTQSPATLSLSPGERATLSCRASQSVSSY LAWYQQKPGQAPRLLIYDASNRATGIPARF SSGSGSGTDFTLTISSLEPEDFAVYYCQQRSN WPFTFGPGTKVDIK SEQIDNo: 159 VH 078 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGCLYHSGNTYY NPSLKSRVTISVDRSKNQFSLKLSSVTAADT AVYYCARSSYDILTDWGQGILVTVSS SEQIDNo: 160 VL078 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQIDNo: 161 VH 084 QLQLQESGSGLVKPSQTLSLTCGVSGGSISS GGHSWSWIRQPPGKGLEWIGCLYHSGNTYY NPSLKSRVTISVDRSKNQFSLKLSSVTAADT AVYYCARSSYDILTDWGQGTLVTVSS SEQ ID No: 162 VL084 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN SFPITFGQGTRLEIK SEQ ID No: 163 VH063 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGCIYHSGNTYD NPSLKSRVTIAVDRSKNQLSLKLSFVTAADT AVYYCARSSYDILTDWGQGTLVTVSS SEQ ID No: 164 VL063 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN GFPITFGQGTRLEIK SEQ ID No: 165 VH 087 QLQLQESGSGLVKPSQTLSLTCAVSGGSISS GGHSWSWIRQPPGKGLEWIGCIYHSGNTYD NPSLKSRVTISVDRSKNQFSLKLSSVTAADT AVYYCARSSYDILTDWGQGTLVTVSS SEQ ID No: 166 VL087 DIQMTQSPSSVSASVGDRVTITCRASQGISS WLAWYQHKPGKAPKLLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQAN GFPITFGQGTRLEIK SEQIDNo: 167 VH016 EVQLVQSGAEVKKPGESLKISCKGSGYIFTS YWIGWVRQMPGKGLEWMGIIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEVTGDFDYWGQGTLVTVSS SEQ ID No: 168 VL016 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PLTFGGGTKVEIK

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SEQIDNo: 169 VH 028 EVQLVQSGGEVKKPGESLKISCKGSGYSFTS YWIGWVRQMPGKGLEWMGIIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEVTGDFDYWGQGTLVTVSS SEQ ID No: 170 VL028 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PLTFGGGTKVEIK SEQIDNo: 171 VH 012 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS YWIGWVRQMPGKGLEWMGIIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEITGEFDYWGQGTLVTVSS SEQ ID No: 172 VL012 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PRTFGQGTKVEIK SEQ ID No: 173 VH 095 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS YWIGWVRQMPGKGLEWMGIIYPGDSNTRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEITGDFDYW GQGTLVTVSS SEQIDNo: 174 VL 095 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYDASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PLTFGGGTKVEIK SEQIDNo: 175 VH 093 EVQLVQSGAEVKKPGESLKISCKGSGYSFTS YWIGWVRQMPGKGLEWMGIIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEITGDFDYW GQGTLVTVSS SEQIDNo: 176 VL093 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPNLLIYAASSLESGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQFNSY PLTFGGGTKVEIK SEQ ID No: 177 VH 104 EVQLVQSGAEVKKPGESLKISCKGSGYSFIS YWIGWVRQMPGKGLEWMGIIYPGDSDTRY SPSFQGQVTISADKSISTAYLQWSSLKASDT AMYYCARQEITGDFDYW GQGTLVTVSS SEQ ID No: 178 VL 104 AIQLTQSPSSLSASVGDRVTITCRASQGISSA LAWYQQKPGKAPKLLIYVASSLESGVPSRFS GSGSGTDFTLTITSLQPEDFATYYCQQFNSY PITFGQGTRLEIK

Table 2. Homologies of mouse origin and heavy chain sequences

Antibody mouse number mouse line germinal VH THIO 16-005 339732 HCo12V, CI IgHVl-69-4 TH1016-006 339732 HCo12V, CI IgHVl-69-4 TH1016-008 339732 HCo12V, CI IgHV5-51-l TH1016-022 339733 HCo12V, CI IgHV3-30-3*l TH1016-024 339733 HCo12V, CI IgHV3-23-l TH1016-035-D09 339732 HCo12V, CI IgHV5-51-l TH 1016-045 339282 HCo17, CI IgHV3-23-l TH1016-058 343191 HCo12V, C2 IgHV3-ll-3 TH 1016-061 348072 NCo20, C2 IgHV4-30-2*l TH1016-062 348072 NCo20, C2 IgHV4-30-2*l TH1016-064 348072 NCo20, C2 IgHV4-30-2*l TH 1016-068 348072 NCo20, C2 IgHV4-30-2*l TH1016-069 348072 NCo20, C2 IgHVl-18-1 TH 1016-096 339732 НСО12В, С1 IgHV5-51-l TH1016-098 347330 HCo20; C2 IgHVl-18-1 TH1016-101 340659 NCo20,C1 IgHVl-18-1 TH1016-181 348072 HCo20', C2 IgHVl-18-1

Table 3. Homologies of mouse origin and light chain sequences

Antibody mouse number mouse line germ line PC 1016-005 339732 HCo12V, CI IGKV1D-16*O1 PC 1016-006 339732 HCo12V, CI IGKV1D-16*O1 PC 1016-008 339732 HCo12V, CI IGKVl-13*02 PC 1016-022 339733 HCo12V, CI IGKV1-12*O1 PC1016-024 339733 HCo12V, CI IGKV1-12*O1 Pl 016-035 339732 HCo12V, CI IGKV1-13*O2 PC1016-045 339282 HCo17, CI IGKV3-11*01 PC1016-058 343191 HCo12V, C2 IGKV1-13*O2 PC1016-061 348072 NCo20, C2 IGKV1-12*O1 PC1016-062 348072 NCo20, C2 IGKV1-12*O1 PC 1016-064 348072 NCo20, C2 IGKV1-12*O1 PC 1016-068 348072 NCo20, C2 IGKV1-12*O1 PC1016-069 348072 NCo20, C2 IGKV1-12*01 PC1016-096 339732 HCo12V, CI IGKV1-13*O2 PC 1016-098 347330 NCo20, C2 IGKV1D-16*O1 PC1016-101 340659 NCo20, CI IGKV3-20*01 PC1016-181 348072 NCo20, C2 IGKV1-12*01

In fig. Figures 1 and 2 show the sequence alignment of HuMabs. Based on these sequences, a consensus sequence for the CDR sequences can be determined. These consensus sequences are presented in Table. 4.

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Table 4. Consensus sequences

SEQID No: 179 005-006 SEQ ID No: 180 005-006 IgHVl-694 IgHVl-694 CDR1 CDR2 SX1X2X3X4 RXIX2PILGX3X4NYAQ X5FQG where X1=Y or F, X2=A or G, X3 = F or I, X4 = S or G. It is preferable if X1=Y or F, X2 = G, X3 - F or I and X4=G. where XI-I or V, X2 - I,S or F, X3=I or T, X4=A or T, X5 = K or M. It is preferable if X1=1 or V, X2=S or F, X3= 1 or T, X4=A or T and X5 = M. SEQ ID No: 181 005-006 IgHVl-694 CDR3 DVGYDX1X2DX3FDI where X1=W or S, X2 = P or A, XZ=T or A SEQ ID No: 182 008-035 IgHV551-1 CDR2 IIYPGDSXITRYSPSFQG where XI = D, E or N SEQ ID No: 183 008-035096 IgHV551-1 CDR3 QEX1TGX2FDY where X1=V or I, X2 = E or D SEQ ID No: 184 022 IgHV330-3*1 CDR2 X1 IS YDGSX2KX3X4AD SVKG where X1=V or F, X2 = N or S, XZ = D or Y, X4=Y or F SEQ ID No: 185 024 IgHV323-1 CDR2 AISGSX1GGSTYYX2DS VKG where XI = S or absence of ak, X2 = V or A SEQID No: 186 045 IgHV323-1 CDR1 X1YAMX2 where XI = S or N, X2=S or T SEQ ID No: 187 045 IgHV323-1 CDR2 X1ISGSGGX2TYYADS X3KG where X1=A or V, X2 = S or I, X3=V or E. Preferably, where X1=A or V, X2 = I and X3=V or E. SEQID No: 188 045 IgHV323-1 CDR3 DRGWGSDX1 where X1=Y or C SEQ ID No: 189 058 IgHV3-ll3 CDR1 DYYMX1 where Xl-Υ or S SEQ ID No: 190 058 IgHV3-ll3 CDR2 X1ISX2X3X4SYTX5YX 6DSVKG where X1=T or Υ, X2 = D or S, X3= D or S, X4 = G or S, Χ5=Υ or Ν, X6 = P or A SEQID No: 191 062-064068 IgHV430-2*1 CDR1 SGGX1SWS where Χ1=Υ or H SEQ ID No: 192 062-064068 IgHV430-2*1 CDR2 X1X2 YHSGX3TYX4NPS LKS where X1=any amino acid, preferably C, Y, S or A, X2 = I or L, X3=S or Ν, X4=Y or D SEQID No: 193 062-064068 IgHV430-2*1 CDR3 SSYDX1LTD where X1=F or I

