Anti-MET antibodies and their uses

Abstract

This invention relates to agonist anti-MET antibodies and their use in the therapeutic treatment of diseases. [Solution] This antibody binds with high affinity to the human and mouse hepatocyte growth factor (HGF) receptor (also known as MET), and is an agonist for both human and mouse MET, producing molecular and cellular effects similar to those of HGF binding.

Description

[Technical Field]

[0001] This invention relates to an antibody and antigen-binding fragment that bind with high affinity to the human and mouse hepatocyte growth factor (HGF) receptor (also known as MET). This antibody and antigen-binding fragment are agonists of both human and mouse MET, producing molecular and cellular effects similar to those of HGF binding. The invention further relates to the therapeutic use of this MET-agonist antibody and antigen-binding fragment. [Background technology]

[0002] HGF is a mesenchymal-derived, multifunctional cytokine that mediates a characteristic range of biological functions, including cell proliferation, motility, differentiation, and survival. HGF receptors (also known as METs) are expressed in a wide variety of tissues, including all epidermal, endothelial, muscle, nerve, osteoblast, hematopoietic, and various components of the immune system.

[0003] HGF and MET signaling plays a crucial role in embryonic development, guiding the migration of progenitor cells and determining cell survival and death. In adults, HGF / MET signaling is usually dormant but is reactivated during wound healing and tissue regeneration. Some cancers and tumors hijack HGF / MET signaling to promote their survival and proliferation within the host organism. Therefore, inhibiting the HGF-MET axis has attracted attention as a target for anti-cancer therapy, although success has been limited.

[0004] Recombinant HGF has been studied as a potential treatment for numerous diseases (including degenerative diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, and transplant-related disorders) due to its role in tissue healing and regeneration. However, recombinant HGF has unsatisfactory pharmacological properties. Specifically, it requires activation by proteolysis to become biologically active; once activated, it has an extremely short half-life in the body; and industrial production is complex and expensive.

[0005] Various agonist anti-MET antibodies that activate MET by mimicking the mechanism of HGF have been proposed as alternatives.

[0006] The following antibodies have been reported that at least partially mimic HGF activity: (i) 3D6 mouse anti-human MET antibody (United States Patent No. 6,099,841); (ii) 5D5 mouse anti-human MET antibody (United States Patent No. 5,686,292); (iii) NO-23 mouse anti-human MET antibody (United States Patent No. 7,556,804 B2); (iv) B7 human naive anti-human MET antibody (United States Patent Application Publication No. 2014 / 0193431 A1); (v) DO-24 mouse anti-human MET antibody (Prat et al., Mol Cell Biol. Vol. 11, pp. 5954-5962, 1991; Prat et al., J Cell Sci. Vol. 111, pp. 237-247, 1998); (vi) DN-30 mouse anti-human MET antibody (Prat et al., Mol Cell Biol. Volume 11, pp. 5954-5962, 1991; Prat et al., J Cell Sci. Volume 111, pp. 237-247, 1998). [Overview of the project]

[0007] For example, the agonist anti-MET antibodies currently being produced, as described in the background technology section, are often obtained as byproducts of processes aimed at identifying antagonist molecules and are not explicitly designed to be therapeutic agonists. Furthermore, the most obvious limitation of prior art anti-MET antibodies is that they have been produced using mouse systems (with the exception of B7, which was identified using a human naive phage library). As a result, these antibodies are unlikely to cross-react with mouse MET. Even if small cross-reactivity to autoantigens is possible in principle, the affinity of that interaction is usually very low.

[0008] While the lack of cross-reactivity is not a problem in mouse models of cancer (because human xenografts are used), antibody cross-reactivity between human METs and mouse METs is a crucial requirement for regenerative medicine or preclinical mouse models of non-tumor human diseases, where the antibodies must function against mouse tissues or cells.

[0009] To evaluate agonist anti-MET antibodies in preclinical models, it is desirable that the antibody not only cross-reacts with mouse MET, but also binds to mouse MET with the same or similar affinity as it does to human MET, and that the antibody induces the same or similar effect in mouse strains as it does in human strains. Otherwise, experiments conducted in preclinical models cannot predict the situation in humans. As shown in the examples, none of the prior art anti-MET agonist antibodies show affinity for mouse MET, and therefore none of the prior art antibodies show the same or equivalent binding and agonist effect in both mouse and human strains.

[0010] This application provides an anti-MET agonist antibody designed and manufactured to bind with high affinity to both human MET and mouse MET. This antibody (i) exhibits agonist activity in human and mouse MET biological systems, that is, it induces MET signaling, and in some cases induces MET signaling with efficacy equivalent to or greater than that of HGF; (ii) comprehensively extracts the biological activity induced by HGF, making it an effective substitute for recombinant HGF; (iii) exhibits superior binding to mouse MET compared directly with antibodies from prior art; (iv) (v) Exhibits biologically important agonist activity at low concentrations of pM; (v) Shows a plasma half-life of several days in mice, reaching pharmacological saturation concentration at a very low dose of 1 μg / kg for a therapeutic antibody; (vi) Maintains renal function and kidney health in a mouse model of acute kidney injury; (vii) Prevents liver failure and antagonizes hepatocyte damage in a mouse model of acute liver injury; (viii) Exhibits anti-fibrotic, anti-inflammatory, and regenerative activity in a mouse model of chronic liver injury; (ix) In mouse models of ulcerative colitis and inflammatory bowel disease (x) In mouse models of the disease, it prevents weight loss, reduces intestinal bleeding, maintains colon health, suppresses inflammation, and promotes epidermal regeneration; (x) In mouse models of type 1 diabetes, it promotes glucose uptake independently of insulin; (xi) In mouse models of type 2 diabetes, it overcomes insulin resistance; (xii) In mouse models of non-alcoholic steatohepatitis (NASH), it improves fatty liver, suppresses fibrosis, and restores liver function; (xiii) In mouse models of diabetic ulcers, it accelerates wound healing; (xiv) It cross-reacts with rat (Rattus norvegicus) MET and cynomolgus monkey (Macaca fascicularis) MET, making it possible to conduct necessary toxicological and pharmacological studies in these two vertebrate species before conducting the first clinical trials in humans; (xv) It recognizes conserved epitopes between humans, mice, rats, and cynomolgus monkeys, thus increasing its usefulness in a variety of animal models.

[0011] Thus, in a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that binds to human MET protein (hMET) with high affinity and also binds to mouse MET protein (mMET) with high affinity. This antibody or its antigen-binding fragment is an hMET agonist and an mMET agonist. In some embodiments, this antibody or its antigen-binding fragment comprises at least one heavy chain variable domain (VH) and at least one light chain variable domain (VL), and when tested as a Fab fragment, the VH domain and the VL domain have an off-rate (k -3 measured by Biacore) in the range of 1×10 -2 / sec to 1×10 -3 / sec, and in some cases in the range of 1×10 -3 / sec to 6×10 off / sec, with respect to hMET, and an off-rate in the range of 1×10 -3 / sec to 1×10 -2 / sec, and in some cases in the range of 1×10 -3 / sec to 6×10 -3 / sec, with respect to mMET. In some embodiments, this antibody or its antigen-binding fragment has equivalent affinity for hMET and mMET.

[0012] In some embodiments, this antibody or its antigen-binding fragment induces phosphorylation of hMET and also induces phosphorylation of mMET. In some embodiments, this antibody or its antigen-binding fragment induces phosphorylation of hMET at an EC 50 of less than 3.0 nM, and in some cases less than 2.0 nM (measured by phospho-MET ELISA), and induces phosphorylation of mMET at an EC 50 of less than 3.0 nM, and in some cases less than 2.0 nM (measured by phospho-MET ELISA). In some embodiments, this antibody or its antigen-binding fragment induces phosphorylation of hMET and mMET equivalently.

