NZ789715A - Anti-met antibodies and uses thereof - Google Patents

Abstract

The invention relates to agonistic anti-MET antibodies and uses thereof in the therapeutic treatment of disease. The antibodies bind with high affinity to the human and mouse hepatocyte growth factor (HGF) receptor, also known as MET, and are agonists of MET in both humans and mice, producing molecular and cellular effects resembling the effects of HGF binding. lar and cellular effects resembling the effects of HGF binding.

Description

ANTI-MET ANTIBODIES AND USES THEREOF The entire content of the complete specification of New Zealand Patent Application No. 749578 as originally filed is incorporated herein by reference.

FIELD OF THE ION The present ion relates to dies and antigen binding fragments that bind with high affinity to the human and mouse hepatocyte growth factor (HGF) receptor, also known as MET. The antibodies and antigen binding fragments are agonists of MET in both humans and mice, ing in molecular and cellular effects resembling the effects of HGF binding. The invention r relates to therapeutic uses of antibodies and antigen g fragments that are agonists of MET.

BACKGROUND HGF is a pleiotropic cytokine of mesenchymal origin that mediates a characteristic array of biological functions including cell proliferation, motility, differentiation and al. The HGF receptor, also known as MET, is expressed by a variety of tissues including all epithelia, the endothelium, muscle cells, neuronal cells, osteoblasts, hematopoietic cells and various components of the immune system.

HGF and MET signalling plays an essential role during embryo development, where it guides ion of precursor cells and determines cell survival or death. In adults, HGF/MET signalling is ordinarily quiescent and is d during wound healing and tissue regeneration. Some cancers and tumours usurp HGF/MET signalling in order to promote the survival and proliferation of the tumour in the host organism. ore, inhibiting the HGF-MET axis has become a popular target for anti-cancer treatment, though with limited success.

Due to its role in tissue healing and regeneration, recombinant HGF has also been investigated as a treatment for a number of conditions, including degenerative diseases, inflammatory es, auto-immune diseases, metabolic diseases, and transplantationrelated disorders. r, recombinant HGF has poor pharmacological properties: it requires proteolytic activation in order to become biologically active; once activated, it has an extremely short half-life in vivo; and its industrial manufacture is complex and expensive. tic anti-MET antibodies which te MET in a manner mimicking that of HGF have been proposed as alternatives.

The following antibodies that mimic HGF activity, at least partially, have been described: (i) the 3D6 mouse anti-human MET dy (U.S. Patent No. 6,099,841); (ii) the 5D5 mouse anti-human MET antibody (U.S. Patent No. 5,686,292); (iii) the NO-23 mouse uman MET antibody (U.S. Patent No. 7,556,804 B2); (iv) the B7 human naïve anti-human MET antibody (U.S. Patent Application No. 2014/0193431 A1); (v) the DO-24 mouse anti-human MET antibody (Prat et al., Mol Cell Biol. 11, 5954-5962, 1991; Prat et al., J Cell Sci. 111, 237-247, 1998); and (vi) the DN-30 mouse anti-human MET antibody (Prat et al., Mol Cell Biol. 11, 5954-5962, 1991; Prat et al., J Cell Sci. 111, 7, 1998).

SUMMARY OF ION Agonistic anti-MET antibodies generated to date, for e those described in the Background n, are frequently obtained as by-products from processes intending to identify antagonistic molecules and are not designed explicitly to become agonistic molecules for therapeutic use. Moreover, the most manifest limit of the prior art anti-MET antibodies is that they have been generated in a mouse system (except for B7 that was identified using a human naïve phage library); as a result, it is unlikely that these antibodies will display cross-reactivity with mouse MET. Even if a minor cross-reactivity with self- antigens is in principle possible, these interactions have normally a very low affinity.

While the absence of reactivity is not a concern for mouse models of cancer (as they employ human xenografts), cross-reactivity of antibodies between human and mouse MET is an important requirement for pre-clinical mouse models of regenerative medicine or non- gical human diseases, which require the antibody to function on mouse tissues and cells.

Not only is it necessary for an agonistic anti-MET antibody to cross-react with mouse MET in order for the antibody to be ted in pre-clinical models, but it is desirable that the antibody binds to mouse MET with an affinity the same or similar to its affinity for human MET, and also that the antibody elicits effects in mouse systems the same or similar to the effects which it evokes in human systems – otherwise the experiments conducted in preclinical models will not be predictive of the human situation. As demonstrated in the Examples, none of the prior art anti-MET agonistic antibodies exhibit affinity for mouse MET, and nly none of the prior art antibodies exhibit the same or similar binding and agonistic effects in both mouse and human systems.

The present application provides anti-MET agonistic antibodies made by design to bind to both human and mouse MET with high affinities. These antibodies: (i) y agonistic ty in both human and mouse MET biological systems – that is they induce MET signalling – some with a potency similar or or to that of HGF; (ii) elicit the full um of HGF-induced biological activities, thus representing valid substitutes for recombinant HGF; (iii) t superior binding to mouse MET when directly compared to prior art antibodies; (iv) display ically significant agonistic activity at trations as low as 1 pM; (v) display a plasma half-life of several days in mice, reaching pharmacologically saturating concentrations already at a dose of 1 µg/kg, which is very low for a therapeutic antibody; (vi) preserve renal function and kidney integrity in a mouse model of acute kidney injury; (vii) t liver failure and antagonize hepatocyte damage in a mouse model of acute liver injury; (viii) display anti-fibrotic, anti-inflammatory and pro- regenerative activity in a mouse model of chronic liver ; (ix) prevent weight loss, attenuate intestinal bleeding, preserve colon integrity, ss inflammation and e epithelial regeneration in a mouse model of ulcerative colitis and a mouse model of inflammatory bowel disease; (x) promote n-independent uptake of glucose in a mouse model of type I diabetes; (xi) overcome insulin resistance in a mouse model of type II diabetes; (xii) ameliorate fatty liver, suppress fibrosis and restore liver function in a mouse model of non-alcoholic steatohepatitis (NASH); (xiii) accelerate wound healing in a mouse model of diabetic ulcer; (xiv) cross-react with Rattus norvegicus MET and Macaca fascicularis MET, thus allowing to conduct toxicological and pharmacological studies in these two rates, required prior to applying for first-in-human trials; (xv) recognise epitopes conserved across human, mouse, rat and cynomolgus macaque, thereby providing greater utility across animal models.

Therefore, in a first aspect, the present invention provides an antibody, or an antigen binding fragment thereof, which binds human MET protein (hMET) with high affinity and binds mouse MET protein (mMET) with high affinity, n the antibody or an antigen binding fragment thereof is a hMET agonist and a mMET agonist. In certain embodiments, the antibody or antigen binding fragment thereof comprises at least one heavy chain variable domain (VH) and at least one light chain variable domain (VL), wherein said VH and VL domain, when tested as a Fab fragment, exhibit an off-rate (koff measured by Biacore) for hMET in the range of from 1 x 10-3 s-1 to 1 x 10-2 s-1, optionally 1 x 10-3 s-1 to 6 x -3 s-1, and exhibit an off-rate (koff measured by Biacore) for mMET in the range of from 1 x -3 s-1 to 1 x 10-2 s-1, ally 1 x 10-3 s-1 to 6 x 10-3 s-1. In certain embodiments, the antibody or antigen binding fragment thereof has equivalent affinity for hMET and mMET.

In certain embodiments, the dy or antigen binding nt thereof induces phosphorylation of hMET and induces phosphorylation of mMET. In certain embodiments, the antibody or antigen binding fragment induces phosphorylation of hMET with an EC50 (as measured by phospho-MET ELISA) of less than 3.0 nM, optionally less than 2.0 nM and induces phosphorylation of mMET with an EC50 (as ed by phospho-MET ELISA) of less than 3.0 nM, optionally less than 2.0 nM. In certain embodiments, the antibody or antigen binding fragment f induces phosphorylation of hMET and mMET lently.

In certain embodiments, the antibody or antigen binding fragment thereof exhibits high phosphorylation potency for hMET and exhibits high orylation potency for mMET. In certain embodiments, the antibody or n binding fragment thereof induces phosphorylation of hMET with an EC50 of less than 1nM and/or an Emax (as a tage of HGF-induced activation in a phospho-MET ELISA) of at least 80%.and induces orylation of mMET with an EC50 of less than 1nM and/or an Emax (as a percentage of HGF-induced tion in a phospho-MET ELISA) of at least 80%. In certain alternative embodiments, the dy or antigen binding fragment thereof exhibits low phosphorylation potency for hMET and exhibits low phosphorylation potency for mMET. In n such embodiments, the antibody or antigen binding fragment thereof induces phosphorylation of hMET with EC50 of 1nM-5nM and/or an Emax (as a percentage of HGF- induced activation in a phospho-MET ELISA) of 60-80% and induces phosphorylation of mMET with EC50 of 1nM-5nM and/or an Emax (as a percentage of HGF-induced activation in a phospho-MET ELISA) of 60-80%.