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SEQID No: 194 069-181 IgHVl-181 CDR1 X1YGIS where X1=8 or N SEQ ID No: 195 069-181 IgHVl-18- 1 CDR2 WISX1YNGX2TNYAQK LQG where X1=A or T, X2 = N or Y. Preferably, where X1=A or T and Χ2=Υ SEQ ID No: 196 069-181 IgHVl-181 CDR3 DLRGTX1YFDY where X1=A or N SEQID No: 197 098 IgHVl-181 CDR1 X1X2GIS where XI = Ν or S, X2 = F or Υ SEQ ID No: 198,098 IgHVl-18- 1 CDR2 WISAX1NGX2TX3YX4 QKX5QG where XI = F or Υ, X2 = N or Ν, ХЗ = D or N, X4=S or A, X5=V or L SEQID No: 199 101 IgHVl-18- 1 CDR1 X1X2GIX3 where XI = R or S, X2 = H or Y, XZ = T or S SEQ ID No: 200 101 IgHVl-18- 1 CDR2 WISAX1NGNTNYAQK X2QX3 where XI = D or Y, X2 = F or L, XZ - D or G SEQ ID No: 201 101 IgHVl-18- 1 CDR3 VX1RYFD WLLX2 YFD Y where XI = F or L, X2 = P or absence of ac SEQ ID No: 202 005-006 IGKV1D16*01 CDR3 QQYNSX1PX2T where X1=Y or F, X2 = P or W. Preferably, where Χ1=Υ or F and X2 = P SEQ ID No: 203 008-035 IGKV113*02 CDR2 X1ASSLES where XI = D, V or A SEQ ID No: 204 008-035 IGKV113*02 CDR3 QQFNSYPLX1T where XI = R, I, L, W or ΜΥ SEQ ID No: 205 022 IGKV112*01 CDR3 QX1X2X3SFX4WT where XI = Q or E, X2 = A or T, XZ = Ν or S; X4 = P or T SEQ ID No: 206 024 IGKV1- 12*01 CDR3 QQANSFPX1T where X1=1 or absence of ak SEQ ID No: 207 058 IGKV1- 13*02 CDR3 QQFX1SYPX2IT where X1=T or Ν, X2 = Q or absence of ac SEQ ID No: 208 062-064068 IGKV112*01 CDR3 QQANX1FPIT where XI = G or S SEQID No: 209 069-181 IGKV112*01 CDR1 RASQGISX1WLA where X1=8 ηπηΝ SEQ ID No: 210 069-181 IGKV112*01 CDR2 AASSLX1S ^eXl=Q or L SEQ ID No: 211 098 IGKV1D16*01 CDR3 X1QYX2SYPWT where X1 = H or Q, X2 = K or N SEQ ID No: 212 101 IGKV320*01 CDR2 GX1X2SRAT where X1=V or A, X2 = F or S

Example 12 Antibody Purification

The culture supernatant was filtered through a 0.2 μm dead-end filter and applied to 5 ml MabSelect SuRe columns (GE Health Care) and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 and dialyzed overnight against 12.6 mM NaH ₂ PO ₄ , 140 mM NaCl, pH 7.4 (B. Braun). Otherwise, after purification, the eluate was applied to a HiPrep Desalting column and the antibody buffer was replaced with 12.6 mM NaH ₂ PO ₄ , 140 mM NaCl, pH 7.4 (B. Braun). After dialysis or buffer exchange, samples were sterile filtered through 0.2 μm dead-end filters. Purity was determined by SDS-PAGE, and concentration was measured by nephelometry and absorbance at 280 nm. Purified antibodies were stored at 4°C. Mass spectrometry was performed to identify the molecular weight of the heavy and light chains of the antibodies expressed by the hybridomas as described in Example 10.

Example 13 Binding of anti-c-Met clones to tumor cells that express membrane-bound c-Met measured by FACS analysis

The binding of c-Met antibodies and their monovalent forms (also referred to herein as UniBody molecules, see Example 5) to A431 cells expressing membrane-bound c-Met (purchased from ATCC, CRL-1555) was tested by flow cytometry (FACS Canto II BD Biosciences). The Qifi assay (Dako, Glostrup, Denmark) revealed that A431 cells express an average of 30

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000 copies of c-Met protein per cell. Binding of anti-c-Met antibodies and UniBody molecules was determined using phycoerythrin-conjugated goat human IgG binding antibodies (Jackson). IgG1-5D5 was used as a positive control antibody, and HuMab-KLH was used as an isotype control antibody. EC ₅₀ values were determined by nonlinear regression (sigmoidal dose response curve with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA).

In fig. Figure 3 shows that all anti-c-Met antibodies and UniBody molecules tested bind c-Met expressed on A431 cells in a dose-dependent manner. EC ₅₀ values for binding range from 0.28-1.92 nM for IgG and 0.52-13.89 nM for UniBody molecules. Interestingly, the IgG1-024 antibody showed high levels of unsaturated binding to A431 cells, which was not observed when testing binding to HT-29 cells (purchased from ATCC, NTB38™) (data not shown). For antibodies 022, 024, 062, 064, 069, 098, 101 and 181, no reduction in EC ₅₀ was observed or less than a 2-fold reduction was observed between IgG1 and UniBody molecules of identical clones. Also, the levels of maximum binding did not change for IgG1 and UniBody molecules. For antibodies 005, 006, 008, 035, 045 and 058, on the other hand, when comparing IgG1 with the equivalent UniBody, a greater than 2-fold decrease in EC ₅₀ value was observed, as well as a decrease in the level of maximum binding. This was most likely due to the lower dissociation rates ( _Kd ) of these antibodies (see example 14).

Example 14: Octet Analysis for Affinity Ranking

Antibody binding to cMetECDHis was analyzed by Bio-Layer Interferometry (BLI) technology on an Octet system (Fortebio, Menlo Park, USA). Biosensors coated with anti-human IgG antibodies (Fc-specific) were used to capture anti-c-Met antibodies according to the procedure recommended by the manufacturer. HEK293 cell-derived cMetECDHis was coated on top of immobilized anti-c-Met antibodies by placing the loaded biosensor in a well containing 10 μL/mL cMetECDHis diluted 10-fold in kinetics measurement buffer (Fortebio). The difference in light reflectance (Δλ, nm) of the biosensor surface due to binding to cMetECDHis was measured in real _time over approximately 10 minutes, and Octet software (V4.0, Fortebio). The loaded biosensor was then placed in a well containing only the kinetics buffer (diluted 10-fold in PBS) to determine the dissociation constant (kd [1/s]). Kinetics analysis was performed to determine affinity (K _D [M]) using a 1:1 (Langmuir) model. 0.2 μg/ml IgG15D5 produced by HEK293 cells was used as a positive control.

In table Figure 5 shows that all anti-c-Met antibodies bind cMetECDHis with nanomolar affinities in the range of 0.6-13.9 nM.

Table 5. Kinetic constants ( _ka , kd and _KD ) of antibodies upon binding to cMetECDHis

Clone K _a [1/Ms] LI1/S] KIM] 5D5 2D4E+05 1.25E-03 5.86E-09 005 ZD8E+05 2.52E-03 7.92E-09 006 4.25E+05 4.20E-03 9.89E-09 008 3.08E+05 1.57E-03 5D2E-09 022 2.36E+05 2.51E-04 1.06E-09 024 1.45E+05 2.28E-04 1.57E-09 035 2.64E+05 3.68E-03 1.39E-08 045 7.21E+05 2.07E-03 2.87E-09 058 4.64E+05 1.25E-03 2.70E-09 061 2.56E+05 1.53E-04 5.96E-10 062 2.73E+05 ZD9E-04 1D7E-09 064 2.84E+05 3.24E-04 1D4E-09 068 3.21E+05 1.35E-03 4.21E-09 069 2D2E+05 2.67E-04 1.26E-09 096 1.96E+05 5.00E-04 2.55E-09 098 L64E+05 2.97E-04 L82E-09 101 1.69E+05 2D4E-04 1.27E-09 181 2.37E+05 5.31E-04 2.23E-09

Example 15 Binding of anti-c-Met antibodies to membrane-bound c-Met on rhesus macaque epithelial cells measured by FACS analysis

To determine cross-reactivity with rhesus monkey c-Met, binding of anti-c-Met antibodies to cMet-positive rhesus monkey epithelial cells (4MBr-5, purchased from ATCC) was tested by flow cytometry (FACS Canto II, BD Biosciences) . Phycoerythrin-conjugated goat anti-human IgG antibody (Jackson) was used as a secondary conjugate. HuMab-KLH was used as an isotype control antibody.