[0013] In some embodiments, this antibody or its antigen-binding fragment exhibits high phosphorylation efficacy against hMET and also exhibits high phosphorylation efficacy against mMET. In some embodiments, this antibody or its antigen-binding fragment phosphorylates hMET to less than 1 nM EC 50 and / or at least 80% E (as the percentage of activation induced by HGC in phospho-MET ELISA) max Inducing this, as well as phosphorylation of mMET at EC levels less than 1 nM 50 and / or at least 80% E (as the percentage of activation induced by HGC in phospho-MET ELISA) max This induces phosphorylation. In some alternative embodiments, this antibody or its antigen-binding fragment exhibits low phosphorylation efficacy against hMET and low phosphorylation efficacy against mMET. In some such embodiments, this antibody or its antigen-binding fragment induces phosphorylation of hMET at 1 nM to 5 nM EC 50 and / or at least 60-80% E (as the percentage of HGC-induced activation in phospho-MET ELISA) max This induces phosphorylation of mMET with 1 nM to 5 nM EC2. 50 and / or at least 60-80% E (as the percentage of HGC-induced activation in phospho-MET ELISA) max Guide them with this.

[0014] In some embodiments, this antibody or its antigen-binding fragment induces an HGF-like cellular response when in contact with human cells and when in contact with mouse cells. In some embodiments, this antibody or its antigen-binding fragment fully induces an HGF-like cellular response when in contact with human cells and when in contact with mouse cells. In some embodiments, the measurement of the full induction of the HGF-like cellular response is (i) In a cell scattering assay, this antibody or its antigen-binding fragment induces cell scattering at a concentration of 0.1–1.0 nM that is comparable to the maximum scattering induced by HGF; (ii) In an anti-apoptotic cell assay, the antibody or its antigen-binding fragment is less than 1.1 times the value of HGF in the EC 50 and / or E (measured as a percentage of the total ATP content of non-apoptotic control cells) greater than 90% of the value observed in HGF max To demonstrate; (iii) In a branching morphogenesis assay, cells treated with this antibody exhibit more than 90% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF. It is possible to use one of them, any two of them, or all of them.

[0015] In some embodiments, this antibody or its antigen-binding fragment partially induces an HGF-like cellular response when in contact with human cells and when in contact with mouse cells. In some embodiments, the partial induction of the HGF-like cellular response is measured as follows: (i) In a cell scattering assay, the antibody or its antigen-binding fragment induces at least 25% of the cell scattering induced by 0.1 nM homologous HGF at a concentration of 1 nM or less; and / or (ii) In an anti-apoptotic cell assay, the antibody or its antigen-binding fragment is 7.0 times or less the value of HGF in EC 50 and / or E at least 50% of the value observed in HGF max To indicate cell viability, and / or; (iii) In a branching morphogenesis assay, cells treated with this antibody exhibit at least 25% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF. It is possible as such.

[0016] In some embodiments, this antibody or its antigen-binding fragment is an HGF competitor. In some embodiments, this antibody or its antigen-binding fragment has an IC50 or less binding to hMET. 50 and / or at least 50% max It competes with hHGF and has an IC of 5 nM or less for coupling to mMET. 50 and / or at least 50% max It competes with mHGF. In some embodiments, this antibody or its antigen-binding fragment competes equally with hMET and mMET. In some embodiments, this antibody or its antigen-binding fragment is a total HGF competitor. In some such embodiments, this antibody or its antigen-binding fragment binds to hMET with an IC50 of less than 2 nM. 50 and / or more than 90% max It competes with hHGF and has an IC of less than 2 nM for binding to mMET. 50 and / or more than 90% max It competes with mHGF. In some embodiments, this antibody or its antigen-binding fragment is a partial HGF competitor. In some such embodiments, this antibody or its antigen-binding fragment binds to hMET with an IC50 of 2 nM to 5 nM. 50 and / or 50% to 90% max It competes with hHGF and has an IC of 2 nM to 5 nM for binding to mMET. 50 and / or 50% to 90% max Therefore, it competes with mHGF.

[0017] The antibody or antigen-binding fragment of the present invention can exhibit cross-reactivity with METs of monkey origin (e.g., cynomolgus monkey (Macaca cynomologus)) and also with METs of rat origin (Rattus norvegicus).

[0018] The antibody or antigen-binding fragment of the present invention can bind to the epitopes of human MET, specifically amino acid residues 123-223 (throughout this document, the numbering of human MET refers to GenBank sequence # X54559). The antibody or antigen-binding fragment of the present invention is also provided, which can bind to the epitopes of human MET, specifically amino acid residues 224-311. The antibody or antigen-binding fragment of the present invention is also provided, which can bind to the epitopes of human MET, specifically amino acid residues 314-372. The antibody or antigen-binding fragment of the present invention is also provided, which can bind to the epitopes of human MET, specifically amino acid residues 546-562.

[0019] The antibody or antigen-binding fragment of the present invention is also provided, which can bind to an epitope of human MET containing the amino acid residue Ile367. The antibody or antigen-binding fragment of the present invention is also provided, which can bind to an epitope of human MET containing the amino acid residue Asp372. In some embodiments, this antibody or antigen-binding fragment binds to an epitope of human MET containing the amino acid residues Ile367 and Asp372.

[0020] The present invention also provides an antibody or antigen-binding fragment thereof that can bind to an epitope of human MET containing the amino acid residue Thr555 of human MET.

[0021] The present invention further includes 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, among which H-CDR1 contains amino acid sequences selected from SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 58, 65, and 72; H-CDR2 contains amino acid sequences selected from SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, and 74; H-CDR3 contains amino acid sequences selected from SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, and 76; L-CDR1 contains amino acid sequences selected from sequence numbers 79, 86, 93, 100, 107, 114, 121, 128, 135, 142, and 149; L-CDR2 contains amino acid sequences selected from sequence numbers 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, and 151; L-CDR3 provides an antibody or antigen-binding fragment containing an amino acid sequence selected from SEQ ID NOs: 83, 90, 97, 104, 111, 118, 125, 132, 139, 146, and 153.

[0022] [71G2] In one embodiment, the present invention includes 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, among which H-CDR1 contains the amino acid sequence shown as SEQ ID NO: 44; H-CDR2 contains the amino acid sequence shown as SEQ ID NO: 46; H-CDR3 contains the amino acid sequence shown as SEQ ID NO: 48; L-CDR1 contains the amino acid sequence shown as Sequence ID No. 121; L-CDR2 contains the amino acid sequence shown as Sequence ID No. 123; L-CDR3 provides an antibody or its antigen-binding fragment containing the amino acid sequence shown as Sequence ID No. 125.

[0023] [71G2] In some such embodiments, the heavy chain variable domain of the antibody or fragment comprises the amino acid sequence of SEQ ID NO: 167 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 168 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence.

[0024] [71D6] In another embodiment, the present invention includes 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, among which H-CDR1 contains the amino acid sequence shown as SEQ ID NO: 30; H-CDR2 contains the amino acid sequence shown as SEQ ID NO: 32; H-CDR3 contains the amino acid sequence shown as SEQ ID NO: 34; L-CDR1 contains the amino acid sequence shown as Sequence ID No. 107; L-CDR2 contains the amino acid sequence shown as Sequence ID No. 109; L-CDR3 provides an antibody or antigen-binding fragment containing the amino acid sequence shown as Sequence ID No. 111.

[0025] [71D6] In some such embodiments, the heavy chain variable domain of the antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 163 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 164 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence.