In certain ments, the antibody or antigen binding nt thereof induces an HGF- like cellular response when contacted with a human cell and induces an HGF-like cellular response when contacted with a mouse cell. In certain embodiments, the antibody or antigen binding fragment thereof fully induces an ke ar response when contacted with a human cell and when ted with a mouse cell. In certain embodiments, full induction of HGF-like cellular response is able as one, any two, or all of: (i) in a cell scattering assay, the antibody or n binding fragment thereof induces cell scattering comparable to maximal HGF-induced scattering when the antibody or antigen binding fragment thereof is at a concentration of 0.1-1.0 nM; (ii) in an poptotic cell assay, the antibody or antigen binding fragment thereof exhibits an EC50 of less than 1.1x that of HGF, and/or with an Emax (measured as a % of total ATP content of optotic control cells) of greater than 90% that observed for HGF; and/or (iii) in a ing morphogenesis assay, cells treated with the antibody exhibit greater than 90% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF.

In certain embodiments, the antibody or antigen binding fragment thereof partially induces an HGF-like cellular response when ted with a human cell and when contacted with a mouse cell. In certain ments, partial induction of an HGF-like cellular response is measurable as: (i) in a cell scattering assay, the antibody or antigen binding fragment thereof induces cell scattering of at least 25% that induced by 0.1 nM homologous HGF when the antibody concentration is 1 nM or lower; (ii) in anti-apoptotic cell assay, the dy or antigen binding fragment thereof exhibits an EC50 no more than 7.0x that of HGF and/or an Emax cellular ity of at least 50% that ed for HGF; and/or (ii) in a branching morphogenesis assay, cells treated with the antibody exhibit at least 25% the number of branches per spheroid induced by the same (non-zero) concentration of HGF; and the antibody or antigen binding fragment does not fully induce an HGF-like cellular response.

In certain embodiments, the antibody or antigen binding fragment thereof is a HGF competitor. In certain embodiments, the antibody or antigen binding fragment thereof es with hHGF binding to hMET with an IC50 of no more than 5nM and/or an Imax of at least 50% and competes with mHGF binding to mMET with an IC50 of no more than 5nM and/or an Imax of at least 50%. In certain embodiments, the antibody or antigen binding nt thereof es with hHGF and mHGF equivalently. In certain embodiments, the antibody or antigen binding fragment thereof is a full HGF competitor. In certain such embodiments, the antibody or antigen binding fragment thereof competes with hHGF with an IC50 of less than 2 nM and/or an Imax of greater than 90% and es with mHGF with an IC50 of less than 2 nM and/or an Imax of greater than 90%. In n embodiments, the antibody or antigen binding fragment f is a partial HGF itor. In n such embodiments, the antibody or antigen binding fragment thereof competes with hHGF with an IC50 of 2-5nM and/or an Imax of 50%-90% and competes with mHGF with an IC50 of 2- 5nM and/or an Imax of 50%-90%.

Antibodies or antigen binding fragment thereof of the invention may t reactivity with MET of simian origin, such as cynomolgus monkey (Macaca cynomolgus) MET, and may exhibit cross-reactivity with MET of rat origin (Rattus norvegicus).

Antibodies or antigen binding fragment thereof of the ion may bind an epitope of human MET from amino acid residue 123 to223 of human MET (throughout the document, numbering of human MET refers to GenBank sequence # X54559). Also provided are antibodies or antigen binding fragment thereof of the invention which may bind an epitope of human MET between amino acids 224-311 of human MET. Also provided are antibodies or antigen binding fragment thereof of the invention which may bind an epitope of human MET between amino acids 314-372 of human MET. Also provided are antibodies or antigen binding fragment thereof of the invention which may bind an epitope of human MET between amino acids 546-562 of human MET.

Also provided are antibodies or antigen binding fragment thereof of the invention which may bind an epitope of human MET comprising the amino acid residue Ile367. Also ed are antibodies or antigen binding fragment thereof of the invention which may bind an epitope of human MET comprising the amino acid residue Asp372 of human MET.

In n embodiments, the antibody or antigen binding fragment thereof binds an e of human MET comprising the amino acid residues Ile367 and Asp372 of human MET.

Also provided are antibodies or antigen g fragment thereof of the invention which may bind an epitope of human MET comprising the amino acid residue Thr555 of human The ion further provides an antibody or antigen binding fragment thereof which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises an amino acid sequence selected from SEQ ID NO:2, 9, 16, 23, 30, 37, 44, 51, 58, 65, and 72; H-CDR2 ses an amino acid sequence selected from SEQ ID NO:4, 11, 18, 25, 32, 39, 46, 53, 60, 67, and 74; H-CDR3 comprises an amino acid sequence selected from SEQ ID NO:6, 13, 20, 27, 34, 41, 48, 55, 62, 69, and 76, L-CDR1 comprises an amino acid sequence selected from SEQ ID NO:79, 86, 93, 100, 107, 114, 121, 128, 135, 142, and 149; L-CDR2 comprises an amino acid sequence selected from SEQ ID NO:81, 88, 95, 102, 109, 116, 123, 130, 137, 144, and 151; and L-CDR3 comprises an amino acid sequence selected from SEQ ID NO:83, 90, 97, 104, 111, 118, 125, 132, 139, 146, and153. [71G2] In one ment, the invention provides an dy or n binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and HCDR3 , and a light chain variable domain comprising , L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:44, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:46, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:48, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:121, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:123, and L-CDR3 ses the amino acid sequence shown as SEQ ID NO:125. [71G2] In certain such embodiments, the heavy chain variable domain of the antibody or fragment comprises the amino acid ce of SEQ ID NO:167, or a sequence at least 90%, 95%, 97% or 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:168, or a sequence at least 90%, 95%, 97% or 99% identical thereto. [71D6] In another embodiment, the invention provides an antibody or n binding fragment which comprises a heavy chain variable domain sing H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid ce shown as SEQ ID NO:30, H-CDR2 ses the amino acid sequence shown as SEQ ID NO:32, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:34, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:107, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:109, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:111. [71D6] In certain such embodiments, the heavy chain variable domain of the antibody or n binding fragment comprises the amino acid sequence of SEQ ID , or a sequence at least 90%, 95%, 97% or 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:164, or a sequence at least 90%, 95%, 97% or 99% identical thereto. [71G3] In a further embodiment, the ion provides an antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and , and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:9, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:11, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:13, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:86, L-CDR2 comprises the amino acid ce shown as SEQ ID NO:88, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:90. [71G3] In certain such embodiments, the heavy chain variable domain of the antibody or antigen binding nt comprises the amino acid sequence of SEQ ID NO:157, or a sequence at least 90%, 95%, 97% or 99% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:158, or a sequence at least 90%, 95%, 97% or 99% identical thereto.

In further embodiments, the invention provides an antibody or antigen binding fragment comprising a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and , wherein H-CDR1, H-CDR2 and H-CDR3 are selected from a set of CDRs (CDR1, CDR2 and CDR3) for a Fab shown in Table 3, and L-CDR1, L-CDR2 and L-CDR3 are the corresponding CDRs (CDR1, CDR2 and CDR3) for the same Fab shown in Table 4.

In certain embodiments, the heavy chain variable domain of the dy or antigen g fragment comprises a VH amino acid sequence from Table 5 or a sequence at least 90%, 95%, 97% or 99% identical thereto, and the light chain variable domain comprises the corresponding VL amino acid sequence in Table 5 or a sequence at least 90%, 95%, 97% or 99% identical thereto.

Embodiments wherein the amino acid sequence of the VH domain exhibits less than 100% sequence identity with a defined VH domain amino acid sequence (e.g. SEQ ID NO: x) may nevertheless comprise heavy chain CDRs which are identical to the HCDR1, HCDR2 and HCDR3 of the VH of SEQ ID NO: x whilst exhibiting amino acid sequence variation within the framework regions. For example, one or more amino acid residues of the framework region may be substituted by an amino acid e which occurs in the equivalent position in a human VH domain encoded by the human germline. Likewise, ments wherein the amino acid sequence of the VL domain exhibits less than 100% sequence identity with a defined VL domain amino acid sequence (e.g. SEQ ID NO:y) may nevertheless comprise light chain CDRs which are identical to the LCDR1, LCDR2 and LCDR3 of the VL of SEQ ID NO:y, whilst exhibiting amino acid ce variation within the framework regions. For example, one or more amino acid residues of the framework region may be substituted by an amino acid e which occurs in the equivalent position in a human VL domain encoded by the human germline.

The invention also es dies and antigen binding fragments comprising humanised/germlined variants of VH and VL domains of the ing antibodies, plus affinity variants and variants containing conservative amino acid substitutions, as defined herein. Specifically ed are chimeric antibodies containing VH and VL domains of the llama-derived Fabs described above, or human ned ts thereof, fused to constant domains of human antibodies, in particular human IgG1, IgG2, IgG3 or IgG4. The heavy and light chain variable domains of the foregoing antibodies, or germlined variants, affinity variants or conserved variants thereof, may be included within a conventional four- chain antibody or other antigen binding proteins, such as for example Fab, Fab', F(ab')2, ific Fabs, and Fv fragments, diabodies, linear dies, single-chain antibody molecules, a single chain variable nt (scFv) and pecific antibodies. The heavy chain variable domains, or germlined variant, affinity variant or conserved variant thereof, can also be utilised as single domain antibodies.

In further s, the invention also provides an isolated polynucleotide which encodes an antibody or antigen binding fragment of the invention, an expression vector comprising said polynucleotide operably linked to regulatory sequences which permit expression of the antibody or antigen binding fragment f in a host cell or cell-free expression system, and a host cell or cell free expression system containing said expression vector. The invention further provides a method of producing a recombinant antibody or antigen binding fragment f which comprises culturing said host cell or cell free expression system under conditions which permit expression of the antibody or antigen binding fragment and recovering the expressed antibody or antigen binding fragment.