In fig. 4 demonstrates that all anti-c-Met antibodies tested are cross-reactive with rhesus monkey c-Met. At both tested concentrations (0.5 and 10 µg/ml)

- 41 045516 anti-c-Met la were able to specifically bind rhesus monkey c-Met. For all antibodies, the signal was at least 5-fold higher than that for the isotype control antibody HuMab-KLH. Interestingly, P1016-035 exhibited much higher upper fluorescence levels (MFI ~200,000) compared to other c-Met-specific antibodies. This difference was not observed in cell lines expressing the human c-Met receptor.

Example 16: Blocking HGF Binding to the Extracellular Domain of c-Met Determined by Enzyme-Linked Immunosorbent Assay (ELISA)

To analyze the ability of anti-c-Met antibodies to block the binding of hepatocyte growth factor (HGF) to the c-Met receptor, ELISA was performed. To do this, the attached extracellular domain of c-Met was incubated with unlabeled anti-c-Met antibody and fluorescently labeled HGF. Non-blocking antibodies do not compete with labeled HGF for binding to c-Met, resulting in maximum fluorescence signal.

Blocking antibodies compete with labeled HGF for binding to c-Met, resulting in a decrease in fluorescent signal.

HGF (ProSpec Tarry, Rehovot, Israel) was fluorescently labeled by conjugation with Europium ³⁺ (PerkinElmer, Turku, Finland). ELISA wells were coated overnight at 4°C with 0.5 μg/ml recombinant human c-Met extracellular domain (R&D systems, Minneapolis, USA) diluted in PBS. ELISA wells were then washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and blocked for 1 h at room temperature (RT) with PBST supplemented with 2% (v/v). ) chicken whey (Gibco, Paisley, Scotland). After washing with PBST, ELISA wells were incubated for 1 h at room temperature in the dark with a mixture of 50 μl of serially diluted anti-c-Met antibody (0.128-10000 ng/ml in 5-fold dilutions) and HGF conjugated to 50 μl of 0. 44 µg/ml Europium ³⁺ in PBST. Unbound Europium ³⁺ -conjugated HGF was then washed with PBST, and bound Europium ³⁺ -conjugated HGF was incubated for 30 min at RT in the dark with Delfia Enhancement Solution (PerkinElmer) to enhance the fluorescent signal. The average fluorescence intensity at 615 nm was measured using an EnVision 2101 Multilabel reader (PerkinElmer) with the following settings: Lance/Delfia pair mirror, 615 emission filter, 340 nm excitation filter, 400 μs delay time, 400 μs window, 100 flashes, 2000 μs per cycle and bidirectional line-by-line readout. To determine _IC50 values, binding curves were analyzed using nonlinear regression (sigmoidal dose-response curve with variable slope, highest values constrained to be common across all data sets) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, California, USA).

In fig. 5 shows representative examples of inhibition curves of HGF binding by anti-c-Met antibodies binding the extracellular domain of recombinant human c-Met. 5D5 was used as a positive control antibody. All anti-c-Met antibodies in the experiment demonstrated their ability to compete with Europium ³⁺ -conjugated HGF for binding to recombinant c-Met. IC ₅₀ values vary in the range of 0.0011-0.0794 μg/ml. Without the addition of HGF conjugated to Europium ³⁺ , approximately 600 relative fluorescent units (RFU) were detected, indicating the signal level when maximum inhibition was achieved. When Europium ³⁺ -conjugated HGF binding was not inhibited, approximately ∼66,000 RFU were detected. Antibodies 005, 006, 058, 101 and the positive control antibody 5D5 were able to inhibit HGF binding to the c-Met receptor by 84.5-92.1%. All other antibodies were able to inhibit the binding of HGF to c-Met by at least 55%. Because HGF can bind the c-Met receptor at both the SEMA and Ig domains, some antibodies may inhibit only one of these interactions. To determine which interaction was inhibited, a cMetSEMAHis-based time-resolved fluorescence resonance energy transfer (TR-FRET) inhibition assay was performed.

Example 17: Competition of anti-c-Met antibodies for binding to soluble cMetECDHis measured by sandwich ELISA.

First, the optimal coverage concentrations of the tested anti-c-Met antibodies and the optimal cMetECDHis concentration were determined. Next, ELISA wells were coated overnight at 4°C with HuMab against c-Met, serially diluted in PBS (8 μg/ml in 2-fold dilutions). ELISA wells were then washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and blocked for 1 h at room temperature (RT) with PBSTC (PBST supplemented with 2% (v/v) vol.) chicken whey [Gibco, Paisley, Scotland]). Thereafter, ELISA wells were washed with PBST and incubated for one hour at room temperature with biotinylated cMetECDHis serially diluted in PBSTC (1 μl/ml in 2-fold dilutions). Unbound biotinylated cMetECDHis was washed with PBST, and bound biotinylated cMetECDHis was incubated for 1 h at RT with 0.1 μg/ml Streptavidin-poly-HRP (Sanquin, Amsterdam, the Netherlands) diluted in PBST.

- 42 045516

After washing, the reaction was visualized after a 15-minute incubation with 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic acid (ABTS: dilute one ABTS tablet in 50 ml ABTS buffer [Roche Diagnostics, Almere, the Netherlands]) at RT with protection from light. Staining was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured using a microtiter plate reader (Biotek Instruments, Winooski, USA). Conditions that resulted in suboptimal (approx. 80%) binding of each antibody were determined and used for subsequent cross-blocking experiments.

ELISA wells were coated with anti-c-Met antibody at a suboptimal dose as described above. After blocking, ELISA wells were incubated with a given concentration of biotinylated cMetECDHis in the presence of excess anti-c-Met antibody. The reaction proceeded as described above. Residual binding was expressed as a percentage relative to the binding observed in the absence of the competitor antibody.

Table 6. When added as a competitor, all anti-c-Met antibodies were able to compete for binding with their immobilized equivalents. 022, 058 and 5D5, when added as competitor antibodies, competed with antibodies 005 and 006. However, the reverse reaction revealed only partial competition between antibodies 005 and 006. These differences may be explained by the lower affinities of antibodies 005 and 006 for biotinylated cMetECDHis. Antibody 5D5, when added as a competitor antibody, also showed partial competition with antibodies 008 and 045, while no or minimal competition was observed in the reverse reaction. Additionally, antibodies 024, 062, 064, 068 and 181, when added as competitor antibodies, showed partial competition with antibody 101, whereas the reverse reaction showed complete inhibition of cMetECDHis binding. Values higher than 100% can be explained by avidity effects and the formation of antibody-cMetECDHis complexes containing two non-competing antibodies.

Antibodies 024, 062, 064, 068, 069, 098, 101 and 181 compete with each other for binding to cMetECDHis. Antibodies 005, 006, 022 and 058 were considered to belong to the same cross-blocking group, which is characterized by complete competition with 005, 006, 022, 058 and 5D5. However, antibody 5D5 was the only antibody that was also able to compete for binding with antibody 045. Another group of antibodies that compete for binding with cMetECDHis is formed from 008, 035, and G11-HZ.

Table 6. Competition of anti-c-Met antibodies for binding to biotinylated cMetECDHis

Competing antibody Immobilized 005 006 008 022 024 035 045 058 antibody 005 7.7 18.2 81.9 4.9 113.5 84.9 116.9 3.6 ±1.1 ±3.6 ±3.1 ±1.3 ±5.0 ±0.2 ±7.0 ±0.1 006 11.3 14.6 58.8 4.6 113.3 67.5 114.5 3.6 ±0.9 ±0.7 ±2.2 ±0.3 ±1.0 ±4.2 ±3.5 ±0.3 008 63.9 47.3 5.4 82.1 103.2 32.9 100.4 40.8 ±3.1 ±1.2 ±0.3 ±3.0 ±0.4 ±1.0 ±3.8 ±0.8 022 37.9 60.5 94.1 3.8 99.4 92.4 95.7 5.8 ±3.9 ±4.0 ±3.5 ±1.2 ±4.8 ±0.4 ±3.5 ±0.0 024 98.4 101.4* 104.2* 100.2* 5.4 108.1* 98.1* 102.8* ±10.4 ±16.7 ±12.7 ±9.0 ±0.5 ±5.8 ±11.9 ±12.8 035 36.7 33.0 7.2 54.6 121.4 10.6 125.0 18.5 ±1.0 ±17.6 ±1.7 ±6.5 ±27.8 ±0.3 ±16.8 ±2.5 045 111.4 110.6 98.5 105.3 102.4 105.4 21.3 115.3* ±1.5 ±3.5 ±3.1 ±2.5 ±5.6 ±5.5 ±0.1 ±6.5 058 31.4 43.6 90.2 6.8 109.0 90.1 111.7 4.0 ±3.6 ±2.1 ±2.5 ±0.3 ±4.1 ±5.4 ±4.9 ±0.2 062 95.8 95.2 97.4 94.6 7.3 90.6 97.0 94.4 ±5.1 ±6.8 ±5.3 ±4.0 ±2.9 ±11.5 ±3.0 ±4.3 064 90.4 90.1* 94.6* 94.2 7.5 83.5 95.0 95.5 ±1.9 ±1.4 ±0.5 ±3.6 ±2.5 ±12.2 ±4.9 ±0.6 068 101.1 98.5 101.7 99.6 4.7 88.6 100.4 101.5 ±7.6 ±6.7 ±5.5 ±4.0 ±2.3 ±12.7 ±9.0 ±5.1 069 102.3 100.3 102.1 97.8 6.6 91.7 99.8 100.6 ±11.2 ±12.3 ±12.8 ±12.5 ±4.1 ±27.3 ±14.4 ±14.1 098 99.6 97.9 99.8 95.8 12.9 89.4 96.7 98.6 ±6.3 ±6.7 ±4.2 ±5.4 ±4.2 ±20.6 ±3.7 ±2.9 101 91.5 89.7 94.0 90.7 40.5 96.7 94.7 93.1 ±7.2 ±7.9 ±6.3 ±5.3 ±5.4 ±1.9 ±5.1 ±5.2 181 95.9 93.7 98.7 92.5 4.3 96.0 96.8 98.9 ±7.8 ±8.4 ±5.8 ±7.4 ±1.9 ±9.6 ±6.7 ±9.8 5D5 42.3 58.8 90.2 12.4 94.2 98.1 83.9 6.6 ±14.7 ±19.4 ±9.9 ±4.7 ±9.7 ±13.4 ±3.2 G11-HZ 50.5 47.7 33.3 54.3 98.8 32.8 72.0 27.6 ±7.6 ±2.9 ±0.2 ±3.7 ±5.6 ±4.0 ±9.9 ±4.3