[0026] [71G3]In yet another embodiment, the present invention includes 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, among which H-CDR1 contains the amino acid sequence shown as SEQ ID NO: 9; H-CDR2 contains the amino acid sequence shown as SEQ ID NO: 11; H-CDR3 contains the amino acid sequence shown as SEQ ID NO: 13; L-CDR1 contains the amino acid sequence shown as SEQ ID NO: 86; L-CDR2 contains the amino acid sequence shown as Sequence ID No. 88; L-CDR3 provides an antibody or antigen-binding fragment containing the amino acid sequence shown as SEQ ID NO: 90.

[0027] [71G3] In some such embodiments, the heavy chain variable domain of the antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 157 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 158 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence.

[0028] In yet another embodiment, the present invention provides an antibody or antigen-binding fragment comprising a heavy chain variable domain containing H-CDR1, H-CDR2, and H-CDR3, and a light chain variable domain containing L-CDR1, L-CDR2, and L-CDR3, wherein H-CDR1, H-CDR2, and H-CDR3 are selected from a group of CDRs (CDR1, CDR2, and CDR3) for the 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.

[0029] In some embodiments, the heavy chain variable domain of the antibody or antigen-binding fragment contains a VH amino acid sequence from Table 5, or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence, and the light chain variable domain contains a corresponding VL amino acid sequence from Table 5, or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence.

[0030] However, in embodiments where the amino acid sequence of the VH domain shows less than 100% sequence matching with a given VH domain amino acid sequence (e.g., sequence number x), the framework region may contain the same heavy chain CDRs as HCDR1, HCDR2, and HCDR3 of the VH domain in sequence number x, while the amino acid sequence may be altered. For example, one or more amino acids in the framework region may be substituted with amino acids at equivalent positions in the human VH domain encoded by the human germline. Similarly, in embodiments where the amino acid sequence of the VL domain shows less than 100% sequence matching with a given VL domain amino acid sequence (e.g., sequence number y), the framework region may contain light chain CDRs that match LCDR1, LCDR2, and LCDR3 of the VL domain in sequence number y, while the amino acid sequence within the framework region may be altered. For example, one or more amino acids in the framework region may be substituted with amino acids at equivalent positions in the human VL domain encoded by the human germline.

[0031] The present invention also provides antibodies and antigen-binding fragments comprising humanized / germ-derived variants of the VH and VL domains of the above-mentioned antibody, as well as affinity variants and variants containing conserved amino acid substitutions as defined herein. In particular, chimeric antibodies are provided that contain the VH and VL domains of the above-mentioned llama-derived Fab, or its human germ-derived variant, and are fused to the constant domain of a human antibody (especially human IgG1, IgG2, IgG3, IgG4). The heavy-chain variable domain and light-chain variable domain of the above-mentioned antibody, or its germ-derived variant, or its affinity variant, or its conserved variant, can be incorporated into conventional quadruple-chain antibodies or other antigen-binding proteins (e.g., Fab, Fab', F(ab')2, bispecific Fab, Fv fragment, dimeric antibody, linear antibody, single-chain antibody molecule, single-chain variable fragment (scFv), multispecific antibody). The heavy-chain variable domain, or its germ-derived variant, or its affinity variant, or its conserved variant, can also be used as a single-domain antibody.

[0032] In another aspect, the present invention also provides isolated polynucleotides encoding the antibody or antigen-binding fragment of the present invention, an expression vector comprising the polynucleotide functionally linked to a regulatory sequence enabling the expression of the antibody or antigen-binding fragment in a host cell or a cell-free expression system, and a host cell or cell-free expression system containing the expression vector. The present invention further provides a method for producing a recombinant antibody or its antigen-binding fragment, comprising culturing a host cell or cell-free expression system under conditions that enable the expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

[0033] In yet another aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention and at least one pharmaceutically acceptable base or excipient.

[0034] In yet another aspect, the present invention provides an antibody or antigen-binding fragment of the present invention, or a pharmaceutical composition of the present invention, for use in therapeutic purposes.

[0035] In yet another aspect, the present invention provides a method for treating or preventing liver injury (possibly acute or chronic liver injury) in human patients, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need. In some embodiments, the MET agonist antibody is the antibody or antigen-binding fragment of the present invention.

[0036] In yet another aspect, the present invention provides a method for treating or preventing renal impairment (in some cases acute or chronic renal impairment) in human patients, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need. In some embodiments, the MET agonist antibody is the antibody or antigen-binding fragment of the present invention.

[0037] In yet another aspect, the present invention provides a method for treating or preventing inflammatory bowel disease (and possibly ulcerative colitis) in human patients, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need. In some embodiments, the MET agonist antibody is the antibody or antigen-binding fragment of the present invention.

[0038] In yet another aspect, the present invention provides a method for treating or preventing diabetes (in some cases type 1 or type 2 diabetes) in human patients, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need. In some embodiments, the MET agonist antibody is the antibody or antigen-binding fragment of the present invention.

[0039] In yet another aspect, the present invention provides a method for treating or preventing non-alcoholic steatohepatitis in human patients, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need. In some embodiments, the MET agonist antibody is the antibody or antigen-binding fragment of the present invention.

[0040] In yet another aspect, the present invention provides a method for treating or promoting wound healing in a human patient (possibly a patient with diabetes) that includes administering a therapeutically effective amount of a MET agonist antibody to a patient in need. In some embodiments, the MET agonist antibody is the antibody or antigen-binding fragment of the present invention. [Brief explanation of the drawing]