In a further aspect, the invention es a pharmaceutical composition comprising an antibody or n binding fragment of the invention and at least one pharmaceutically acceptable carrier or excipient.

In a further aspect, the invention es an antibody or antigen binding fragment of the invention, or the pharmaceutical composition of the invention, for use in therapy.

In a further aspect, the invention provides a method of treating or preventing liver damage in a human patient, optionally acute liver damage or chronic liver damage, which comprises stering to a patient in need thereof a therapeutically effective amount of a MET agonist antibody. In certain embodiments, the MET agonist dy is an antibody or antigen binding fragment ing to the invention.

In a further aspect, the invention es a method of treating or preventing kidney damage in a human patient, optionally acute kidney damage, which comprises administering to a patient in need thereof a eutically effective amount of a MET agonist antibody. In certain embodiments, the MET agonist antibody is an antibody or antigen binding fragment according to the invention.

In a further aspect, the invention provides a method of treating or preventing inflammatory bowel disease in a human patient, optionally ulcerative colitis, which comprises administering to a t in need thereof a therapeutically effective amount of a MET t antibody. In certain embodiments, the MET t antibody is an antibody or antigen binding fragment according to the invention.

In a further aspect, the ion provides a method of treating or preventing diabetes in a human t, optionally type I or type II diabetes, which comprises administering to a patient in need thereof a therapeutically effective amount of a MET agonist antibody.

In n embodiments, the MET t antibody is an dy or antigen binding fragment according to the invention.

In a further aspect, the invention provides a method of treating or preventing non-alcoholic steatohepatitis in a human t, which comprises administering to a t in need thereof a therapeutically effective amount of a MET agonist antibody. In certain embodiments, the MET agonist antibody is an antibody or antigen binding fragment according to the invention.

In a further aspect, the invention provides a method of treating or promoting wound healing in a human patient, optionally a patient having diabetes, which comprises administering to a patient in need thereof a therapeutically effective amount of a MET agonist antibody. In certain embodiments, the MET agonist antibody is an antibody or n binding fragment according to the invention.

DRAWINGS Figure 1. Immune response of llamas zed with human MET-Fc as determined by ELISA. Human MET ECD (hMET) or mouse MET ECD (mMET) inant protein was immobilized in solid phase and exposed to serial dilutions of sera from llamas before (PRE) or after (POST) immunization. Binding was revealed using a mouse anti-llama IgG1 and a njugated donkey anti-mouse antibody. OD, optical density; AU, arbitrary units.

Figure 2. Schematic drawing of the human MET deletion mutants used for identifying the domains of MET responsible for mAb binding. ECD, extra-cellular domain; aa, amino acid; L. peptide, leader peptide; SEMA, semaphorin homology domain; PSI or P, plexin-semaphorin-integrin homology domain; IPT, Immunoglobulin-transcription factorplexin homology domain. On the right, the corresponding es of human MET are reported according to UniProtKB # P08581.

Figure 3. Schematic drawing of the llama-human chimeric MET proteins used for finely mapping the epitopes recognized by anti-MET antibodies. The extracellular portions of llama MET and human MET are composed of 931 and 932 amino acids (aa), respectively (llama MET has a 2 aa shorter leader peptide but has an insertion after aa 163). Both receptor mains comprise a leader peptide, a semaphorin homology domain (SEMA), a plexin-semaphorin-integrin homology domain (PSI or P) and four immunoglobulin-transcription factor-plexin homology domains (IPT). Chimeras CH1-5 have a N-terminal llama portion followed by a C-terminal human portion. Chimeras CH6-7 have an N-terminal human n followed by a inal llama n.

Figure 4. Agonistic activity of human/mouse lent anti-MET antibodies in human and mouse cells as measured by Western blotting. A549 human lung carcinoma cells and MLP29 mouse liver precursor cells were serum-starved and then stimulated with increasing concentrations of mAbs or recombinant human HGF (hHGF; A549) or mouse HGF (mHGF; MLP29). MET auto-phosphorylation was determined by Western blotting using anti-phospho-MET dies (tyrosines 1234-1235). The same cell lysates were also ed by Western blotting using otal human MET antibodies (A549) or anti-total mouse MET antibodies (MLP29).

Figure 5. Biological activity of human/mouse equivalent anti-MET antibodies as measured by a branching morphogenesis assay using LOC human kidney epithelial cells and MLP29 mouse liver precursor cells. Cell ids were seeded inside a collagen layer and then d to increasing concentrations of mAbs or recombinant human HGF (LOC) or mouse HGF ). Branching morphogenesis was followed over time by microscopy, and colonies were photographed after 5 days.

Figure 6. Comparison with prior art antibodies: human-mouse cross-reactivity.

Human or mouse MET ECD was immobilized in solid phase and exposed to increasing concentrations of antibodies (all in a mouse IgG/λ format) in solution. Binding was revealed by ELISA using HRP-conjugated anti-mouse Fc antibodies.

Figure 7. Comparison with prior art antibodies: MET auto-phosphorylation. A549 human lung oma cells and MLP29 mouse liver precursor cells were deprived of serum growth factors for 48 hours and then ated with increasing trations of antibodies. After 15 minutes of stimulation, cells were lysed, and phospho-MET levels were determined by ELISA using anti-MET dies for capture and anti-phospho-tyrosine antibodies for ing.

Figure 8. Comparison with prior art antibodies: branching morphogenesis. LOC human kidney epithelial cell spheroids were seeded in a collagen layer and then incubated with increasing concentrations of mAbs. Branching morphogenesis was followed over time by microscopy, and colonies were photographed after 5 days.

Figure 9. Comparison with prior art antibodies: branching morphogenesis. MLP29 mouse liver precursor cell spheroids were seeded in a collagen layer and then incubated with increasing trations of mAbs. ing morphogenesis was followed over time by microscopy, and colonies were photographed after 5 days.

Figure 10. Plasma stability of human/mouse equivalent anti-MET antibodies. A single bolus of 1 mg/kg or 10 mg/kg antibody was injected i.p. and blood s were taken from the tail vein at 3, 6, 12 and 24 hours post-injection. Blood s were processed and antibody concentration in plasma was determined by ELISA. (A) Peak and trough levels of injected antibodies. (B) dy plasma half-life was calculated by linear fitting of the antibody concentration Ln transforms.

Figure 11. Acute liver failure model: plasma concentration of liver function markers.

Acute liver damage was induced in BALB/c mice by subcutaneous injection of a CCl4 solution. Soon after intoxication, mice were randomized into 4 arms which received a single bolus of 71G3, 71D6, 71G2 or vehicle only (PBS). Antibodies were administered by i.p. injection at a dose of 5 mg/kg. Each arm comprised three groups of mice that were sacrificed at different times post-intoxication (12, 24 and 48 hours). Blood samples were taken at different times post-injection (0, 12, 24 and 48 hours). At autopsy, blood and livers were collected for analysis. Plasma levels of the hepatic markers aspartate transaminase (AST), alanine aminotransferase (ALT) and bin (BIL) was determined by standard clinical biochemistry methods.

Figure 12. Acute liver failure model: histological examination of liver ns. Acute liver damage was induced in BALB/c mice as described in Figure 11 legend. At autopsy, livers were extracted and embedded in paraffin for ogical analysis. Sections were d with hematoxylin and eosin and examined by microscopy. A representative image for each treatment arm is shown. Magnification: 100X.

Figure 13. Chronic liver damage model: plasma concentration of liver function markers. Liver injury and fibrosis in BALB/c mice was induced by chronic exposure to CCl4 for several weeks. Soon after the first CCl4 injection, mice were randomized into 4 arms which ed treatment with 71G3, 71D6, 71G2 or vehicle only (PBS), respectively.

Antibodies were administered three times a week by i.p. injection at a dose of 1 mg/kg. An additional, fifth control arm received no CCl4 or antibody and served as healthy control.

Mice were sacrificed after 6 weeks of chronic CCl4 intoxication. At autopsy, blood and livers were collected for analysis. Plasma levels of the hepatic s aspartate transaminase (AST) and e aminotransferase (ALT) were determined by standard clinical biochemistry methods.

Figure 14. Chronic liver damage model: ogical examination of liver sections stained with Picro Sirius red. Liver injury and fibrosis in BALB/c mice were induced by chronic exposure to CCl4 as described in Figure 13 legend. At autopsy, livers were extracted and ed in paraffin for immuno-histochemical analysis. Sections were stained with Picro Sirius red. A entative image for each treatment arm is shown.

Magnification: 100X.

Figure 15. c liver damage model: histological examination of liver sections stained with anti-alpha smooth muscle actin (α-SMA) antibodies. Liver injury and fibrosis in BALB/c mice were induced by chronic exposure to CCl4 as described in Figure 13 legend. At autopsy, livers were extracted and embedded in paraffin for immuno- histochemical analysis. Sections were stained with anti-alpha smooth muscle actin (α-SMA) dies. A representative image for each treatment arm is shown. Magnification: 100X.