Competition 75 -> 100%

Competition 25-74% competition 0-24%

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Immobilized antibody 062 064 K 068 onkurg 069 ruyushch 098 ;e anti 101 body 181 5D5 G11-HZ 005 117.7 ±10.7 118.2 ±7.8 128.7 ±9.5 124.0 ±8.0 110.4 ±7.6 103.2 ±5.0 131.0 ±7.7 2.9 ±0.1 76.8 ±4.4 006 118.8 ±8.4 122.2 ±5.3 128.6 ±6.5 124.5 ±1.0 110.6 ±2.3 105.9 ±4.1 123.5 ±6.1 3.1 ±0.0 54.0 ±35.1 008 100.5 ±2.5 107.1 ±6.2 112.2 ±5.1 104.1 ±4.4 106.6 ±2.6 101.0 ±2.5 111.3 ±1.3 32.4 ±0.8 2.7 ±0.2 022 99.4 ±2.0 101.9 ±3.2 104.1 ±3.3 99.6 ±6.0 104.8 ±4.0 103.6 ±5.1 107.1 ±5.2 4.2 ±2.1 85.9 ±8.3 024 2.3 ±0.6 2.3 ±0.6 12.0 ±5.5 2.9 ±0.5 10.4 ±4.2 4.8 ±1.0 7.1 ±2.8 95.5* ±1.1 98.2* ±1.3 035 119.6 ±11.2 131.7 ±20.0 175.1 ±30.2 150.9 ±24.9 126.2 ±19.9 113.0 ±4.6 159.1 ±12.9 25.5 ±9.9 7.8 ±3.2 045 103.1 ±3.5 103.7 ±5.7 113.1 ±1.4 97.0 ±5.2 76.4 ±11.7 101.5 ±5.1 99.4 ±3.8 27.8 ±3.9 99.3 ±5.3 058 109.1 ±4.6 108.8 ±4.4 118.8 ±4.2 112.6 ±4.0 111.8 ±6.2 104.4 ±0.8 121.3 ±3.1 2.8 ±0.4 81.5 ±8.6 062 2.4 ±0.5 2.2 ±0.2 14.2 ±1.8 2.9 ±0.1 13.2 ±0.9 7.8 ±1.1 9.4 ±1.6 97.7 ±8.5 101.3 ±0.9 064 2.2 ±0.6 2.0 ±0.2 13.0 ±0.9 2.7 ±0.2 14.7 ±1.2 7.6 ±0.8 10.1 ±3.0 94.9* ±4.6 102.0 ±10.5 068 069 098 101 2.0 ±0.3 2.2 ±0.4 8.8 ±0.6 36.9 ±3.3 2.0 ±0.3 2.3 ±0.5 9.3 ±1.3 37.4 ±3.7 6.6 ±0.7 10.1 ±2.6 18.0 ±2.5 45.9 ±4.3 2.4 ±0.4 2.4 ±0.7 3.4 ±0.6 9.5 ±1.2 8.2 ±1.3 12.5 ±3.1 2.6 ±0.4 9.7 ±1.5 4.8 ±0.7 3.9 ±0.5 4.0 ±0.6 3.7 ±2.4 5.2 ±0.6 6.3 ±1.0 12.0 ±2.1 41.9 ±0.8 94.8 ±2.7 99.4 ±16.2 94.9 ±1.2 97.2 ±4.6 110.3 ±6.6 110.4 ±13.2 99.6 ±1.2 98.3 ±2.1 181 2.0 ±0.2 2.1 ±0.3 6.5 ±1.1 2.2 ±0.3 5.1 ±1.1 2.4 ±0.2 3.6 ±0.2 94.2 ±4.5 98.7 ±6.7 5D5 97.6 ±8.1 97.1 ±12.7 97.8 ±6.6 99.6 ±3.9 97.6 ±4.9 97.9 ±10.6 103.4 ±4.3 4.1 ±1.5 97.3 G11-HZ 95.3 ±3.1 99.2 ±0.6 102.6 ±1.3 95.0 ±8.4 96.2 ±11.8 90.1 ±6.8 101.1 ±5.2 29.1 ±9.2 2.6 ±0.4

Data shown are percent binding inhibition ± st. off 3 independent experiments. For antibodies 035, 5D5 and G11-HZ, cross-blocking ELISA was performed only twice. In addition, several competitive reactions (*) gave Optimal Density values higher than 5.0, which are above the detection limit of the ELISA reader. These results were excluded from the analysis due to repeated measures.

Example 18 Blocking HGF Binding to cMetSEMA-567His8 Determined by Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay

HGF can bind the c-Met receptor in both the SEMA and IgG domains. However, only the binding of HGF to the SEMA domain appears to be critical for receptor activation. For this reason, the interaction of anti-c-Met antibodies with the SEMA domain of the c-Met receptor was investigated using TR-FRET technology. To perform this homogeneous proximity assay, hepatocyte growth factor (HGF, ProSpec Tarry, Rehovot, Israel) was conjugated to the fluorescent dye acceptor AlexaFluor-647 (Invitrogen, Breda, the Netherlands). cMetSEMA-567His8 was labeled with a fluorescent donor molecule directed against the histidine tag (Anti-6xhis Europium, PerkinElmer, Turku, Finland). Binding of AlexaFluor-647-conjugated HGF to Europium ³⁺ -labeled cMetSEMA-567His8 allows energy transfer from the donor molecule (excitation 340 nm) to the acceptor molecule (emission 665 nm). The average fluorescence intensity at 665 nm was measured using an EnVision 2101 Multilabel reader (PerkinElmer). Competition of unlabeled anti-c-Met antibodies with HGF conjugated to AlexaFluor-647 was measured by the decrease in TR-FRET signal at 665 nm, due to the fact that in the unbound state the distance between the donor and acceptor fluorophores is too great for energy transfer to occur.

All dilutions were made in 0.5x LANCE Detection Buffer (PerkinElmer) supplemented with 2.67% Stabilizing Solution (PerkinElmer) and 0.03% (v/v) Tween-20 (Riedel de Haen, Selze, Germany). 25 μl of cMetSEMA-567His8 was added to 25 μl of AlexaFluor-647-conjugated HGF, 35 μl of Anti-6xhis Europium ³⁺ , and 25 μl of unlabeled anti-c-Met antibody in an opti-white 96-well plate (PerkinElmer). A final concentration of 2.93 μl/ml cMetSEMA567His8, 0.96 μl/ml HGF conjugated to AlexaFluor-647 and 0.4 μl/ml Anti-6xhis Europium ³⁺ was obtained. A 4-fold serial dilution of unlabeled anti-c-Met antibody was tested in the range of 0.49-8000 ng/ml. After incubation at 4°C in the dark overnight. The average fluorescence intensity at 665 nm was measured using an EnVision 2101 Multilabel reader (PerkinElmer) with the following set parameters: Lance/Delfia pair mirror, 615-665 nm emission filter, WHO filter

- 44 045516 320 nm wake-up, 60 µs latency, 100 µs window, 100 flashes, 2000 µs per cycle and bidirectional line readout. To determine _IC50 values, binding curves were analyzed using nonlinear regression (sigmoidal dose response curve with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA).