[0041] [Figure 1]The immune response of llamas immunized with human MET-Fc was investigated by ELISA. Human MET ECD (hMET) recombinant protein or mouse MET ECD (mMET) recombinant protein was immobilized on a solid phase and exposed to serial dilutions of serum from llamas either before (PRE) or after (POST) immunization. Binding was elucidated using mouse anti-llama IgG1 and HRP-labeled donkey anti-mouse antibody. OD: optical density; AU: arbitrary units. [Figure 2] Schematic diagram of a human MET deletion mutant used to identify MET domains important for mAb binding. ECD, extracellular domain; aa, amino acid; L. peptide, leader peptide; SEMA, semaphorin homologous domain; PSI or P, plexin-semaphorin-integrin homologous domain; IPT, immunoglobulin-transcription factor-plexin homologous domain. On the right, the corresponding residues of human MET are shown according to UniProtKB # P08581. [Figure 3] Schematic diagram of the llama-human chimeric MET protein used for detailed mapping of epitopes recognized by anti-MET antibodies. The extracellular portions of llama MET and human MET consist of 931 and 932 amino acids (aa), respectively (llama MET has a leader peptide that is two amino acids shorter, but has one insertion after amino acid 163). The receptor internal domain of both proteins contains 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). Chimeric CH1-CH5 have a C-terminal human portion after the N-terminal llama portion. Chimeric CH6-CH7 have a C-terminal llama portion after the N-terminal human portion. [Figure 4]The agonist activity of human / mouse equivalent anti-MET antibodies in human and mouse cells was measured by Western blotting. Human lung cancer cells (A549) and mouse liver progenitor cells (MLP29) were serum-starved and then stimulated with increasing concentrations of mAbs or recombinant human HGF (hHGF; A549) or mouse HGF (mHGF; MLP29). MET autophosphorylation was examined by Western blotting using anti-phospho-MET antibodies (tyrosine 1234-1235). The same cell lysates were also analyzed by Western blotting using anti-whole-human MET antibodies (A549) or anti-whole-human MET antibodies (MLP29). [Figure 5] The bioactivity of human / mouse equivalent anti-MET antibodies was measured using LOC human kidney epidermal cells and MLP29 mouse liver progenitor cells via a branching morphogenesis assay. Cell spheroids were seeded in a collagen layer and then exposed to increasing concentrations of mAb or recombinant human HGF (LOC) or mouse HGF (MLP29). The time course of branching morphogenesis was observed under a microscope, and colonies were photographed after 5 days. [Figure 6] Comparison with prior art antibodies: Human-mouse cross-reactivity. Human MET ECD and mouse MET ECD were immobilized in a solid phase and exposed to increasing concentrations of antibodies in solution (all in mouse IgG / λ format). Binding was determined by ELISA using HRP-labeled anti-mouse Fc antibodies. [Figure 7] Comparison with prior art antibodies: MET autophosphorylation. Human lung cancer cells (A549) and MLP29 mouse liver progenitor cells were deprived of serum growth factor for 48 hours, and then stimulated with increasing antibody concentrations. After 15 minutes of stimulation, the cells were lysed, and phospho-MET levels were determined by ELISA using an anti-MET antibody for capture and an anti-phospho-tyrosine antibody for proof. [Figure 8] Comparison with prior art antibodies: Branching morphogenesis. LOC human kidney epidermal cell spheroids were seeded in a collagen layer and incubated with increasing concentrations of mAbs. The time course of branching morphogenesis was tracked by microscopy, and colonies were photographed after 5 days. [Figure 9]Comparison with prior art antibodies: Branching morphogenesis. MLP29 mouse liver progenitor cell spheroids were seeded in a collagen layer and incubated with increasing concentrations of mAbs. The time course of branching morphogenesis was tracked 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 was injected intraperitoneally, and blood samples were collected from the tail vein 3, 6, 12, and 24 hours after injection. Blood samples were processed, and the concentration of antibody in plasma was determined by ELISA. (A) Peak and trough levels of the injected antibody. (B) The plasma half-life of the antibody was calculated by linear fitting of logarithmically transformed antibody concentrations. [Figure 11] Acute liver failure model: Plasma concentrations of liver function markers. Acute liver injury was induced in BALB / c mice by subcutaneous injection of CCl4 solution. Immediately after poisoning, mice were randomly divided into four groups and injected with a single bolus of either 71G3, 71D6, 71G2, or vehicle only (PBS). Antibodies were administered by intraperitoneal injection at a dose of 5 mg / kg. Each group contained three groups of mice, which were euthanized at different time points after poisoning (12 hours, 24 hours, 48 ​​hours). Blood samples were collected at different time points after injection (0 hours, 12 hours, 24 hours, 48 ​​hours). At necropsy, blood and liver were collected for analysis. Plasma levels of the liver markers aspartate transaminase (AST), alanine aminotransferase (ALT), and bilirubin (BIL) were determined by standard clinical biochemistry methods. [Figure 12] Acute liver failure model: Histological examination of liver sections. Acute liver injury was induced in BALB / c mice as described in the caption for Figure 11. At necropsy, the liver was removed and embedded in paraffin for histological analysis. Sections were stained with hematoxylin and eosin and examined under a microscope. Representative images for each treatment group are shown. Magnification: 100x. [Figure 13]Chronic liver injury model: Plasma concentrations of liver function markers. Liver damage and fibrosis were induced in BALB / c mice by chronic exposure to CCl4 over several weeks. Immediately after initial CCl4 injection, mice were randomly divided into four groups, each treated with either 71G3, 71D6, 71G2, or vehicle only (PBS). Antibodies were administered by intraperitoneal injection at a dose of 1 mg / kg three times per week. An additional fifth control group received neither CCl4 nor antibodies and was used as a healthy control. Mice were euthanized six weeks after chronic CCl4 poisoning. Blood and liver were collected for analysis at necropsy. Plasma levels of the liver markers aspartate transaminase (AST) and alanine aminotransferase (ALT) were determined by standard clinical biochemistry methods. [Figure 14] Chronic liver injury model: Histological examination of liver sections stained with picrosilius red. Liver damage and fibrosis were induced in BALB / c mice by chronic exposure to CCl4 as described in the caption for Figure 13. At necropsy, the livers were removed and embedded in paraffin for immunohistochemical analysis. The sections were stained with picrosilius red. Representative images for each treatment group are shown. Magnification: 100x. [Figure 15] Chronic liver injury model: Histological examination of liver sections stained with anti-alpha smooth muscle actin (α-SMA) antibody. Liver damage and fibrosis were induced in BALB / c mice by chronic exposure to CCl4 as described in the caption for Figure 13. At necropsy, the livers were removed and embedded in paraffin for immunohistochemical analysis. The sections were stained with anti-alpha smooth muscle actin (α-SMA) antibody. Representative images for each treatment group are 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 a single intraperitoneal bolus of HgCl2. Immediately after HgCl2 poisoning, mice were randomly divided into four groups and treated with either 71G3, 71D6, 71G2, or vehicle alone (PBS). Antibodies were administered by intraperitoneal injection at a dose of 5 mg / kg every 24 hours. Mice were euthanized 72 hours after HgCl2 injection. Blood and kidneys were collected for analysis at necropsy. Plasma levels of blood urea nitrogen (BUN) and creatinine (CRE) were determined by standard biochemical methods. [Figure 17] Acute kidney injury model: Histological analysis of kidney sections. Acute renal failure was induced in BALB / c mice by injecting HgCl2 as described in the caption for Figure 16. At necropsy, the kidneys were removed and embedded in paraffin for histological analysis. Kidney sections were stained with hematoxylin and eosin. Representative images for each treatment group are shown. Magnification: 400x. [Figure 18]Ulcerative colitis model: body weight, disease activity index (DAI), colon length. Ulcerative colitis was induced in BALB / c mice by adding dextran sulfate sodium (DSS) to drinking water for 10 days. On day 10, DSS treatment was discontinued and the mice were returned to normal water. From day 1, the mice were randomly divided into seven groups and treated with either 71G3, 71D6, 71G2 (dose of 1 mg / kg or 5 mg / kg), or vehicle alone (PBS). An additional eighth control group was used as a healthy control without receiving DSS or antibodies. On day 12, i.e., two days after discontinuation of DSS administration, the mice were euthanized. At necropsy, the colon was collected, washed, and its length was measured using a ruler. After measurement, the colon was embedded in paraffin for histological analysis. Throughout this experiment, the body weight of the mice was regularly monitored, and the clinical symptoms of ulcerative colitis were evaluated by examining fecal occult blood, rectal bleeding, and fecal consistency. Each parameter was assigned a score from 0 (asymptomatic) to 3 (maximum symptom). The scores for each parameter were summed to obtain a DAI ranging from 0 to 9. (A) Change in body weight over time (percentage relative to time 0). (B) Change in DAI over time. (C) Colon length at autopsy. For clarity, data for the 1 mg / kg group and the 5 mg / kg group are shown in separate graphs. [Figure 19] Ulcerative colitis model: Histological analysis of colon