Figure 16. Acute kidney injury model: plasma levels of renal function markers. Acute renal failure was induced in BALB/c mice by i.p. injection of a single bolus of HgCl2. Soon after HgCl2 intoxication, mice were ized into 4 arms which were subjected to treatment with 71G3, 71D6, 71G2 or vehicle only (PBS). Antibodies were administered by i.p. injection every 24 hours at a dose of 5 mg/kg. Mice were sacrificed 72 hours after HgCl2 injection. At y, blood and kidneys were collected for analysis. Blood urea nitrogen (BUN) and creatinine (CRE) plasma levels were determined by standard clinical biochemistry methods.

Figure 17. Acute kidney injury model: histological analysis of kidney sections. Acute renal failure was induced in BALB/c mice by HgCl2 injection as described in Figure 16 legend. At autopsy, s were extracted and embedded in paraffin for histological analysis. Kidney sections were d with hematoxylin and eosin. A representative image for each treatment arm is shown. Magnification: 400X.

Figure 18. Ulcerative colitis model: body weight, Disease Activity Index (DAI), and colon length. Ulcerative colitis was induced in BALB/c mice by addition of dextran sodium sulphate (DSS) to the ng water for 10 days. On day 10, DSS treatment was interrupted and mice were put back on normal water. Starting from day 1, mice were randomized into 7 arms which received treatment with 71G3, 71D6, 71G2 (at a dose of 1 mg/kg or 5 mg/kg) or vehicle only (PBS). An additional, eighth control arm ed no DSS or antibody and served as healthy l. Mice were iced on day 12, i.e. 2 days after DSS stration was interrupted. At autopsy, colons were collected, washed through, and their length was ined using a ruler. Following measurement, colons were embedded in paraffin and processed for ogical analysis. During the whole course of the experiment, mouse weight was monitored on a regular basis, and the clinical symptoms of ulcerative colitis were assessed by determining faecal blood, rectal bleeding and stool consistency. Each parameter was given a score from 0 (absence of the symptom) to 3 (maximal manifestation of the symptom). Scores relative to the single parameters were summed together to give rise to the DAI ranging from 0 to 9. (A) Body weight over time (% relative to time 0). (B) DAI over time. (C) Colon length at autopsy. Data of the 1 mg/kg arms and of the 5 mg/kg arms are shown in separate graphs for clarity.

Figure 19. Ulcerative colitis model: histological analysis of colon sections. Ulcerative colitis was induced in BALB/c mice by exposure to dextran sodium sulphate (DSS) as described in Figure 18 legend. At autopsy, colons were collected, measured, and then embedded in paraffin and sed for histological analysis. Colon sections were stained with hematoxylin and eosin, examined by microscopy, and raphed. Experimental arm, antibody dose and magnification are indicated close to each image. Please refer to the main text for image analysis.

Figure 20. Inflammatory bowel disease model: body weight and colon length. Colon injury and inflammation was d in S/J mice by intra-rectal injection of 2,4,6- trinitrobenzenesulfonic acid (TNBS) dissolved in ethanol. Soon after TNBS administration, mice were randomized into 4 arms which received treatment with 71G3, 71D6, 71G2 or e only (PBS). An additional, fifth control arm received no TNBS or antibody and served as healthy l. Mice were sacrificed 5 days after TNBS administration. At autopsy, colons were collected and measured. Following measurement, colons were embedded in in and processed for histological analysis. During the whole course of the experiment, mouse weight was measured every day. (A) Body weight over time (% relative to time 0). (B) Colon length at autopsy.

Figure 21. Inflammatory bowel disease model: histological analysis of colon sections. Colon injury and mation was induced in BALB/c mice by intra-rectal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as described in Figure 20 legend. At autopsy, colons were collected and measured. Following measurement, colons were embedded in paraffin and processed for histological analysis. Colon sections were stained with hematoxylin and eosin, examined by microscopy, and photographed. Please refer to the main text for image is.

Figure 22. Type I diabetes model: ion of glucose uptake and cooperation with insulin in diabetic mice. Pancreatic β-cell degeneration was induced in BALB/c mice by i.p. injection of streptozotocin (STZ). STZ-treated mice displayed a mean basal glycemy two times higher compared to untreated mice. STZ-treated mice were randomized into 4 arms, which received treatment with 71G3, 71D6, 71G2 or vehicle only (PBS), respectively.

An additional, fifth control arm received no STZ or antibody and served as healthy control.

Blood glucose concentration in fasting ions was monitored over time for 5 weeks. At the end of week 5, a glucose tolerance test (GTT) and an insulin tolerance test (ITT) were performed. (A) Analysis over time of basal blood glucose levels in fasting conditions. (B) GTT: following oral administration of glucose to a fasting animal, blood e levels are monitored over time. (C) ITT: following i.p. injection of insulin to a partially fasting animal, blood glucose levels are monitored over time.

Figure 23. Type I es model: promotion of glucose uptake and co-operation with n in cultured cells. C2C12 mouse myoblast cells were induced to differentiate into myocytes and then incubated with human/mouse equivalent agonistic anti-MET antibodies (71G3, 71D6, 71G2). After 24 hours, antibody-treated cells were d into 3 arms, which were subjected to acute stimulation with 0 nM, 100 nM or 1000 nM human recombinant insulin for 1 hour in the presence of the fluorescent glucose analogue 7-Nitrobenz oxa-1,3-diazolyl)Amino)Deoxyglucose G). 2-NBDG uptake was determined by flow cytometry. (A) Induction of 2-NBDG uptake by human/mouse equivalent agonistic anti- MET antibodies or insulin. (B) Induction of 2-NBDG uptake by 71G3 in the absence or ce of insulin. (C) Induction of 2-NBDG uptake by 71D6 in the absence or presence of insulin. (D) Induction of 2-NBDG uptake by 71G2 in the absence or presence of insulin.

Figure 24. Type II diabetes model: blood glucose level normalization and insulin resistance overcoming in db/db mice. At the age of 8 weeks, female db/db mice (a C57BLKS/J variant bearing a point mutation in the leptin or gene lepr) were randomized into 4 arms that received treatment with 71G3, 71D6, 71G2 or vehicle only (PBS), respectively. Antibodies were administered two times a week by i.p. ion at a dose of 1 mg/kg. Blood glucose concentration in fasting conditions was monitored every 10 days for 7 weeks. At the end of the treatment, i.e. when mice were 15 weeks old, a glucose tolerance test (GTT) and an insulin tolerance test (ITT) were performed using age-matched wild-type C57BLKS/J mice as control. (A) Blood e concentration over time. (B) GTT: following oral administration of glucose to a fasting animal, blood glucose levels are monitored over time. (C) ITT: ing i.p. ion of insulin to a partially fasting animal, blood glucose levels are monitored over time.

Figure 25. Mouse model of non-alcoholic steatohepatitis (NASH): fatty liver amelioration as determined by histology. Eight week-old female db/db mice were randomized into 4 arms that received treatment with 71G3, 71D6, 71G2 or vehicle only (PBS), respectively. Antibodies were stered two times a week by i.p. injection at a dose of 1 mg/kg. After 8 weeks of treatment, mice were sacrificed and subjected to autopsy. Blood was collected for analysis of hepatic function markers. Livers were extracted, embedded in in and sed for histological examination. Liver sections were stained with hematoxylin and eosin. The cytoplasm of fatty cells appears empty and white because lipids are washed away during alcohol processing of the specimen. A representative image for each treatment arm is shown. Magnification: 200X.

Figure 26. Mouse model of non-alcoholic steatohepatitis (NASH): suppression of fibrosis as determined by Picro Sirius red staining. Eight week-old female db/db mice were randomized and treated as described in Figure 25 legend. At autopsy, livers were processed for histological examination. Liver sections were stained with Sirius red to highlight is. A representative image for each treatment arm is shown. Magnification: 200X.

Figure 27. Mouse model of non-alcoholic steatohepatitis (NASH): normalization of liver function markers. Eight week-old female db/db mice were treated with ed 71G3, 71D6, 71G2 or vehicle only as described in Figure 25 legend. After 7 weeks of ent, blood was collected for analysis of the hepatic function markers. (A) Plasma levels of aspartate transaminase (AST). (B) Plasma levels of alanine ransferase (ALT).

Figure 28. Mouse model of diabetic ulcers: accelerated healing of wounds. Eight week-old db/db diabetic mice were subjected to anaesthesia and then cut with a 0.8 cmwide circular punch blade for skin biopsies to create a round wound in the right posterior flank. The entire epidermal layer was d. The day after surgery, mice were randomized into 4 arms that ed treatment with ed 71G3, 71D6 and 71G2 or vehicle only (PBS). Antibodies were delivered every second day by i.p injection at a dose of mg/kg. Wound diameter was measured every day using a calliper. (A) Wound area over time. (B) Mean re-epithelization rate as determined by averaging the daily % of wound closure.

Figure 29. Rattus norvegicus and Macaca fascicularis cross-reactivity as determined by ELISA. In order to test pan-species cross-reactivity, a cted panel of antibodies representative of both SEMA binders (71D6, 71C3, 71D4, 71A3, 71G2) and PSI s (76H10, 71G3) was selected. The 5D5 prior art antibody was used as control. Human, mouse, rat or monkey MET ECD was immobilized in solid phase and exposed to increasing concentrations of mAbs (in their human IgG1/λ format) in solution. Binding was revealed using HRP-conjugated anti-human Fc antibodies.