In fig. 6 shows the binding inhibition curves of various Met-binding antibodies upon binding to cMetSEMA_567His8 tested by TR-FRET. With the exception of antibodies 008, 035, and 096, all antibodies were able to compete with AlexaFluor647-conjugated HGF for binding to cMetSEMA-567His8. Antibody 022 was able to inhibit ~80% of HGF binding, while antibodies 005, 006, 024, 045, 058, 061, 062, 064, 068, 069, 101, 181 and the positive control antibody 5D5 were able to inhibit >90% of HGF binding with cMetSEMA-567His8. IC ₅₀ values were determined to be in the range of 0.082-0.624 μg/ml.

Table 7. _IC50 values (mg/ml) and percent inhibition of cMetSEMA-567His8 ligand binding by anti-c-Met antibodies determined by TR-FRET

mat IC50 % inhibition 005 0.16 92 006 0.16 92 008 But. 4 022 0.37 77 024 0.39 95 035 But. 19 045 0.17 92 058 0.15 99 061 0.49 96 062 0.58 97 064 0.07 97 068 0.26 96 069 0.54 97 096 But. 16 098 0.55 98 101 0.53 96 181 0.34 93 5D5 0.2 95

Data shown are the average MFI of three independent experiments.

Example 19 KP4 Viability Assay

Antibodies against c-Met were tested for their ability to inhibit KP4 cell viability (Riken BioResource Center Cell Bank, RCB1005). KP4 cells, which express high levels of both c-Met and HGF in an autocrine manner, were seeded in a 96-well culture plate (Greiner bio-one, Frickenhausen, Germany) (10,000 cells/well) in serum-free medium (1 part HAM's F12K [ Cambrex, East Rutherford, NJ] and 1 part DMEM [Cambrex]). A 66.7 nM dilution of c-Met antibody was prepared in serum-free medium and added to the cells. After 3 days of incubation, the number of viable cells was counted using Alamarblue (BioSource International, San Francisco, USA) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard parameters for Alamarblue. The Alamarblue signal of antibody-treated cells is plotted as a percentage of signal compared to untreated cells.

In fig. 7 depicts the percentage inhibition of viable KP4 cells after treatment with c-Met binding antibody compared to untreated cells (0%). Boxed clones are antibodies that cross-competite with each other as described in Example 17. Interestingly, antibodies 024, 062, 064, 068, 069, 098, 101 and 181, which belong to the same cross-blocking group, were all able inhibit the viability of KP4 (18-46%), both as IgG1 and UniBody molecules. Also, IgG1 antibody molecules 008, 061 and 096 were able to inhibit KP4 viability. In contrast, antibody 045 does not inhibit the viability of KP4 either as IgG1 or as the UniBody molecule. For Uni-1016-045-TE, this is possibly due to its low apparent affinity for membrane-bound c-Met as measured by FACS analysis (Example 13). IgG1 antibodies clones 005, 006, 022 and 058 did not significantly inhibit KP4 viability, whereas Uni-1016-022-TE, Uni-1016-058-TE and IgG1-1016-058-wtFab inhibited KP4 viability at 57, 38 and 44% respectively. Uni-1016-005 and Uni-1016-006 also cross-competed with clones 022 and 058 but did not significantly inhibit KP4 viability. This is possibly due to their low apparent affinities as measured by FACS analysis (Example 13). Interestingly, IgG4-1016-058 also showed some inhibition of KP4 viability. This was not observed with IgG4-5D5).

Overall, the data indicate that in some cross-blocking groups, monovalent binding is required to inhibit KP4 viability, whereas in others cross-linking

- 45 045516 blocking groups KP4 viability can be inhibited by both monovalently and bivalently binding antibodies.

Example 20 KP4 Xenograft Tumor Model in SCID Mice

A xenograft tumor model in SCID mice was used to determine the effectiveness of HuMab against c-Met in inhibiting tumor growth in vivo. Seven to eleven week old female SCID mice, strain C.B-17/IcrPrkdc-scid/CRL, were purchased from Charles River Laboratories Nederland (Maastricht, The Netherlands) and maintained under sterile conditions in overhead filter cages with food and water offered ad libitum . Microchips (PLEXX BV, Elst, The Netherlands) were used to identify mice. All experiments were approved by the animal ethics committee of the University of Utrecht.

On day 0, 10x106 KP4 cells were inoculated subcutaneously in 200 μl PBS in the right flank. Mice were monitored at least twice a week for clinical signs of disease. Tumor size was determined at least once a week. Volumes (mm3) were calculated after measurements with a caliper (PLEXX) as 0.52 x (length) x (width)2 from day 16 onwards. On day 9, the average tumor sizes were measured and the mice were divided into 8 groups of 7 mice each. Antibodies against c-Met (008, 058, 069, and 098) were injected i.p. Antibody G11-HZ was used as a positive control antibody, while 5D5 and isotype control antibodies were used as negative control antibodies. Mice received a loading dose of 400 μg/mouse followed by a weekly maintenance dose of 200 μg/mouse for 7 weeks.

Additionally, plasma samples were collected before maintenance doses 1, 3 and 5, and then mice were sacrificed and the presence of human IgG was determined using latex beads on a BNII nephelometer (Dade Behring, Atterbury, UK).

In fig. 8 and 9 show that tumor growth of KP4 cells was inhibited by HuMab 008, 069, 098 and the positive control G11-HZ. Inhibition was compared with isotype control antibody treatment. Tumor growth of KP4 cells was slowed but not completely inhibited by the control antibody G11-HZ. Clones 069 and 098 showed more potent inhibition compared to clones 008 and G11-HZ. Antibodies 5D5 and 058 did not inhibit tumor growth. This was consistent with the in vitro data described in Example 19. Taken together, these data indicate that in certain cross-blocking groups, bivalent binding antibodies can inhibit KP4 tumor growth.

Example 21 Tumor model based on MKN45 xenograft

The MKN45 human gastric adenocarcinoma xenograft tumor model in nude mice was used to determine the effectiveness of HuMab against c-Met in inhibiting tumor growth in vivo.

Human gastric adenocarcinoma cells MKN45 were cultured at 37°C and 5% CO ₂ in RPMI-1640 medium containing 100 units/ml sodium penicillin G, 100 μg/ml streptomycin sulfate, 25 μg/ml gentamicin, 20% fetal bovine serum and 2 mM glutamine. Seven to eight week old female nude mice (nu/nu, Harlan) (body weight ranging from 17.0 to 26.4 g at baseline) were used. Animals received water and food ad libitum. Mice were housed under conditions that met the recommendations of the Guide for the Care and Use of Laboratory Animals. The animal care and utilization program has been accredited by AAALAC. On day 0, 1x10e ⁷ MKN45 cells were inoculated subcutaneously in 200 μl of 50% Matrigel in PBS into the flank of each mouse. On day 7, animals were sorted into five groups (n=10) with an average tumor volume of 80 to 120 mm3 and treatment began. Antibodies against c-Met (008, 058, 069) were injected into the tail vein (i.v.). The G11-HZ antibody was used as a positive control antibody, and the isotype control antibody was used as a negative control antibody. All mice received 40 mg/kg antibody on day 7 and 20 mg/kg antibody on days 14, 21, and 28.

Tumors were measured twice weekly using calipers until a final tumor volume of 700 mm ³ or until the end of the study (day 62). In fig. 10 and 11 show that tumor growth of MKN45 cells was significantly slowed by antibodies 008, 058, 069, and control antibody G11-HZ compared to treatment with isotype control antibody.

Example 22 Reduction of residual agonist activity of anti-c-Met IgG1 antibodies by reducing conformational flexibility

The natural ligand of c-Met, HGF, is a functional dimer that induces dimerization of two c-Met molecules. Subsequent intracellular phosphorylation of the intracellular domain of c-Met leads to the activation of several signaling pathways that are involved in cell proliferation, invasion, and survival. Most bivalent antibodies raised against c-Met exhibit effects comparable to HGF on cell guidance, especially when the antibody-binding epitopes in c-Met are located near or in the SEMA domain.

To minimize potential residual agonist activity of bivalent IgG1 antibodies, a strategy to reduce conformational flexibility was used. In IgG1, Fab arms have

- 46 045516 greater degree of freedom for movement relative to the Fc domain. The largest conformational changes result from the flexibility of the hinge, which allows a wide range of Fab-Fc angles (Ollmann Saphire, E., R.L. Stanfield, M.D.M. Crispin, P.W.H.I. Parren, P.M. Rudd, R.A. Dwek, D.R. Burton and L.A. Wilson. 2002. Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 319: 9-18). One way to reduce the flexibility of the Fab arm in immunoglobulins is to prevent the formation of disulfide bonds between the light and heavy chains through genetic modification. In the natural IgG1 antibody, the light chain is linked covalently to the heavy chain through a disulfide bond connecting the C-terminal cysteine of the light chain to the cysteine at position 220 (C220 in EU numbering) in the heavy chain Fc hinge. Either by mutating amino acid C220 to serine or any other naturally occurring amino acid, or by removing the hinge entirely, or by replacing the IgG1 hinge with an IgG3 hinge, a molecule is produced in which the light chains are connected through their C-terminal cysteines, similar to the situation found in the human isotype IgA2m(1). This results in reduced flexibility of the Fab relative to the Fc and consequently reduced cross-linking ability, as shown in comparative studies of the c-Met agonist antibody (5D5) in IgA2m(1) format and IgG1 in the KP4 viability assay (Fig. 12).