sections. Ulcerative colitis was induced in BALB / c mice by exposure to dextran sulfate sodium (DSS) as described in the caption for Figure 18. The colon was collected at autopsy, measured, and then embedded in paraffin for histological analysis. Colon sections were stained with hematoxylin and eosin, examined under a microscope, and photographed. The experimental group, antibody dose, and magnification are indicated near each image. See the main text for details on image analysis. [Figure 20]Inflammatory Bowel Disease Model: Body Weight and Colon Length. Colon injury and inflammation were induced in C57BLKS / J mice by intrarectal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in ethanol. Immediately after TNBS administration, mice were randomly divided into four groups and treated with either 71G3, 71D6, 71G2, or vehicle only (PBS). An additional fifth control group was used as a healthy control without TNBS or antibodies. Mice were euthanized 5 days after TNBS administration. The colon was collected and measured at necropsy. After measurement, the colon was embedded in paraffin for histological analysis. Throughout the experiment, the body weight of the mice was measured daily. (A) Change in body weight over time (%) relative to time 0. (B) Colon length at necropsy. [Figure 21] Inflammatory Bowel Disease Model: Histological Analysis of Colon Sections. Colon injury and inflammation were induced in BALB / c mice by intrarectal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as described in the caption for Figure 20. The colon was collected at necropsy and measured. After measurement, the colon was embedded in paraffin for histological analysis. Colon sections were stained with hematoxylin and eosin, examined under a microscope, and photographed. See the main text for details on image analysis. [Figure 22]Type 1 diabetes model: Enhanced glucose uptake and synergy with insulin in diabetic mice. Pancreatic β-cell degeneration was induced in BALB / c mice by intraperitoneal injection of streptozotocin (STZ). STZ-treated mice had twice the mean basal blood glucose level compared to untreated mice. STZ-treated mice were randomly divided into four groups, each treated with either 71G3, 71D6, 71G2, or vehicle alone (PBS). An additional fifth control group was used as a healthy control without STZ or antibodies. Blood glucose levels were monitored under fasting conditions for 5 weeks. After 5 weeks, a glucose tolerance test (GTT) and an insulin tolerance test (ITT) were performed. (A) Time course of analysis of basal blood glucose levels under fasting conditions. (B) GTT: Blood glucose levels were monitored over time after oral administration of glucose to fasting mice. (C)ITT: After injecting insulin intraperitoneally into slightly fasted mice, the changes in blood glucose levels over time are monitored. [Figure 23] Type 1 diabetes model: Enhancement of glucose uptake in cultured cells and its interaction with insulin. Myoblasts from C2C12 mice were induced to differentiate into muscle cells and then incubated with human / mouse equivalent anti-MET antibodies (71G3, 71D6, 71G2). After 24 hours, antibody-treated cells were divided into three groups and acutely stimulated for 1 hour with 0 nM, 100 nM, or 1000 nM human recombinant insulin in the presence of the fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). 2-NBDG uptake was examined by flow cytometry. (A) Induction of 2-NBDG uptake by human / mouse equivalent anti-MET antibodies or insulin. (B) Induction of 2-NBDG uptake by 71G3 in the absence or presence 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: Normalization of blood glucose levels and overcoming insulin resistance in db / db mice. Female db / db mice (C57BLKS / J variant with a point mutation in the leptin receptor gene lepr) were randomly divided into four groups at 8 weeks of age, and each group was treated with either 71G3, 71D6, 71G2, or vehicle alone (PBS). Antibodies were administered twice a week by intraperitoneal injection at a dose of 1 mg / kg. Blood glucose levels were monitored every 10 days for 7 weeks under fasting conditions. At the end of treatment, i.e., when the mice were 15 weeks of age, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed using age-matched wild-type C57BLKS / J mice as controls. (A) Time course of blood glucose levels. (B) GTT: After oral administration of glucose to fasting mice, the time course of blood glucose levels was monitored. (C)ITT: After injecting insulin intraperitoneally into slightly fasted mice, the changes in blood glucose levels over time are monitored. [Figure 25] Mouse model of non-alcoholic steatohepatitis (NASH): Improvement of fatty liver examined by histology. Eight-week-old female db / db mice were randomly divided into four groups and treated with either 71G3, 71D6, 71G2, or vehicle alone (PBS). Antibodies were administered twice weekly by intraperitoneal injection at a dose of 1 mg / kg. After 8 weeks of treatment, the mice were euthanized and necropsy was performed. Blood was collected for analysis of liver function markers. The liver was removed for histological analysis and embedded in paraffin. Liver sections were stained with hematoxylin and eosin. The cytoplasm of adipocytes appears empty and white because lipids are washed away during the alcohol treatment of the sample. Representative images of each treatment group are shown. Magnification: 200x. [Figure 26] Mouse model of non-alcoholic steatohepatitis (NASH): Suppression of fibrosis investigated by picrosilius red. Eight-week-old female db / db mice were randomly treated as described in the caption for Figure 25. At necropsy, the livers were processed for histological examination. Liver sections were stained with picrosilius red to highlight fibrosis. Representative images from each treatment group are 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 either purified 71G3, 71D6, 71G2, or vehicle alone, as described in the caption for Figure 25. After 7 weeks of treatment, blood was collected for analysis of liver function markers. (A) Plasma levels of aspartate transaminase (AST). (B) Plasma levels of alanine aminotransferase (ALT). [Figure 28] Mouse model of diabetic ulcer: Accelerated wound healing. Eight-week-old female db / db mice were anesthetized, and a circular incision was made in the right posterior flank using a 0.8 cm wide circular punch blade for skin biopsy, creating a round wound. The entire epidermal layer was removed. The day after surgery, the mice were randomly divided into four groups and treated with either purified 71G3, 71D6, 71G2, or vehicle only (PBS). Antibodies were delivered every two days by intraperitoneal injection at a dose of 5 mg / kg. Wound diameter was measured daily with calipers. (A) Change in wound area over time. (B) Mean epidermal reformation rate, calculated by averaging the percentage of wound repair per day. [Figure 29] Cross-reactivity between rats (Rattus norvegicus) and cynomolgus monkeys (Macaca fascicularis) was investigated by ELISA. To examine cross-reactivity across all species, a limited antibody population representative of both SEMA conjugates (71D6, 71C3, 71D4, 71A3, 71G2) and PSI conjugates (76H10, 71G3) was selected. Prior art 5D5 antibodies were used as controls. Human, mouse, rat, and monkey MET ECDs were immobilized on a solid phase and exposed to increasing concentrations of mAbs (in human IgG / λ format) in solution. Binding was elucidated using HRP-labeled anti-human Fc antibodies. [Figure 30]Alignment of amino acid sequences between MET ECD domains from human (H. sapiens), mouse (M. musculus), rat (R. norvegicus), cynomolgus monkey (M. fascicularis), and llama (L. glama). (A) Sequence alignment of the region recognized by SEMA-conjugated antibodies (71D6, 71C3, 71D4, 71A3, 71G2) (sequence number 239, which is the human MET sequence; sequence number 240, which is the mouse MET sequence; sequence number 241, which is the rat MET sequence; sequence number 242, which is the cynomolgus monkey MET sequence; sequence number 243, which is the llama MET sequence). Amino acids identified by the human-llama chimeric approach shown in Table 12 are underlined. Within this region, there are five residues that are conserved between human MET and mouse MET but not in llama MET (Ala327, Ser336, Phe343, Ile367, Asp372). These amino acids are indicated by black rectangles and increasing numbers 1-5. Four of these residues are conserved in rat MET and cynomolgus monkey MET (Ala327, Ser336, Ile367, Asp372). Amino acids that are very important for binding to SEMA-binding antibodies are indicated with "S" (meaning SEMA). (5D5) Amino acids that are very important for binding to onartuzumab are indicated with "O" (meaning onartuzumab). (B) Sequence alignment of the region recognized by the PSI-conjugating antibodies 76H10 and 71G3 (sequence number 244, which is the human MET sequence; sequence number 245, which is the mouse MET sequence; sequence number 246, which is the rat MET sequence; sequence number 247, which is the cynomolgus monkey MET sequence; sequence number 248, which is the llama MET sequence). Amino acids identified by the human-llama chimeric approach shown in Table 12 are underlined. Within this region, there are three residues that are conserved between human MET and mouse MET but not in llama MET (Arg547, Ser553, Thr555). These amino acids are indicated by black rectangles and increasing numbers 6-8. Two of these residues are also conserved in rat MET and cynomolgus monkey MET (Ser553, Thr555).The amino acids that are crucial for binding to PSI-binding antibodies are indicated with "P" (meaning PSI). [Figure 31] Schematic diagram of MET mutants used for detailed epitope mapping. Using human MET ECD as a template, mutants A to L were created by mutating key residues, indicated by numbers 1 to 8 in Figure 30, in different combinations. Each of these mutants is entirely human except for the llama residues shown. [Modes for carrying out the invention]