Figure 30. Amino acid sequence ent among the MET ECD domains of from H. sapiens, M. musculus, R. norvegicus, M. fascicularis and L. glama. (A) Sequence ent relative to the region recognized by the SEMA-binding antibodies (71D6, 71C3, 71D4, 71A3 and 71G2) (human MET sequence SEQ ID NO: 239; mouse MET sequence SEQ ID NO: 240; rat MET sequence SEQ ID NO: 241, cyno MET sequence SEQ ID NO: 242, Llama MET sequence SEQ ID NO: 243). The amino acids identified by the humanllama chimera approach shown in Table 12 are underlined. Within this region there are five es that are conserved in human and mouse MET but not in llama MET (Ala 327, Ser 336, Phe 343, Ile 367, Asp 372). These amino acids are indicated with a black box and the ssive numbers 1-5. Of these, four residues are also conserved in rat and cynomolgus monkey MET (Ala 327, Ser 336, Ile 367, Asp 372). Amino acids responsible for binding to the SEMA-binding dies are indicated with an “S” (for SEMA). Amino acids sible for binding to 5D5/Onartuzumab are indicated with an “O” (for Onartuzumab). (B) Sequence alignment relative to the region recognized by the PSI- binding antibodies 76H10 and 71G3 (human MET sequence SEQ ID NO: 244; mouse MET sequence SEQ ID NO: 245; rat MET sequence SEQ ID NO: 246, cyno MET sequence SEQ ID NO: 247, Llama MET sequence SEQ ID NO: 248). The amino acids identified by the human-llama chimera approach shown in Table 12 are underlined. Within this region there are three residues that are conserved in human and mouse MET but not in llama MET (Arg 547, Ser 553, Thr 555). These amino acids are indicated with a black box and the progressive numbers 6-8. Of these, two es are also conserved in rat and cynomolgus monkey MET (Ser 553 and Thr 555). The amino acid responsible for binding to the PSI-binding antibodies is ted with a “P” (for PSI).

Figure 31. Schematic representation of the MET mutants used for fine epitope mapping. Using human MET ECD as a template, the key residues indicated with the progressive numbers 1-8 in Figure 30 were mutagenized in different ations, ting mutants A-L. Each of these mutants is fully human except for the indicated residues, which are llama.

DETAILED DESCRIPTION As used herein, the term "immunoglobulin" includes a polypeptide having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity. "Antibodies" refers to such assemblies which have significant known specific immunoreactive activity to an antigen of interest (e.g. MET). The term “MET antibodies” or “anti-MET antibodies” are used herein to refer to dies which exhibit immunological specificity for MET protein. Antibodies and immunoglobulins comprise light and heavy chains, with or t an interchain nt linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.

The generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. Although all five classes of antibodies are within the scope of the present invention, the following discussion will generally be ed to the IgG class of immunoglobulin molecules. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region.

The light chains of an antibody are classified as either kappa or lambda (, ). Each heavy chain class may be bound with either a kappa or lambda light chain. In l, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent ide linkages or non-covalent linkages when the immunoglobulins are generated by B cells or cally engineered host cells. In the heavy chain, the amino acid sequences run from an inus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those d in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (, , , , ) with some sses among them (e.g., 1-4). It is the nature of this chain that determines the " of the antibody as IgG, IgM, IgA, IgD or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known to confer functional specialization.

Modified versions of each of these classes and es are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.

As indicated above, the variable region of an antibody allows the antibody to selectively recognize and ically bind epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary dy ure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary ining regions (CDRs) on each of the VH and VL chains.

As used herein, the terms "MET protein" or "MET antigen" or "MET" are used hangeably and refer to the receptor tyrosine kinase that, in its wild-type form, binds Hepatocyte Growth Factor (HGF). The terms “human MET protein” or “human MET or” or “human MET” or “hMET” are used interchangeably to refer to human MET (GenBank accession : X54559), including the native human MET protein naturally expressed in the human host and/or on the surface of human cultured cell lines, as well as recombinant forms and nts thereof and also naturally occurring mutant forms. The terms “mouse MET protein” or “mouse MET receptor” or “mouse MET” or “mMET” are used interchangeably to refer to mouse MET (GenBank accession number: NM_008591), including the native mouse MET protein naturally expressed in the mouse host and/or on the surface of mouse cultured cell lines, as well as recombinant forms and fragments thereof and also naturally occurring mutant forms.

As used herein, the term ng site” comprises a region of a polypeptide which is responsible for selectively binding to a target antigen of interest (e.g. hMET). g domains comprise at least one binding site. Exemplary binding domains include an antibody variable domain. The antibody les of the invention may comprise a single binding site or multiple (e.g., two, three or four) binding sites.

As used herein the term ed from" a designated protein (e.g. a MET antibody or antigen-binding fragment thereof) refers to the origin of the polypeptide. In one embodiment, the polypeptide or amino acid sequence which is derived from a particular starting polypeptide is a CDR sequence or sequence related thereto. In one embodiment, the amino acid sequence which is derived from a particular starting polypeptide is not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are derived from a starting dy. In one embodiment, the polypeptide or amino acid sequence which is d from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is essentially identical to that of the starting sequence, or a portion thereof wherein the portion ts of at least 3-5 amino acids, at least 5-10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence. In one embodiment, the one or more CDR sequences d from the starting antibody are altered to produce variant CDR sequences, e.g. affinity variants, n the variant CDR sequences maintain MET binding ty. id-Derived” --- In certain preferred ments, the MET antibody molecules of the invention se framework amino acid sequences and/or CDR amino acid sequences derived from a camelid conventional antibody raised by active immunisation of a camelid with a MET-derived antigen. However, MET antibodies comprising camelidderived amino acid sequences may be engineered to comprise framework and/or constant region sequences derived from a human amino acid sequence (i.e. a human antibody) or other non-camelid mammalian species. For example, a human or non-human primate framework region, heavy chain portion, and/or hinge portion may be included in the subject MET antibodies. In one embodiment, one or more non-camelid amino acids may be present in the framework region of a id-derived” MET antibody, e.g., a camelid framework amino acid sequence may comprise one or more amino acid mutations in which the corresponding human or non-human primate amino acid residue is t. Moreover, camelid-derived VH and VL domains, or humanised variants thereof, may be linked to the constant domains of human antibodies to produce a chimeric molecule, as extensively described elsewhere herein.

As used herein, a "conservative amino acid substitution" is one in which the amino acid e is replaced with an amino acid residue having a r side chain. es of amino acid residues having similar side chains have been defined in the art, ing basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, cine) and ic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Thus, a nonessential amino acid residue in an immunoglobulin polypeptide may be ed with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally r string that s in order and/or ition of side chain family members.

As used herein, the term “heavy chain portion” includes amino acid sequences derived from the constant domains of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, an antibody or antigen binding fragment of the invention may comprise the Fc portion of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, and a CH3 domain). In another embodiment, an antibody or antigen binding nt of the invention may lack at least a portion of a nt domain (e.g., all or part of a CH2 domain). In certain embodiments, at least one, and preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain portion comprises a fully human hinge domain. In other red embodiments, the heavy chain portion comprises a fully human Fc portion (e.g., hinge, CH2 and CH3 domain sequences from a human immunoglobulin).

In certain embodiments, the constituent constant domains of the heavy chain portion are from different immunoglobulin molecules. For example, a heavy chain n of a polypeptide may comprise a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domains are ic domains comprising portions of different immunoglobulin molecules.

For example, a hinge may comprise a first portion from an IgG1 molecule and a second portion from an IgG3 or IgG4 molecule. As set forth above, it will be understood by one of ordinary skill in the art that the nt domains of the heavy chain portion may be modified such that they vary in amino acid sequence from the naturally occurring (wildtype ) immunoglobulin le. That is, the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the heavy chain nt domains (CH1, hinge, CH2 or CH3) and/or to the light chain constant region domain (CL).

Exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more s.

As used herein, a "chimeric" protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences may normally exist in separate proteins that are brought er in the fusion polypeptide or they may normally exist in the same protein but are placed in a new arrangement in the fusion polypeptide. A chimeric protein may be created, for example, by al synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. ary chimeric MET antibodies include fusion proteins comprising camelid-derived VH and VL domains, or humanised variants thereof, fused to the constant domains of a human antibody, e.g. human IgG1, IgG2, IgG3 or IgG4, or fused to the constant domains of a mouse antibody, e.g. mouse IgG1, IgG2a, IgG2b, IgG2c or IgG3.

As used , the terms "variable region" and "variable domain" are used interchangeably and are intended to have equivalent meaning. The term ble" refers to the fact that certain portions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly buted throughout the variable domains of antibodies. It is concentrated in three segments called variable loops" in each of the VL domain and the VH domain which form part of the antigen binding site. The first, second and third hypervariable loops of the VLambda light chain domain are referred to herein as L1(λ), L2(λ) and L3(λ) and may be defined as comprising residues 24-33 (L1(λ), consisting of 9, 10 or 11 amino acid es), 49-53 (L2(λ), consisting of 3 residues) and 90-96 (L3(λ), consisting of 5 es) in the VL domain (Morea et al., Methods 20, 267-279, 2000). The first, second and third hypervariable loops of the VKappa light chain domain are referred to herein as L1(κ), L2(κ) and L3(κ) and may be defined as sing residues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(κ), ting of 3 residues) and 90-97 (L3(κ), consisting of 6 residues) in the VL domain (Morea et al., Methods 20, 267-279, 2000). The first, second and third hypervariable loops of the VH domain are referred to herein as H1, H2 and H3 and may be defined as comprising residues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et al., Methods 20, 267-279, 2000).