Another strategy to reduce the flexibility of the IgG1 molecule is to replace the IgG1 hinge with an IgG2 hinge or an IgG2-like hinge. (Dangl et al. EMBO J. 1988; 7:1989-94). This hinge region has two properties that are different from those of the IgG1 hinge, which are thought to make the molecule less flexible. First, compared to the IgG1 hinge, the IgG2 hinge is 3 amino acids shorter. Second*, the IgG2 hinge contains an additional cysteine, thus forming three, instead of two, disulfide bridges within the heavy chain. Alternatively, a variant IgG1 hinge may be introduced that is similar to the IgG2 hinge. This mutant (TH7A6-9) (WO2010063746) contains the T223C mutation and two deletions (K222 and T225) to form a shorter hinge with an additional cysteine.

Example 23: Preparation of IgG1 Molecules with Reduced Flexibility (Rigid IgG1 Molecules)

Cloning and expression

Mutant IgG1 antibodies were developed and cloned using standard molecular biology techniques. An overview of the sequences of all hinge region mutations obtained is presented in Table 1. 8 below.

Table 8. Amino acid sequence of the hinge of mutant IgG1 antibodies. Deletions are marked with '-' and mutations are underlined.

IgGl Wild Type

IgGl ma _P HHp-IgG2

IgGl AC220

IgGl C220S

IgGl ΤΗ7Δ6-9

IgGl with hinge removed (Uni-IgGl)

IgGl IIIa _P HHp-IgG3

EPKSCDKTHTCPPCP ERKCCVE—СРРСС EPKS-DKTHTCPPCP EPKSSDKTHTCPPCP EPKSCD-CH-CPPCP

ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCP EPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

To express the resulting IgG1 rigid antibodies in mammalian cells, the HC constant region from IgG1 containing mutations in the hinge region (see Table 8 above) was synthesized as a codon optimized construct in the mammalian expression vector pcDNA3.3 (Invitrogen). A separate vector was constructed by inserting a codon optimized constant region into the human kappa light chain region of pcDNA3.3. The VH and VL regions of clone 069 and the control antibody 5D5 were inserted into a HC constant region plasmid and a kappa light chain plasmid to provide vectors for the expression of the (mutated) heavy and light chains of specific antibodies. Cotransfection of specific antibody heavy and light chain vectors into HEK-293F cells (Invitrogen) resulted in transient production of mutant antibodies. Antibody purification was carried out using Protein A affinity column chromatography (as described in Example 11).

Determination of biochemical characteristics

Temporal Expression

All mutants were expressed at sufficient levels and did not exhibit aberrant multimer formation as determined by MS (>99% purity) and SDS-PAGE.

The results of SDS-PAGE are presented in Fig. 13. In C220 mutants (C220S and DS220) and hinge-deleted IgG1 variants (hinge-deleted IgG1 variants are also called UniBody-IgG1 or Uni-IgG1), light chain pairing was observed, visible as a protein band of about 50 kDa when analyzed in non-reducing SDS -PAAG. The IgG3 hinge variant also showed light chain pairing, whereas the IgG2 hinge variant and the IgG1 mutant TH7A6-9 showed normal light and heavy chain pairing.

Example 24 Binding properties of c-Met mutants

The c-Met binding properties of the mutants were tested by ELISA. ELISA plate wells were coated overnight at 4°C with rhHGF R/Fc Chimera (R&D Systems; Cat.358MT/CF) in PBS (1 μg/ml). The ELISA wells were then washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht,

- 47 045516

Netherlands]) and blocked for one hour at room temperature (RT) with PBSTC (PBST supplemented with 2% (v/v) chicken serum [Gibco, Paisley, Scotland]). Thereafter, wells were washed with PBST and incubated for 1 h at room temperature with antibodies against c-Met and variants serially diluted in PBSTC (10 μl/ml in 4-fold dilutions). Unbound antibody was washed with PBST, and coating bound antibody was detected by incubation for one hour at RT with goat F(ab')2-HRP anti-human IgG diluted in PBST (Jackson cat. no. 109035-097). After washing, the reaction was visualized by 15 min incubation with 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic acid (ABTS: dilute one ABTS tablet in 50 ml ABTS buffer [Roche Diagnostics, Almere, the Netherlands]) at RT with protection from light. Staining was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured using a microtiter plate reader (Biotek Instruments, Winooski, USA). All mutants bound to c-Met with comparable observed affinity (EC50) (Fig. 14) Table 10 shows the EC50 values of the mutants obtained in this experiment.

Table 9. EC ₅₀ values determined using ELISA

IgGl1016069 IgGl1016069DS220 IgGl1016069- C220S IgGl1016069 Sharni P IgG2 IgG21016069 IgGl1016069 ΤΗ7Δ69 Uni1016069TE IgGl1016069 IgGl1016069 Sharni p!gG3 UniIgGl1016069 EC50 (ng/ml) 49.5 18.87 15.56 23.03 29.61 18.81 30.08 45.43 14.18 15.39

Example 25 Reduced agonist effect of rigid IgG1 antibodies against c-Met

Phosphorylation of receptors

To determine the agonistic properties of the protected antibodies, the effect of the antibodies on c-Met phosphorylation was examined. Following dimerization of two adjacent c-Met receptors by either the natural HGF ligand or most bivalent antibodies, three tyrosine residues (positions 1230, 1234, and 1235) in the c-Met intracellular domain become cross-phosphorylated, followed by further phosphorylation of several other amino acids in the intracellular domain and activation of several signaling cascades. Dimerization and activation of c-Met can therefore be monitored using antibodies specific to the receptor phosphorylated at these positions and thus can be used as data on the potential agonism of anti-c-Met antibodies.

A549, CCL-185 cells obtained from ATCC were grown in serum containing DMEM until 70% confluence was achieved. After trypsinization and washing, the cells were seeded in a 6-well culture plate at 1*10e6 cells/well in a culture medium containing serum. After overnight incubation, cells were treated with either HGF (R&D systems; cat. 294-HG) (50 ng/ml) or a panel of antibodies (30 μg/ml) and incubated for 15 min at 37°C. Cells were then washed twice with ice-cold PBS and lysed with lysis buffer (Cell Signaling; cat. 9803) supplemented with protease inhibitor cocktail (Roche; cat. 11836170001), and samples were stored at -80°C. Receptor activation was determined by measuring phosphorylation by Western blotting using phospho-c-Met-specific antibodies. Proteins in the cell lysate were separated on a 4-12% SDS-PAGE gel and transferred to a nitrocellulose membrane, which was then stained with an antibody specific for phosphorylated c-Met (Y1234/1235) (Cell Signaling, cat: 3129). Antibodies against total c-Met and beta-actin were used to control gel loading. The results of Western blotting are presented in Fig. 15.

Tissue culture media controls and cells treated with monovalent UniBody antibody 5D5 format did not demonstrate receptor phosphorylation. In contrast, Western blot analysis of cells treated with the positive control HGF or the agonist antibody IgG1-1016-058 demonstrated a clear band of the expected mass. Antibody IgG1-1016-069 showed low but detectable phosphorylation of the receptor, indicating that some cross-linking of the receptor occurs. However, variants that were designed to reduce the flexibility of the antibody molecule showed minimal receptor activation, to levels comparable to those detected in cells treated with the monovalent control Uni-5D5-TE (Fig. 15).

Effect of c-Met antibodies on NCI-H441 proliferation in vitro

The potential proliferative agonist activity of anti-c-Met antibodies was tested using the lung adenocarcinoma cell line NCI-H441 (ATCC, NTV-174™), which expresses high levels of c-Met but does not produce its ligand HGF. NCI-H441 cells were seeded into 96-well tissue culture plates (Greiner bio-one, Frickenhausen, Germany) (5000 cells/well) in RPMI (Lonza) without serum. Dilutions of anti-c-Met antibody (66.7 nM) were prepared in serum-free RPMI and added to the cells. After 7 days of incubation at 37°C/5% CO ₂ , the number of viable cells was counted using Alamarblue (BioSource International, San Francisco, USA) according to the manufacturer's instructions. Fluorescence was monitored using an EnVision 2101 Multilabel reader (PerkinElmer, Turku, Finland) with standard parameters for Alamarblue.

As can be seen from Fig. 17 proliferation of NCI-H441 cells is strongly induced by agonistic con

- 48 045516 troll mAbs IgG1-058 and IgG1-5D5. The IgG1-1016-069 antibody also demonstrated an agonistic effect compared to cells treated with isotype control. The agonistic activity of IgG1-1016-069 can be completely eliminated by introducing mutations in C220, C220S and -del, and partially by variants with the IgG2 hinge and TH7A6-9 or with the IgG2 frame. Control samples treated with isotype control and the monovalent version of 5D5 (Uni-5D5-TE) did not induce cell growth.