[0042] In this specification, the term “immunoglobulin” includes polypeptides having a combination of two heavy chains and two light chains, regardless of whether they possess any specific immunoreactivity related to the subject. “Antibody” refers to such a structure that possesses known, large specific immunoreactive activity against an antigen of interest (e.g., MET). In this specification, the terms “MET antibody” or “anti-MET antibody” refer to an antibody that exhibits immunological specificity against the MET protein. Both antibodies and immunoglobulins contain light and heavy chains, but the interchain covalent bonds between them may or may not be present. The basic structure of immunoglobulins is relatively well understood.

[0043] The general term "immunoglobulin" encompasses five distinct classes that can be identified biochemically. While all five classes of antibodies are within the scope of this invention, the following discussion generally focuses on the IgG class of immunoglobulin molecules. An immunoglobulin called IgG contains two identical polypeptide light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with a molecular weight of approximately 53,000–70,000. These four chains are joined by disulfide bonds to form a "Y" configuration, in which the light chains surround the heavy chains, which begin at the opening of the "Y" and continue into a variable region.

[0044] Antibody light chains are classified as kappa or lambda (κ, λ). Each class of heavy chain can bind to either a kappa or lambda light chain. Generally, the light and heavy chains are covalently bonded to each other, with the "tails" of the two heavy chains joining either by a covalent disulfide bond or by a non-covalent bond when the immunoglobulin is produced by B cells or genetically engineered host cells. In the heavy chain, the amino acid sequence begins at the N-terminus at the fork-shaped end of a Y configuration and ends at the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, and epsilon (γ, μ, α, δ, ε), with subclasses (e.g., γ1-γ4) between them. The properties of this chain determine the "class" of the antibody, which is IgG, IgM, IgA, IgD, and IgE, respectively. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, and IgA1, are well-defined and known to have specialized functions. Modified versions of these classes and isotypes are readily identifiable to those skilled in the art by referring to this disclosure and are therefore included within the scope of the present invention.

[0045] As described above, the variable region of an antibody enables it to recognize and specifically bind to epitopes on an antigen. In other words, the VL and VH domains of the antibody combine to form a variable region that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding sites located at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) in both the VH and VL chains.

[0046] In this specification, the terms “MET protein,” “MET antigen,” or “MET” are interchangeable and refer to the receptor tyrosine kinase that binds to hepatocyte growth factor (HGF) in the wild type. The terms “human MET receptor,” “human MET,” or “hMET” are interchangeable and refer to human MET (GenBank registry number: X54559), which includes the native human MET protein spontaneously expressed in the human host and / or on the surface of human cultured cell lines, as well as recombinant forms and their fragments, and naturally occurring variant forms. The terms “mouse MET protein,” “mouse MET receptor,” “mouse MET,” or “mMET” are interchangeable and refer to mouse MET (GenBank registry number: NM_008591), which includes the native mouse MET protein spontaneously expressed in the mouse host and / or on the surface of mouse cultured cell lines, as well as recombinant forms and their fragments, and naturally occurring variant forms.

[0047] In this specification, the term “binding site” refers to a region within a polypeptide that is critically important for selective binding to a target antigen of interest (e.g., hMET). A binding domain includes at least one binding site. An example of a binding domain is an antibody variable domain. The antibody molecule of the present invention may include a single binding site or multiple (e.g., two, three, or four) binding sites.

[0048] In this specification, the expression "derived from" a specified protein (e.g., a MET antibody or its antigen-binding fragment) means the origin of the polypeptide. In one embodiment, the polypeptide or amino acid sequence derived from a particular starting polypeptide is a CDR sequence or a sequence related thereto. In one embodiment, the amino acid sequences derived from a particular starting polypeptide are not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are derived from the starting antibody. In one embodiment, the polypeptide or amino acid sequence derived from a particular starting polypeptide or amino acid sequence has an amino acid sequence that is substantially identical to the starting sequence or a portion thereof. If a portion is substantially identical, that portion consists of at least 3 to 5 amino acids, or at least 5 to 10 amino acids, or at least 10 to 20 amino acids, or at least 20 to 30 amino acids, or at least 30 to 50 amino acids, or a portion that a person skilled in the art can otherwise identify as originating from the starting sequence. In one embodiment, one or more CDR sequences derived from the starting antibody are modified to produce a variant CDR sequence (e.g., an affinity variant) that maintains MET-binding activity.

[0049] "Camellid-derived" - In some preferred embodiments, the MET antibody molecule of the present invention comprises a framework amino acid sequence and / or CDR amino acid sequence derived from a conventional antibody of the camelid family, which is produced by actively immunizing a camelid animal with a MET-derived antigen. However, a MET antibody comprising a camelid-derived amino acid sequence can be manipulated to include a framework region and / or constant region sequence derived from a human amino acid sequence (e.g., a human antibody) or another non-camelid mammalian species. For example, a human or non-human primate framework region and / or heavy chain portion and / or hinge portion can be included in the MET antibody of interest. In one embodiment, one or more non-camelid amino acids may be present in the framework region of the "camellid-derived" MET antibody, for example, the amino acid sequence of the camelid framework region may include one or more amino acid mutations in which the corresponding human or non-human primate amino acid residues are located. Furthermore, the camelid-derived VH domain and VL domain, or a humanized variant thereof, can be ligated to the constant domain of a human antibody to create chimeric molecules as described in detail elsewhere herein.

[0050] In this specification, a “conservative amino acid substitution” is a substitution in which an amino acid residue is replaced by an amino acid having a similar side chain. Families of amino acid residues having similar side chains have been clearly defined in the prior art and include 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in immunoglobulin polypeptides can be replaced by other amino acid residues from the same side chain family. In another embodiment, the amino acid sequence can be replaced with an amino acid sequence that is structurally similar but differs in the order and / or composition of the side-chain family members.

[0051] In this specification, the term “heavy chain portion” includes amino acid sequences derived from the constant region of an immunoglobulin heavy chain. A polypeptide comprising a heavy chain portion includes at least one of the following: a CH1 domain, a hinge domain (e.g., an upper hinge region, and / or a central hinge region, and / or a lower hinge region), a CH2 domain, a CH3 domain, or variants or fragments thereof. In one embodiment, the antibody or antigen-binding fragment of the present invention may include the Fc portion of an immunoglobulin heavy chain (e.g., a hinge portion, a CH2 domain, or a CH3 domain). In another embodiment, the antibody or antigen-binding fragment of the present invention may lack at least a portion of the constant domain (e.g., all or part of the CH2 domain). In some embodiments, at least one, preferably all, of the constant domains are derived from a human immunoglobulin heavy chain. For example, in one preferred embodiment, the heavy chain portion includes the entire human hinge domain. In another preferred embodiment, the heavy chain portion includes the entire human Fc portion (e.g., a sequence of the hinge domain, CH2 domain, or CH3 domain from human immunoglobulin). In some embodiments, the constant domains constituting the heavy chain portion are derived from different immunoglobulin molecules. For example, the heavy chain portion of a polypeptide may include a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In another embodiment, the constant domain is a chimeric domain containing portions from different immunoglobulin molecules. For example, the hinge may include a first portion from an IgG1 molecule and a second portion from an IgG3 or IgG4 molecule. As described above, those skilled in the art will understand that the constant domains of the heavy chain portion may be modified to alter the amino acid sequence from that of the natural (wild-type) immunoglobulin molecule. That is, the polypeptides of the present invention disclosed herein may include alterations or modifications to one or more of the heavy chain constant domains (CH1, hinge, CH2, CH3) and / or light chain constant domains (CL). Examples of modifications include the addition, deletion, or substitution of one or more amino acids in one or more domains.