Unless otherwise indicated, the terms L1, L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and ass hypervariable loops obtained from both Vkappa and Vlambda isotypes. The terms H1, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops ed from any of the known heavy chain isotypes, including γ, ε, δ, α or μ.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise part of a "complementarity determining region" or "CDR", as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly mous, since the hypervariable loops (HVs) are defined on the basis of ure, whereas complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) and the limits of the HVs and the CDRs may be ent in some VH and VL domains.

The CDRs of the VL and VH domains can typically be defined as comprising the following amino acids: residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 ) in the light chain variable domain, and residues 31-35 or 31-35b (CDRH1), 50-65 ) and 95- 102 (CDRH3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological st, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991). Thus, the HVs may be comprised within the corresponding CDRs and references herein to the variable loops" of VH and VL s should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable s are called the framework region (FR), as defined below. The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a β-sheet configuration, connected by the three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, with the ariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies.

Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227, 799-817, 1992; Tramontano et al., J. Mol. Biol, 215, 175-182, 1990).

Despite their high sequence variability, five of the six loops adopt just a small repertoire of main-chain conformations, called “canonical ures”. These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the y to assume l main-chain conformations.

As used herein, the term "CDR" or "complementarity determining region" means the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J.

Biol. Chem. 252, 616, 1977, by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991, by a et al., J. Mol. Biol. 196, 901-917, 1987, and by MacCallum et al., J. Mol. Biol. 262, 732-745, 1996, where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as d by each of the above cited references are set forth for comparison. Preferably, the term “CDR” is a CDR as defined by Kabat based on sequence isons.

Table 1: CDR definitions.

CDR tions Kabat1 Chothia2 MacCallum3 VH CDR1 31-35 26-32 30-35 VH CDR2 50-65 53-55 47-58 VH CDR3 95-102 96-101 93-101 VL CDR1 24-34 26-32 30-36 VL CDR2 50-56 50-52 46-55 VL CDR3 89-97 91-96 89-96 1Residue numbering follows the nomenclature of Kabat et al., supra ue numbering follows the nomenclature of Chothia et al., supra 3Residue numbering follows the nomenclature of MacCallum et al., supra As used herein, the term “framework region” or “FR region” es the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g., using the Kabat definition of CDRs). Therefore, a variable region framework is between about 100- 120 amino acids in length but es only those amino acids outside of the CDRs. For the specific example of a heavy chain variable domain and for the CDRs as d by Kabat et al., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1-30; framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49; framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acids 103 to the end of the le region. The framework regions for the light chain are similarly separated by each of the light claim variable region CDRs. Similarly, using the definition of CDRs by Chothia et al. or um et al. the framework region ries are separated by the respective CDR termini as described above. In preferred embodiments the CDRs are as defined by Kabat.

In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous ces of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three dimensional uration in an aqueous environment. The remainder of the heavy and light variable domains show less inter-molecular variability in amino acid sequence and are termed the ork regions.

The ork regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, these framework regions act to form a ld that provides for oning the six CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen e. The on of CDRs can be readily identified by one of ordinary skill in the art.

As used herein, the term “hinge region” includes the portion of a heavy chain molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately residues and is flexible, thus allowing the two N-terminal antigen binding regions to move independently. Hinge regions can be subdivided into three distinct domains: upper, middle, and lower hinge s (Roux et al., J. Immunol. 161, 4083-4090, 1998). MET antibodies comprising a “fully human” hinge region may contain one of the hinge region sequences shown in Table 2 below.

Table 2: Human hinge sequences.

IgG Upper hinge Middle hinge Lower hinge EPKSCDKTHT APELLGGP CPPCP IgG1 (SEQ ID (SEQ ID (SEQ ID NO:228) NO:227) NO:229) ELKTPLGDTTHT APELLGGP CPRCP (EPKSCDTPPPCPRCP)3 IgG3 (SEQ ID (SEQ ID (SEQ ID NO:231) NO:230) NO:232) ESKYGPP APEFLGGP CPSCP IgG4 (SEQ ID (SEQ ID (SEQ ID NO:234) NO:233) NO:235) ERK APPVAGP IgG42 CCVECPPPCP (SEQ ID (SEQ ID (SEQ ID NO:237) NO:236) ) As used herein the term “CH2 domain” includes the portion of a heavy chain molecule that extends, e.g., from about residue 244 to residue 360 of an antibody using conventional ing schemes (residues 244 to 360, Kabat numbering system; and residues 231- 340, EU numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N- linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It is also well documented that the CH3 domain extends from the CH2 domain to the C-terminal of the IgG molecule and comprises approximately 108 As used herein, the term “fragment” refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. The term en-binding fragment” refers to a ptide nt of an globulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding to hMET and mMET). As used herein, the term “fragment” of an antibody molecule includes antigen-binding fragments of antibodies, for example, an antibody light chain variable domain (VL), an dy heavy chain le domain (VH), a single chain antibody (scFv), a F(ab’)2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, and a single domain antibody fragment (DAb). Fragments can be obtained, e.g., via chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.

As used herein the term “valency” refers to the number of ial target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or specific site on a target molecule. When a polypeptide ses more than one target binding site, each target binding site may specifically bind the same or ent molecules (e.g., may bind to different ligands or different antigens, or different epitopes on the same antigen).

The subject binding molecules have at least one binding site specific for hMET.

As used herein, the term “specificity” refers to the ability to bind (e.g., immunoreact with) a given target, e.g., hMET, mMET. A polypeptide may be monospecific and contain one or more binding sites which specifically bind a target or a polypeptide may be multispecific and contain two or more binding sites which specifically bind the same or different targets.

In one embodiment, an antibody of the invention is specific for more than one target. For example, in one embodiment, a multispecific binding molecule of the invention binds hMET and a second target molecule. In this context, the second target molecule is a molecule other than hMET or mMET.

The term "epitope" refers to the portion(s) of an antigen (e.g. human MET) that contact an antibody. es can be linear, i.e., involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in s s of the antigen that may not necessarily be contiguous. The antibodies provided herein may bind to different (overlapping or non-overlapping) epitopes within the ellular domain of the human MET protein.

As used herein the term “synthetic” with respect to polypeptides includes polypeptides which comprise an amino acid sequence that is not naturally ing. For e, nonnaturally occurring polypeptides which are modified forms of lly occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion) or which comprise a first amino acid sequence (which may or may not be lly occurring) that is linked in a linear sequence of amino acids to a second amino acid sequence (which may or may not be naturally occurring) to which it is not naturally linked in nature.

As used herein the term “engineered” es manipulation of c acid or ptide molecules by synthetic means (e.g. by inant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques).

Preferably, the antibodies of the invention are engineered, including for example, humanized and/or chimeric antibodies, and antibodies which have been engineered to e one or more properties, such as antigen binding, stability/half-life or effector function.

As used herein, the term “modified dy” includes synthetic forms of antibodies which are altered such that they are not naturally ing, e.g., antibodies that se at least two heavy chain portions but not two complete heavy chains (such as, domain deleted antibodies or minibodies); multispecific forms of dies (e.g., bispecific, trispecific, etc.) altered to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules joined to scFv molecules and the like. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019. In addition, the term “modified antibody” includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen). In another embodiment, a modified antibody of the invention is a fusion protein comprising at least one heavy chain portion lacking a CH2 domain and comprising a binding domain of a ptide comprising the binding portion of one member of a receptor ligand pair.

The term “modified antibody” may also be used herein to refer to amino acid sequence variants of a MET dy of the invention. It will be understood by one of ordinary skill in the art that a MET antibody of the invention may be modified to produce a variant MET antibody which varies in amino acid ce in comparison to the MET antibody from which it was derived. For e, nucleotide or amino acid substitutions leading to conservative substitutions or changes at "non-essential" amino acid residues may be made (e.g., in CDR and/or framework residues). Amino acid substitutions can include replacement of one or more amino acids with a naturally occurring or non-natural amino acid.

As used herein, the term “humanising tutions” refers to amino acid substitutions in which the amino acid residue t at a particular position in the VH or VL domain of a MET antibody of the invention (for example a camelid-derived MET antibody) is replaced with an amino acid residue which occurs at an equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be a VH or VL domain encoded by the human germline. Humanising substitutions may be made in the framework s and/or the CDRs of a MET antibody, defined .

As used herein the term “humanised variant” refers to a variant antibody which contains one or more “humanising substitutions” compared to a reference MET dy, wherein a portion of the reference antibody (e.g. the VH domain and/or the VL domain or parts thereof containing at least one CDR) has an amino acid sequence derived from a non- human s, and the ising substitutions” occur within the amino acid sequence derived from a non-human species.

The term “germlined variant” is used herein to refer specifically to “humanised variants” in which the “humanising substitutions” result in replacement of one or more amino acid residues present at a particular position (s) in the VH or VL domain of a MET antibody of the invention (for example a camelid-derived MET antibody) with an amino acid residue which occurs at an lent position in a reference human VH or VL domain encoded by the human ne. It is typical that for any given “germlined variant”, the ement amino acid residues substituted into the germlined variant are taken exclusively, or predominantly, from a single human germline-encoded VH or VL domain. The terms “humanised variant” and “germlined variant” are often used interchangeably herein.