KP4 Viability Assay

The ability to inhibit HGF-dependent cells was also determined for anti-c-Met antibody mutants in the KP4 viability assay (see Example 19 for experimental procedures). The results are presented in Fig. 17. The effectiveness of IgG1-1016-069 mutants was completely preserved or was slightly better in C220 mutants. Notably, introducing a mutation at C220 into the agonistic antibody 5D5 resulted in a significant reduction in KP4 viability. The lack of agonistic effect of antibodies 058 and 5D5 in IgG1 format was observed due to the high expression of HGF by KP4 cells (HGF autocrine loop).

Negative modulation

Negative modulation induced by antagonistic antibodies is the mechanism of action of therapeutic antibodies against c-Met. Accordingly, in one embodiment, antibodies are sought that have reduced agonistic properties but retain the ability to induce negative modulation of c-Met. To determine antibody negative potential, A549 cells (CCL-185, obtained from ATCC) were seeded into 6-well tissue culture plates (500,000 cells/well) in cell culture medium with serum and cultured overnight at 37°C. The next morning, anti-c-Met antibody was added at a final concentration of 10 μg/ml and the plate was incubated for the next 2 days at 37°C. After washing with PBS, cells were lysed by incubation for 30 min at room temperature with 250 μl of lysis buffer (Cell signaling, Danvers, USA). Total protein was calculated using bicinchoninic acid (BCA) protein reagent (Pierce) following the manufacturer's protocol. c-Met protein levels in cell lysates were quantified using a c-Met-specific sandwich ELISA. To this end, ELISA plate wells were coated overnight at 4°C with goat anti-human c-Met antibody directed against the extracellular domain of c-Met (R&D systems) diluted in PBS (1 μg/ml). ELISA wells were then washed with PBST (PBS supplemented with 0.05% Tween-20 [Sigma-Aldrich, Zwijndrecht, The Netherlands]) and blocked for 1 h at room temperature (RT) with PBSTC (PBST supplemented with 2% (v/v) vol.) chicken whey [Gibco, Paisley, Scotland]). Undiluted cell lysates (100 μl) were added and incubated for one hour at RT. After washing with PBST, the wells were incubated for one hour at room temperature with a mouse antibody against the intracellular tyrosine residue -1234 of human c-Met (Cell signaling) diluted 1:1000 in PBSC. Wells were again washed with PBST and incubated for 1 h at RT with goat anti-mouse Fc-HRP antibody (Jackson) diluted 1:5000 in PBSC. After washing with PBST, the reaction was visualized after 30 min incubation with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS: dilute one ABTS tablet in 50 ml ABTS buffer [Roche Diagnostics, Almere, The Netherlands]) at CT with protection from light. Staining was stopped by adding an equal volume of oxalic acid (Sigma-Aldrich, Zwijndrecht, The Netherlands). Fluorescence at 405 nm was measured using a microtiter plate reader (Biotek Instruments, Winooski, USA). As seen in Fig. 18 all antibody 069 mutants were able to induce negative modulation.

Example 26 Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

MKN45 cells (purchased from RIKEN BioResource Center, Tsukuba, Japan, RCB1001) were collected (5x106 cells), washed (twice in PBS, 1500 rpm, 5 min) and collected in 1 ml RPMI 1640 medium supplemented with 10% CCS serum (cosmic calf serum) (HyClone, Logan, Utah, USA), to which 200 μCi of ⁵¹ Cr (Chrome-51; Amersham Biosciences Europe GmbH, Roosendaal, the Netherlands) was added. The mixture was incubated in a stirred water bath for 1.5 h at 37°C. After washing the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI 1640 medium supplemented with 10% CCS, counted for trypan blue incorporation, and diluted to a concentration of 1x105 cells/ml.

In this case, peripheral blood mononuclear cells were isolated from fresh buffy coats (Sanquin, Amsterdam, the Netherlands) using standard Ficoll density gradient centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France). After resuspension in RPMI 1640 medium supplemented with 10% CCS, cells were counted by trypan blue incorporation and concentrated to 1x107 cells/ml.

For each ADCC experiment, 50 μl of ⁵¹ Cr-labeled MKN45 cells (5000 cells) were preincubated with 15 μg/ml anti-c-Met antibody in a final volume of 100 μl of RPMI medium supplemented with 10% CCS in a 96-well microtiter plate. After 15 minutes at RT, 50 pL of PBMC (500,000 cells) was added, resulting in an effector to target ratio of 100:1. The maximum amount of cell lysis was determined by incubating 50 μl of ⁵¹ Cr-labeled MKN45 cells (5000 cells) with 100

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Claims (39)