[0052] In this specification, a “chimeric” protein contains a first amino acid sequence bound to a second amino acid sequence that does not naturally occur in nature. These amino acid sequences normally exist as separate proteins and can combine to form a fusion polypeptide. Alternatively, these amino acid sequences may normally exist within the same protein but can be arranged in a new configuration within the fusion polypeptide. Chimeric proteins can be produced, for example, by chemical synthesis, or by creating and translating polynucleotides in which multiple peptide regions encode each other in a desired relationship. An example of a chimeric MET antibody is a fusion protein containing camelid-derived VH and VL domains, or their humanized variants, fused to the constant domain of a human antibody (e.g., human IgG1, IgG2, IgG3, IgG4) or a mouse antibody (e.g., mouse IgG1, IgG2a, IgG2b, IgG2c, IgG3).

[0053] In this specification, the terms "variable region" and "variable domain" are used interchangeably and are assumed to have equivalent meanings. The term "variable" refers to the fact that the sequences of several parts of the variable domains VH and VL differ widely among antibodies, and that this is utilized for the binding and specificity of individual antibodies to their corresponding target antigens. However, variability is not uniformly distributed throughout the variable domain of the antibody. Variability is concentrated in three compartments called "hypervariable loops" that form antigen-binding sites within the VL and VH domains, respectively. In this specification, the first, second, and third hypervariable loops of the V-lambda light chain domain are referred to as L1(λ), ​​L2(λ), and L3(λ), and can be defined as containing residues 24-33 (L1(λ) consisting of 9, 10, or 11 amino acid residues), 49-53 (L2(λ) consisting of 3 residues), and 90-96 (L3(λ) consisting of 5 residues) within the VL domain (Morea et al., Methods Vol. 20, pp. 267-279, 2000). In this specification, the first, second, and third hypervariable loops of the V-kappa light chain domain are referred to as L1(κ), L2(κ), and L3(κ), and can be defined as containing residues 25-33 (L1(κ) consisting of 6, 7, 8, 11, 12, or 13 amino acid residues), 49-53 (L2(κ) consisting of 3 residues), and 90-97 (L3(κ) consisting of 6 residues) within the VL domain (Morea et al., Methods Vol. 20, pp. 267-279, 2000). The first, second, and third hypervariable loops of the VH domain are referred to herein as H1, H2, and H3, and can be defined as containing residues 25-33 (H1 consisting of 7, 8, or 9 residues), 52-56 (H2 consisting of 3 or 4 residues), and 91-105 (H3 of which has a very variable length) within the VH domain (Morea et al., Methods Vol. 20, pp. 267-279, 2000).

[0054] Unless otherwise specified, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops of the VL domain, respectively, and encompass hypervariable loops derived from both the V-kappa isotype and the V-lambda isotype. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops of the VH domain, respectively, and encompass hypervariable loops derived from any known heavy-chain isotype (including γ, ε, δ, α, and μ).

[0055] The hypervariable loops L1, L2, L3, H1, H2, and H3 each contain a "complementarity-determining region," or "CDR," as defined below. The terms "hypervariable loop" and "complementarity-determining region" are not strictly synonymous. This is because hypervariable loops (HV) are determined based on structure, while complementarity-determining regions (CDR) are determined based on sequence variability (Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991), and the restrictions of HV and CDR may differ in some VH and VL domains.

[0056] The CDRs of the VL and VH domains are typically defined to include the following amino acids: residues 24–34 (CDRL1), 50–56 (CDRL2), and 89–97 (CDRL3) of the light chain variable domain, and residues 31–35 or 31–35b (CDRH1), 50–65 (CDRH2), and 95–102 (CDRH3) of the heavy chain variable domain (Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991). Therefore, HV can be included in the corresponding CDR, and when referring to the "hypervariable loop" of the VH and VL domains herein, unless otherwise specified, it should be interpreted that the corresponding CDR is also included, and vice versa.

[0057] The more conserved portion of the variable domain is called the framework region (FR), as defined below. The variable domains of the native heavy and light chains each primarily employ a β-sheet configuration and contain four FRs (FR1, FR2, FR3, and FR4, respectively) connected by three hypervariable loops. The hypervariable loops within each chain are held in close proximity to hypervariable loops from other chains by the FRs, contributing to the formation of the antibody's antigen-binding site. Structural analysis of antibodies has revealed the relationship between the sequence and the shape of the binding site formed by the complementarity-determining region (Chothia et al., J. Mol. Biol. Vol. 227, pp. 799-817, 1992; Tramontano et al., J. Mol. Biol. Vol. 215, pp. 175-182, 1990). Five of the six loops, despite their high sequence variability, employ a very limited repertoire of main-chain configurations, and these configurations are called "canonical structures." These arrangements are determined first by the length of the loop, and second by the presence of key residues in predetermined positions within the loop and framework regions, whose arrangements are determined by packing density, hydrogen bonding, and the ability to adopt unusual backbone configurations.

[0058] In this specification, the term “CDR,” or “complementarity-determining region,” refers to discontinuous antigen-binding sites found within the variable regions of both heavy-chain and light-chain polypeptides. These special regions are described in Kabat et al., J. Biol. Chem. Vol. 252, pp. 6609–6616, 1977; Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991; Chothia et al., J. Mol. Biol. Vol. 196, pp. 901–917, 1987; and MacCallum et al., J. Mol. Biol. Vol. 262, pp. 732–745, 1996. The definitions in these publications include overlaps or subsets of amino acid residues when compared to one another. For comparison, the amino acid residues encompassing the CDRs defined by each of the above cited publications are shown below. The term "CDR" preferably refers to the CDR defined by Kabat based on sequence comparison.

[0059] [Table 1]

[0060] In this specification, the term “framework region,” or “FR region,” includes amino acid residues that are part of the variable region but not part of the CDR (for example, using Kabat’s definition for CDRs). Therefore, the variable region framework is approximately 100-120 amino acids long but contains only amino acids outside the CDR. With regard to specific examples of heavy chain variable domains and CDRs as defined by Kabat et al., framework region 1 corresponds to the variable region domain encompassing amino acids 1-30, framework region 2 corresponds to the variable region domain encompassing amino acids 36-49, framework region 3 corresponds to the variable region domain encompassing amino acids 66-94, and framework region 4 corresponds to the domain from amino acid 103 to the end of the variable region. The framework regions of the light chain are similarly separated by their respective light chain variable regions (CDRs). Similarly, using the definition of CDRs by Chothia et al. or MacCallum et al., the boundaries of the framework regions are separated by their respective CDR terminals as described above. In preferred embodiments, CDRs are defined by Kabat.

[0061] In natural antibodies, the six CDRs present on the surface of each monomeric antibody are short, discontinuous amino acid sequences in specific positions that form the antigen-binding site when the antibody takes on a three-dimensional configuration in an aqueous environment. The remainder of the heavy-chain and light-chain variable domains, with less intermolecular amino acid sequence variability, are called the framework region. The framework region primarily employs a β-sheet configuration, with the CDRs forming loops that connect the β-sheet structures, and in some cases even forming parts of the β-sheet structures. Thus, these framework regions form a scaffold, positioning the six CDRs in the correct orientation through interchain non-covalent interactions. The antigen-binding site formed by the positioned CDRs defines a surface complementary to the epitope on the surface of the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to the immunoreactive antigen epitope. The positions of the CDRs can be easily identified by those skilled in the art.

[0062] In this specification, the term “hinge region” includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antibody-binding regions to move independently. The hinge region can be divided into three distinct domains: the upper hinge, the central hinge, and the lower hinge. (Roux et al., J. Immunol. Vol. 161, pp. 4083-4090, 1998). A MET antibody containing a “fully human” hinge region may contain one of the hinge region sequences shown in Table 2 below.