Introduction of one or more ising tutions” into a camelid-derived (e.g. llama derived) VH or VL domain results in production of a “humanised variant” of the camelid (llama)-derived VH or VL domain. If the amino acid residues substituted in are derived predominantly or exclusively from a single human germline-encoded VH or VL domain sequence, then the result may be a “human germlined variant” of the camelid (llama)- derived VH or VL domain.

As used herein, the term “affinity variant” refers to a variant antibody which ts one or more changes in amino acid sequence compared to a reference MET antibody of the invention, wherein the affinity variant exhibits an altered affinity for hMET and/or mMET in ison to the reference antibody. Preferably the affinity variant will exhibit improved affinity for hMET and/or mMET, as compared to the reference MET antibody. The ement may be nt as a lower KD for hMET and/or for mMET, or a slower offrate for hMET and/or for mMET. Affinity variants typically t one or more changes in amino acid sequence in the CDRs, as ed to the reference MET antibody. Such substitutions may result in replacement of the original amino acid present at a given position in the CDRs with a different amino acid residue, which may be a naturally occurring amino acid residue or a non-naturally occurring amino acid residue. The amino acid substitutions may be conservative or non-conservative.

As used herein, antibodies having “high human homology” refers to antibodies comprising a heavy chain variable domain (VH) and a light chain le domain (VL) which, taken together, exhibit at least 90% amino acid sequence identity to the closest matching human germline VH and VL sequences. Antibodies having high human homology may include dies comprising VH and VL domains of native non-human antibodies which exhibit sufficiently high % sequence identity to human germline sequences, including for example antibodies comprising VH and VL domains of d conventional antibodies, as well as engineered, ally humanised or germlined, variants of such antibodies and also “fully human” antibodies.

In one embodiment the VH domain of the antibody with high human homology may exhibit an amino acid sequence identity or sequence homology of 80% or greater with one or more human VH s across the framework regions FR1, FR2, FR3 and FR4. In other embodiments the amino acid sequence identity or sequence homology between the VH domain of the ptide of the invention and the closest matching human germline VH domain sequence may be 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%.

In one embodiment the VH domain of the antibody with high human homology may contain one or more (e.g. 1 to 10) amino acid sequence mis-matches across the framework regions FR1, FR2, FR3 and FR4, in comparison to the closest matched human VH sequence.

In another embodiment the VL domain of the antibody with high human gy may t a sequence identity or sequence homology of 80% or r with one or more human VL domains across the framework regions FR1, FR2, FR3 and FR4. In other ments the amino acid sequence identity or sequence homology between the VL domain of the polypeptide of the invention and the closest matching human germline VL domain sequence may be 85% or greater 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%.

In one ment the VL domain of the antibody with high human homology may contain one or more (e.g. 1 to 10) amino acid sequence mis-matches across the framework regions FR1, FR2, FR3 and FR4, in comparison to the closest matched human VL sequence.

Before ing the percentage sequence identity between the antibody with high human homology and human germline VH and VL, the canonical folds may be determined, which allows the identification of the family of human germline segments with the identical ation of canonical folds for H1 and H2 or L1 and L2 (and L3). Subsequently the human germline family member that has the t degree of sequence homology with the variable region of the antibody of interest is chosen for scoring the sequence homology.

Procedures for determining the closest ng human germline, and determining % ce identity/homology, are well-known to the d . dies with high human homology may comprise hypervariable loops or CDRs having human or human-like canonical fold structures. In one embodiment at least one hypervariable loop or CDR in either the VH domain or the VL domain of the dy with high human homology may be obtained or derived from a VH or VL domain of a nonhuman antibody, for example a conventional antibody from a species of Camelidae, yet exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies. In one embodiment, both H1 and H2 in the VH domain of the dy with high human homology exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.

Antibodies with high human homology may comprise a VH domain in which the hypervariable loops H1 and H2 form a combination of canonical fold structures which is identical to a ation of canonical structures known to occur in at least one human germline VH domain. It has been ed that only certain combinations of canonical fold structures at H1 and H2 actually occur in VH domains encoded by the human germline. In an embodiment H1 and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g. a Camelidae species, yet form a combination of predicted or actual canonical fold structures which is identical to a combination of canonical fold structures known to occur in a human germline or somatically mutated VH domain. In non-limiting embodiments H1 and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g. a Camelidae species, and form one of the following canonical fold combinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1 and 3-5. An antibody with high human homology may contain a VH domain which exhibits both high sequence identity/sequence gy with human VH, and which ns hypervariable loops exhibiting structural homology with human VH.

It may be advantageous for the canonical folds present at H1 and H2 in the VH domain of the antibody with high human homology, and the combination thereof, to be "correct" for the human VH germline sequence which represents the closest match with the VH domain of the antibody with high human homology in terms of l primary amino acid sequence identity. By way of example, if the closest sequence match is with a human ne VH3 domain, then it may be advantageous for H1 and H2 to form a combination of canonical folds which also occurs naturally in a human VH3 domain. This may be particularly important in the case of antibodies with high human homology which are derived from non- human species, e.g. antibodies ning VH and VL domains which are derived from camelid conventional antibodies, especially antibodies containing humanised camelid VH and VL s.

Thus, in one embodiment the VH domain of the MET antibody with high human homology may exhibit a sequence identity or sequence gy of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VH domain across the framework s FR1, FR2 , FR3 and FR4, and in addition H1 and H2 in the same dy are obtained from a non-human VH domain (e.g. derived from a Camelidae species, preferably llama), but form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VH domain.

In other embodiments, L1 and L2 in the VL domain of the antibody with high human homology are each ed from a VL domain of a non-human species (e.g. a camelid- derived VL ), and each exhibits a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.

L1 and L2 in the VL domain of an antibody with high human gy may form a combination of predicted or actual canonical fold structures which is identical to a combination of cal fold structures known to occur in a human germline VL domain.

In non-limiting embodiments L1 and L2 in the a domain of an antibody with high human homology (e.g. an antibody containing a camelid-derived VL domain or a sed variant thereof) may form one of the following canonical fold combinations: 11- 7, 13-7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (as defined in Williams et al., J. Mol.

Biol. 264, 220-232, 1996, and as shown on http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVL.html). In non-limiting embodiments L1 and L2 in the Vkappa domain may form one of the following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (as defined in Tomlinson et al., EMBO J. 14, 4628- 4638, 1995 and as shown on http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVK.html). In a further embodiment, all three of L1, L2 and L3 in the VL domain of an antibody with high human

Claims (39)

CLAIMS:

1. An antibody, or an antigen binding fragment thereof, which binds human MET protein (hMET) with high affinity and binds mouse MET protein (mMET) with high affinity, 5 wherein the antibody or an antigen binding fragment thereof is a hMET agonist and a mMET agonist.

2. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof meets one or more of the following conditions: 10 - it comprises at least one heavy chain variable domain (VH) and at least one light chain variable domain (VL), wherein said VH and VL domain, when tested as a Fab fragment, exhibit an off-rate (koff measured by Biacore) for hMET in the range of from 1 x 10-3 s-1 to 1 x 10-2 s-1, optionally 1 x 10-3 s-1 to 6 x 10-3 s-1 , and exhibit an off-rate (koff measured by Biacore) for mMET in the range of from 1 x 10-3 s-1 to 1 x 10-2 s-1, optionally 1 x 10-3 s-1 to 6 x 10-3 s-1 15 ; - it has equivalent affinity for hMET and mMET; - it induces phosphorylation of hMET and induces phosphorylation of mMET; - it induces phosphorylation of hMET with an EC50 (as measured by phosphoMET ELISA) of less than 3.0 nM, optionally less than 2.0 nM and induces phosphorylation 20 of mMET with an EC50 (as measured by phospho-MET ELISA) of less than 3.0 nM, optionally less than 2.0 nM; - it induces phosphorylation of hMET and mMET equivalently; - it exhibits high phosphorylation potency for hMET and exhibits high phosphorylation potency for mMET; 25 - it induces phosphorylation of hMET with an EC50 of less than 1nM and/or an Emax (as a percentage of HGF-induced activation in a phospho-MET ELISA) of at least 80%.and induces phosphorylation of mMET with an EC50 of less than 1nM and/or an Emax (as a percentage of HGF-induced activation in a phospho-MET ELISA) of at least 80%; - it exhibits low phosphorylation potency for hMET and exhibits low 30 phosphorylation potency for mMET; - it induces phosphorylation of hMET with EC50 of 1nM-5nM and/or an Emax (as a percentage of HGF-induced activation in a phospho-MET ELISA) of 60-80% and induces phosphorylation of mMET with EC50 of 1nM-5nM and/or an Emax (as a percentage of HGFinduced activation in a phospho-MET ELISA) of 60-80%;

3. The antibody or antigen binding fragment thereof of claim 1 or 2, wherein the antibody or antigen binding fragment thereof induces an HGF-like cellular response when contacted with a human cell and induces an HGF-like cellular response when contacted with a mouse cell. 5

4. The antibody or antigen binding fragment thereof of claim 3, wherein the antibody or antigen binding fragment thereof fully induces an HGF-like cellular response when contacted with a human cell and when contacted with a mouse cell, or partially induces an HGF-like cellular response when contacted with a human cell and when contacted with a 10 mouse cell.