µl 5% Triton-X100. The amount of spontaneous lysis was determined by incubating 5000 ⁵¹ Cr-labeled MKN45 cells in 150 μl of medium, without antibody or effector cells. The level of antibody-independent cell lysis was determined by incubating 5000 MKN45 cells with 500,000 PBMCs without antibody. Then the cells were incubated for 4 hours at 37°C, 5% CO2. Cells were centrifuged (1200 rpm, 3 min) and 75 μl of supernatant was transferred into micron tubes, after which ⁵¹ Cr was counted using a gamma counter. The measured counts per minute (cpm) were used to calculate the percentage of antibody-mediated lysis as follows: (cpm/min sample - cpm/min antibody-independent lysis)/(cpm/min max, lysis - cpm/min spontaneous lysis) x 100% Various publications have demonstrated a correlation between reduced core phycosylation and increased ADCC activity in vitro (Shields RL. 2002 JBC; 277:26733-26740, Shinkawa T. 2003 JBC; 278(5):3466-3473). In fig. 19 shows that antibody 069 does not induce lysis of MKN45 cells through ADCC. However, when core fucosylation was reduced by the presence of kifunensine during mAb production in HEK cells, antibody 069 was able to induce more than 30% lysis of MKN45 cells. Moreover, lysis has already been observed at antibody concentrations below 0.01 μg/ml. The values presented are the average maximum percentages of ⁵¹ Cr released ± std. off from one representative in vitro ADCC experiment with MKN45 cells. Low-fucose antibody 069 was produced in HEK 293 cells in the presence of kyfunesin, resulting in ~99.5% non-core fucosylation (i.e., no fucose). Antibody 069 with high levels of fucose was produced in HEK 293 cells lacking kyfunensine, resulting in ∼2.11% non-core fucosylation as determined by high performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) (data not shown). Example 27 Loss of Anti-c-Met Antibody Binding to Human Peripheral Blood Cells To determine the binding of clone 069 to three cell types (B cells, monocytes and granulocytes) present in peripheral blood, FACS binding analysis was performed. Fluorescently labeled clone 069 was used for direct FACS measurement without the use of secondary detection antibodies. Cell populations in the blood were identified in the assay using fluorescent commercial antibodies against specific markers on the cells of interest. Peripheral blood from healthy volunteers (Utrecht University Medical Center) was diluted tenfold in FACS buffer (PBS + 0.4% BSA + 0.02% NaN ₃ ) and incubated with anti-c-Met antibodies conjugated to Alexa ⁴⁸⁸ , and FITC-conjugated anti-CD19, CD16, and CD14 antibodies (final concentration 10 pg/ml) and phycoerethrin-labeled anti-CD19, CD16, and CD14 antibodies (BD Biosciences, San Jose, CA) to identify cell populations (resp. B- cells, granulocytes and monocytes) in a final volume of 100 µl. After 30 minutes at 4°C, the samples were centrifuged (300 g, 3 min), the supernatant was removed, and the red blood cells were lysed by incubation (10 min, 4°C) with 200 μl of Ery-lysis solution (155 mM NH ₄ CI, 10 mM KHCO3, 0.1 mM EDTA [pH 7.4]), and samples were washed twice in FACS buffer. Samples were resuspended in 100 μl of FACS buffer and analyzed using a FACS Canto II (BD Biosciences). Fig. 20 is a FACS plot showing that 069 conjugated to Alexa ⁴⁸⁸ did not bind the B cell population (CD19-PE+ cells within the lymphocyte window). Binding of Alexa ^488- conjugated rituximab was used as a positive control. Binding to other cell populations was analyzed in the same manner, and representative results for one of the 3 donors are also plotted in FIG. 21. Antibody 069-Alexa ⁴⁸⁸ does not bind B cells, monocytes or granulocytes, whereas positive control antibodies showed specific binding. CLAIM 1. A monoclonal antibody that binds human c-Met, wherein the antibody comprises a VH region comprising CDR1, 2 and 3 of SEQ ID NOs: 194, 195 and 196, and a VL region comprising CDR1, 2 and 3 of SEQ ID NOs: 194, 195 and 196 NO: 209, 210 and 104. 2. Antibody according to claim 1, containing: a) a VH region comprising CDR1, 2 and 3 with SEQ ID NOs: 98, 99 and 100, and a VL region comprising CDR1, 2 and 3 with SEQ ID NOs: 102, 103 and 104, or b) the VH region comprising CDR1, 2 and 3 with the sequences SEQ ID NOs: 130, 131 and 132, and the VL region containing CDR1, 2 and 3 with the sequences SEQ ID NOs: 134, 135 and 136. 3. Antibody according to claim 1 or 2, containing: a) a VH region containing the sequence SEQ ID NO: 97, and preferably a VL region containing the sequence SEQ ID NO: 101, b) a VH region containing the sequence SEQ ID NO: 129, and preferably a VL region, - 50 045516 containing the sequence SEQ ID NO: 133, or c) a variant of any of these antibodies, wherein said variant preferably has no more than 1, 2 or 3 conservative amino acid modifications. 4. A monoclonal antibody that binds human c-Met, wherein the antibody contains a VH region containing CDR1, 2 and 3 with the sequences of SEQ ID NOs: 189, 190 and 60, and a VL region containing CDR1, 2 and 3 with the sequences of SEQ ID NO: 62, 63 and 207. 5. The antibody of claim 4, comprising a VH region containing CDR1, 2 and 3 with the sequence SEQ ID NOs: 58, 59 and 60, and a VL region containing CDR1, 2 and 3 with the sequences SEQ ID NOs: 62, 63 and 64. 6. Antibody according to claim 4 or 5, containing: a) a VH region comprising the sequence SEQ ID NO: 57, and preferably a VL region comprising the sequence SEQ ID NO: 61, or b) a variant of any of these antibodies, wherein said variant preferably has no more than 1, 2 or 3 conservative amino acid modifications. 7. An antibody according to any one of claims 1 to 6 that binds the SEMA domain of c-Met, preferably the antibody is capable of inhibiting the binding of HGF to the SEMA domain with an IC50 of less than 10 μg/ml, for example less than 2 μg/ml. 8. An antibody according to any of the preceding claims that binds to A431 cells with an EC50 of 10 nM or less, for example an EC50 of 2 nM or less. 9. The antibody of claim 1, wherein the antibody is a bivalent antibody. 10. An antibody according to any of the preceding claims that binds to c-Met with an affinity constant (KD) of 20 nM or less, such as an affinity of 5 nM or less. 11. An antibody according to any of the preceding claims that binds to rhesus c-Met, preferably for which the antibody binding signal to rhesus c-Met is at least 5 times that of the negative control antibody. 12. An antibody as claimed in any one of the preceding claims, wherein the antibody inhibits binding of HGF to the extracellular domain of c-Met, preferably wherein the antibody inhibits binding by more than 40%, e.g., more than 50%, e.g., more than 60%, e.g., more than 70%, for example more than 80%, for example more than 90%. 13. An antibody according to any of the preceding claims, which is capable of inhibiting the viability of KP4 cells, preferably wherein the antibody is capable of inhibiting the viability by more than 10%, for example more than 25%, for example more than 40%. 14. An antibody according to any of the previous claims, wherein the antibody is a full-length antibody, preferably an IgG1 antibody, in particular an IgG1,K antibody. 15. An antibody according to any of the previous claims, which is an antibody with deficiency of effector function, for example, a stabilized human IgG4 antibody, such as an antibody in which the arginine at position 409 in the human IgG4 heavy chain constant region is replaced by lysine, threonine, methionine or leucine, preferably to lysine, and/or where the hinge region contains the sequence Cys-Pro-Pro-Cys. 16. An antibody according to any of the previous paragraphs, which is a monovalent antibody. 17. Antibody according to claim 16, where the monovalent antibody contains: (i) a variable region of an antibody according to any one of claims 1 to 6; and (ii) a C _H region of an immunoglobulin or a fragment thereof, including C _H 2 and C _H 3 regions, wherein the C _H region or fragment thereof is modified such that the region corresponding to hinge region, and, if the immunoglobulin is not of the IgG4 isotype, then other CH regions, such as the CH3 region, do not contain amino acid residues that are capable of forming disulfide bonds with the identical CH region or other covalent or stable non-covalent bonds between heavy chains with identical CH region in the presence of polyclonal human IgG. 18. The antibody according to claim 17, where the immunoglobulin specified in step (ii) belongs to the IgG4 subtype. 19. The antibody of claim 17 or 18, wherein the heavy chain has been modified such that the entire hinge has been removed. 20. The antibody of any one of claims 1 to 17, wherein the antibody has been modified to make it less flexible, for example by mutations in the hinge region. 21. The antibody according to claim 20, where the antibody is of the IgG1 subtype and the hinge region is modified by: (i) removing the hinge region of the sequence EPKSCDKTHTCPPCP and replacing it with an IgG2 hinge region of the sequence: ERKCCVECPPCP (IgG1-hinge-IgG2); (ii) removing position 220 so that the modified hinge region has the sequence EPKSDKTHTCPPCP (IgG1 AC220); (iii) replacing the cysteine at position 220 with any other naturally occurring amino acid (X) to modi - 51 045516 the fixed hinge region had the sequence EPKSXDKTHTCPPCP (IgG1 C220X); (iv) deleting the hinge region of the sequence EPKSCDKTHTCPPCP (UniBody IgG1); (v) removing the hinge region with the sequence EPKSCDKTHTCPPCP and replacing it with an IgG3 hinge region with the sequence ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP (IgG1-hinge-IgG3) or (vi) replacing the threonine at position 223 with a cysteine and removing the lysine at position 222 and the threonine at position 225 so the modified hinge region has the sequence EPKSCDCHCPPCP (IgG1 TH7A6-9). 22. The antibody of claim 21, wherein the hinge region is modified by replacing the cysteine at position 220 with a serine such that the modified hinge region has the sequence EPKSSDKTHTCPPCP (IgG1 C220S). 23. Antibody according to claim 20, where the antibody is of the IgG2 subtype. 24. The antibody of any one of the preceding claims, wherein the antibody has been modified to reduce core fucosylation to less than 10%, such as less than 5%, as determined by high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). 25. A bispecific antibody comprising a c-Met binding site as defined in the preceding paragraphs and a second antigen binding site having a different binding specificity. 26. The bispecific antibody of claim 25, comprising a VH region containing CDR1, 2 and 3 with the sequences SEQ ID NOs: 98, 99 and 100, and a VL region containing CDR1, 2 and 3 with the sequences SEQ ID NOs: 102, 103 and 104, and a second antigen binding site having a different binding specificity. 27. The bispecific antibody of claim 25 or 26, comprising a first antigen binding site of an anti-c-Met antibody, and a second antigen binding site specific for epidermal growth factor receptor, wherein the first antigen binding site comprises a VH region containing CDR1, 2 and 3 c the sequences SEQ ID NOs: 98, 99 and 100, and the VL region containing CDR1, 2 and 3 with the sequences SEQ ID NOs: 102, 103 and 104. 28. The bispecific antibody of claim 25 or 26, wherein the second antigen binding site has specificity for binding to a human effector cell, a human Fc receptor, a B cell receptor, or a non-overlapping c-Met epitope. 29. Nucleotide sequence encoding the VH and VL regions of the antibody according to any of the previous paragraphs. 30. An expression vector comprising the nucleotide sequence of claim 29, wherein the vector further comprises a nucleotide sequence encoding an operably linked light chain constant region and a heavy chain constant region of the antibody. 31. A recombinant eukaryotic or prokaryotic host cell containing the expression vector of claim 30 and which produces an antibody according to any one of claims 1 to 28. 32. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 28 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is used for treating a disorder associated with c-Met expressing cells. 33. Use of an antibody according to any one of claims 1 to 28 as a medicament for the treatment of a disorder associated with c-Met expressing cells. 34. Use according to claim 33, wherein the disease is cancer. 35. Use of an antibody according to any one of claims 1 to 28 for the treatment of cancer, where the cancer is selected from the group consisting of bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, gallbladder cancer, prostate cancer, thyroid cancer, osteosarcoma, rhabdomyosarcoma, synovial sarcoma, Kaposi's sarcoma, leiomyosarcoma, malignant fibrous histiocytoma, fibrosarcoma, acute myelogenous leukemia, adult T-cell leukemia, chronic myeloid leukemia, lymphoma, multiple myeloma, glioblastoma, astrocytoma, melanoma, mesothelioma, Wilms tumor and MiT tumors, including clear cell sarcoma (CCS), alveolar soft tissue sarcoma (AS PS ) and translocation-associated renal cell carcinoma. 36. The use of claim 35, wherein the antibody is for treating cancer in combination with one or more additional therapeutic agents, such as chemotherapeutic agents. 37. A method of inhibiting the growth and/or proliferation of tumor cells expressing c-Met, comprising administering to an individual in need thereof an antibody according to any one of claims 1 to 28. 38. A method for producing an antibody according to any one of claims 1 to 28, including the steps of: a) culturing the host cell according to claim 31 and b) purification of the antibody from the culture medium. 39. A method for detecting the presence of c-Met in a sample, comprising bringing the sample into contact with the antibody according to any one of claims 1-28 under conditions that ensure -