5. The antibody or antigen binding fragment thereof of claim 4, wherein: - full induction of HGF-like cellular response is measurable as one, any two, or all of: (i) in a cell scattering assay, the antibody or antigen binding fragment thereof 15 induces cell scattering comparable to maximal HGF-induced scattering when the antibody or antigen binding fragment thereof is at a concentration of 0.1-1.0 nM; (ii) in an anti-apoptotic cell assay, the antibody or antigen binding fragment thereof exhibits an EC50 of less than 1.1x that of HGF, and/or with an Emax (measured as a % of total ATP content of non-apoptotic control cells) of greater than 90% that observed for 20 HGF; and/or (iii) in a branching morphogenesis assay, cells treated with the antibody exhibit greater than 90% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF; and - partial induction of an HGF-like cellular response is measurable as: 25 (i) in a cell scattering assay, the antibody or antigen binding fragment thereof induces cell scattering of at least 25% that induced by 0.1 nM homologous HGF when the antibody concentration is 1 nM or lower; (ii) in anti-apoptotic cell assay, the antibody or antigen binding fragment thereof exhibits an EC50 no more than 7.0x that of HGF and/or an Emax cellular viability of at least 30 50% that observed for HGF; and/or (iii) in a branching morphogenesis assay, cells treated with the antibody exhibit at least 25% the number of branches per spheroid induced by the same (non-zero) concentration of HGF; and the antibody or antigen binding fragment does not fully induce an HGF-like cellular 35 response.

6. The antibody or antigen binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen binding fragment thereof is a HGF competitor.

7. The antibody or antigen binding fragment thereof of claim 6, wherein the antibody or 5 antigen binding fragment thereof meets one or more of the following conditions: - it competes with hHGF binding to hMET with an IC50 of no more than 5nM and/or an Imax of at least 50% and competes with mHGF binding to mMET with an IC50 of no more than 5nM and/or an Imax of at least 50%; - it competes with hHGF and mHGF equivalently; 10 - it is a full HGF competitor; - it is a full HGF competitor which competes with hHGF with an IC50 of less than 2 nM and/or an Imax of greater than 90% and competes with mHGF with an IC50 of less than 2 nM and/or an Imax of greater than 90%; - it is a partial HGF competitor; 15 - it is a partial HGF competitor which competes with hHGF with an IC50 of 2-5nM and/or an Imax of 50%-90% and competes with mHGF with an IC50 of 2-5nM and/or an Imax of 50%-90%.

8. An antibody or antigen binding fragment thereof which recognizes an epitope of 20 human MET located in a region from amino acid residue 123 to residue223 of human MET, from amino acid residue 224 to residue311 of human MET, from residue 314to residue 372 of human MET or from residue 546 to residue562 of human MET.

9. The antibody or antigen binding fragment thereof of claim 8 which: 25 - recognizes an epitope in the extracellular domain of MET that comprises one or more amino acids conserved across human and mouse MET; - recognizes an epitope of human MET comprising the amino acid residue Ile367 and/or Asp372 of human MET, optionally wherein the epitope is located in the region from amino acid residue 314 to residue 372 of human MET; or 30 - recognizes an epitope of human MET comprising the amino acid residue Thr555 of human MET optionally wherein the epitope is located in the region from amino acid residue 546 to residue 562 of human MET.

10. The antibody according to any one of claims 8-9 that is an antibody or antigen 35 binding fragment according to any one of claims 1-7.

11. [71G2] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: 5 H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:44, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:46, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:48, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:121, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:123, and 10 L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:125.

12. [71G2] The antibody or antigen binding fragment according to claim 11, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:167, or a sequence at least 90% identical thereto, and the light chain variable domain comprises the 15 amino acid sequence of SEQ ID NO:168, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

13.[ 71G3] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable 20 domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:9, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:11, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:13, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:86, 25 L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:88, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:90.

14. [71G3] The antibody or antigen binding fragment according to claim 13, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:157, or a 30 sequence at least 90% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:158, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

15. [76H10] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:2, 5 H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:4, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:6, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:79, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:81, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:83. 10

16. [76H10] The antibody or antigen binding fragment according to claim 15, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:155, or a sequence at least 90% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:156, or a sequence at least 90% identical thereto, 15 or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

17. [71C3] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: 20 H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:16, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:18, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:20, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:93, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:95, and 25 L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:97.

18. [71C3] The antibody or antigen binding fragment according to claim 16, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:159, or a sequence at least 90% identical thereto, and the light chain variable domain comprises the 30 amino acid sequence of SEQ ID NO:160, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

19. [71D4] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable 35 domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:23, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:25, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:27, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:100, 5 L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:102, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:104.

20. [71D4] The antibody or antigen binding fragment according to claim 19, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:161, or a 10 sequence at least 90% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:162, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

21. [71A3] An antibody or antigen binding fragment which comprises a heavy chain 15 variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:37, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:39, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:41, 20 L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:114, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:116, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:118.

22. [71A3] The antibody or antigen binding fragment according to claim 21, wherein the 25 heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:165, or a sequence at least 90% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:166, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7. 30

23. [71G7] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:51, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:53, 35 H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:55, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:128, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:130, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:132. 5

24. [71G7] The antibody or antigen binding fragment according to claim 23, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:169, or a sequence at least 90% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:170, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7. 10

25. [71G12] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:58, 15 H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:60, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:62, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:135, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:137, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:139. 20

26. [71G12] The antibody or antigen binding fragment according to claim 25, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:171, or a sequence at least 90% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:172, or a sequence at least 90% identical thereto, 25 or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

27. [74C8] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: 30 H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:65, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:67, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:69, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:142, L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:144, and 35 L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:166.

28. [74C8] The antibody or antigen binding fragment according to claim 27, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:173, or a sequence at least 90% identical thereto, and the light chain variable domain comprises the 5 amino acid sequence of SEQ ID NO:174, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

29. [72F8] An antibody or antigen binding fragment which comprises a heavy chain variable domain comprising H-CDR1, H-CDR2 and H-CDR3, and a light chain variable 10 domain comprising L-CDR1, L-CDR2 and L-CDR3, wherein: H-CDR1 comprises the amino acid sequence shown as SEQ ID NO:72, H-CDR2 comprises the amino acid sequence shown as SEQ ID NO:74, H-CDR3 comprises the amino acid sequence shown as SEQ ID NO:76, L-CDR1 comprises the amino acid sequence shown as SEQ ID NO:149, 15 L-CDR2 comprises the amino acid sequence shown as SEQ ID NO:151, and L-CDR3 comprises the amino acid sequence shown as SEQ ID NO:153.

30. [72F8] The antibody or antigen binding fragment according to claim 29, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:175, or a 20 sequence at least 90% identical thereto, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:176, or a sequence at least 90% identical thereto, or wherein the antibody or antigen binding fragment is according to any one of claims 1-7.

31. An affinity variant or human germlined variant of an antibody or antigen binding 25 fragment according to any of claims 1-30.

32. The antibody or antigen binding fragment of any one of claims 1-31, which meets one of more of the following conditions: - it contains the hinge region, CH2 domain and/or CH3 domain of a human 30 IgG, optionally IgG1; - it has a sequence at least 90%, 95%, 97% or 99% identical to a human IgG, preferably IgG1, optionally wherein the CH2 and/or CH3 domain has been modified in order to reduce or substantially eliminate one or more antibody effector functions; - its VH and/or VL domains or one or more of the CDRs are derived from an animal of the Camelidae family;- its VH and/or VL domains or one or more of the CDRs are derived from an animal of the Camelidae family wherein the animal is a llama. 5

33. An isolated polynucleotide which encodes the antibody or antigen binding fragment of any of claims 1-32.

34. An expression vector comprising the polynucleotide of claim 33 operably linked to regulatory sequences which permit expression of the antibody or antigen binding fragment 10 thereof in a host cell or cell-free expression system.

35. A host cell or cell-free expression system containing the expression vector of claim 34. 15

36. A method of producing a recombinant antibody or antigen binding fragment thereof which comprises culturing the host cell or cell free expression system of claim 35 under conditions which permit expression of the antibody or antigen binding fragment and recovering the expressed antibody or antigen binding fragment. 20

37. A pharmaceutical composition comprising an antibody or antigen binding fragment according to any of claims 1 to 32 and at least one pharmaceutically acceptable carrier or excipient.

38. An antibody or antigen binding fragment according to any one of claims 1-32, or the 25 pharmaceutical composition of claim 37, in the preparation of a medicament for use in therapy.

39. The antibody or antigen binding fragment according to any one of claims 1-32, or the pharmaceutical composition according to claim 37, in the preparation of a medicament 30 for: - treating or preventing liver damage in a human patient, optionally acute liver damage or chronic liver damage; - treating or preventing kidney damage in a human patient, optionally acute kidney damage; - treating or preventing inflammatory bowel disease in a human patient, optionally ulcerative colitis; - treating or preventing diabetes in a human patient, optionally type I or type II diabetes; 5 - treating or preventing non-alcoholic steatohepatitis in a human patient; or - treating or promoting wound healing in a human patient, optionally a patient having diabetes