[0063] [Table 2]

[0064] In this specification, the term "CH2 domain" includes the portion of the heavy chain molecule that extends approximately from residue 244 to residue 360 ​​of the antibody, using the standard numbering scheme (residues 244-360 in the Kabat numbering system; residues 231-340 in the EU numbering system; Kabat et al., *Sequences of Proteins of Immunological Interest*, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)). The CH2 domain is unique in that it does not form a close pair with other domains. Rather, two N-linked branched hydrocarbon chains are inserted between the two CH2 domains of the complete native IgG molecule. The literature also clearly states that the CH3 domain extends from the CH2 domain of the IgG molecule to the C-terminus and contains approximately 108 residues.

[0065] In this specification, the term “fragment” means a portion or part of an antibody or antibody chain that contains fewer amino acid residues than a complete or complete antibody or antibody chain. The term “antigen-binding fragment” means a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with a complete antibody (i.e., the source complete antibody) for binding to an antigen (i.e., specific binding to hMET and mMET). In this specification, the term “fragment” of an antibody molecule includes antigen-binding fragments of an antibody (e.g., antibody light chain variable domain (VL), antibody heavy chain variable domain (VH), single-chain antibody (scFv), F(ab')2 fragment, Fab fragment, Fd fragment, Fv fragment, single-domain antibody fragment (DAb)). Fragments can be obtained, for example, by chemical or enzymatic treatment of a complete or complete antibody or antibody chain, or by recombinant means.

[0066] In this specification, the term “valence” refers to the number of sites in a peptide that can potentially be target binding sites. Each target binding site specifically binds to one target molecule or to a specific site on a target molecule. If a polypeptide contains two or more target binding sites, each target binding site can specifically bind to the same molecule or different molecules (for example, to different ligands or different antigens, or to different epitopes on the same antigen). The target binding molecule has at least one binding site specific to hMET.

[0067] In this specification, the term “specificity” means the ability to bind to a given target (e.g., hMET, mMET) (e.g., to react with a given target). A polypeptide can be monospecific and may contain one or more binding sites that specifically bind to one target. Alternatively, a polypeptide can be polyspecific and may contain two or more binding sites that specifically bind to the same or different targets. In one embodiment, the antibody of the present invention has specificity for two or more targets. For example, in one embodiment, the polyspecific binding molecule of the present invention binds to hMET and a second target molecule. In this context, the second target molecule is a molecule other than hMET or mMET.

[0068] The term "epitope" refers to the portion of an antigen (human MET) that comes into contact with an antibody. Epitopes can be linear, i.e., involved in binding to a single amino acid sequence, or conformal, i.e., involved in binding to two or more amino acid sequences located in various regions of the antigen that may not necessarily be continuous. The antibodies presented herein can bind to different (overlapping or non-overlapping) epitopes within the extracellular domain of the human MET protein.

[0069] In this specification, the term "synthetic" in relation to polypeptides includes polypeptides containing amino acid sequences that do not occur naturally. For example, this includes unnatural polypeptides that are modified forms of natural polypeptides (including mutations such as addition, substitution, and deletion), and unnatural polypeptides that contain a first amino acid sequence (which may be natural or non-natural) linked to a second amino acid sequence (which may be natural or non-natural) that is not naturally linked, with the amino acids arranged in a linear fashion.

[0070] In this specification, the term “manipulated” includes manipulating nucleic acid molecules or polypeptide molecules by synthetic means (e.g., by recombinant techniques, by in vitro peptide synthesis, by enzymes, by chemical coupling of peptides, or by any combination of these techniques). The antibodies of the present invention are preferably manipulated, and include humanized antibodies and / or chimeric antibodies, antibodies in which one or more properties (e.g., binding to antigens, stability / half-life, effector function) have been manipulated.

[0071] In this specification, the term “modified antibody” includes antibodies in synthetic forms that have been modified in a way that does not occur naturally (e.g., antibodies containing at least two heavy chain moieties but not two complete heavy chains (e.g., domain deletion antibodies or minibodies)); antibodies in polyspecific forms (e.g., bispecific, tripspecific, etc.) that bind to two or more different antigens or to different epitopes on a single antigen; and heavy chain molecules conjugated to scFv molecules, which are known in the art and are described, for example, in U.S. Patent No. 5,892,019. In addition, the term “modified antibody” includes antibodies in polyvalent forms (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antibody). In another embodiment, the modified antibody of the present invention is a fusion protein comprising a polypeptide binding domain containing at least one heavy chain moiety lacking a CH2 domain, and a binding moiety for one member of a receptor ligand pair.

[0072] The term “modified antibody” may also, as used herein, mean an amino acid sequence variant of the MET antibody of the present invention. Those skilled in the art will understand that the MET antibody of the invention can be modified to produce a variant MET antibody in which the amino acid sequence is altered compared to the source MET antibody. For example, nucleotide or amino acid substitutions (e.g., at CDR and / or framework residues) can be implemented leading to conservative substitutions or changes at the positions of “non-essential” amino acid residues. Amino acid substitutions include replacing one or more amino acids with natural or non-natural amino acids.

[0073] In this specification, the term "humanization substitution" means an amino acid substitution in which an amino acid residue located at a specific position in the VH domain or VL domain of the MET antibody of the present invention (e.g., a MET antibody derived from a camelid) is replaced with an amino acid residue located at an equivalent position in a reference human VH domain or VL domain. The reference human VH domain or VL domain may be a VH domain or VL domain encoded by the human germline. Humanization substitution can be carried out in the framework region and / or CDR of the MET antibody as defined herein.

[0074] In this specification, the term "humanized variant" means a variant antibody that contains one or more "humanization substitutions" compared to a reference MET antibody, wherein a portion of the reference antibody (e.g., the VH domain and / or the VL domain, or a portion of that domain containing at least one CDR) has an amino acid sequence derived from a non-human species, and the "humanization substitution" occurs within the amino acid sequence derived from the non-human species.

[0075] In this specification, the term “germline variant” means, in particular, a “humanized variant” in which one or more amino acid residues located at specific positions in the VH domain or VL domain of the MET antibody of the present invention (e.g., a MET antibody derived from a camelid) are replaced with amino acid residues at equivalent positions in the human VH domain or VL domain encoded by the human germline as a result of “humanization substitution.” For any given “germline variant,” the substituted amino acid residues that become germline variants are typically taken only from a single human germline-encoded VH domain or VL domain, or primarily from that VH domain or VL domain. The terms “humanized variant” and “germline variant” are often used interchangeably in this specification. Introducing one or more “humanization substitutions” into a camelid-derived (e.g., llama-derived) VH domain or VL domain produces a “humanized variant” of the camelid-derived (e.g., llama-derived) VH domain or VL domain. If the amino acids introduced by substitution originate primarily from a VH domain sequence or VL domain sequence encoded by a single human germline, or solely from that VH domain sequence or VL domain sequence, then it can become a "human germline variant" of a VH domain or VL domain derived from a camelid (e.g., a llama).

[0076] In this specification, the term "affinity variant" means a variant antibody whose amino acid sequence has been altered at one or more positions compared to the reference MET antibody of the present invention, and which exhibits altered affinity for hMET and / or mMET compared to the reference antibody. Preferably, the affinity variant exhibits improved affinity for hMET and / or mMET compared to the reference MET antibody. The improvement is due to a lower K for hMET and / or mMET. DThis may manifest as an affinity variant, or as a slower offrate for hMET and / or mMET. Affinity variants typically have one or more amino acid sequence changes in the CDR compared to the reference MET antibody. Such substitutions can replace the original amino acid at a given position in the CDR with a different amino acid residue. That different amino acid residue may be a native or non-native amino acid residue. The amino acid substitution may be conserved or non-conserved.

[0077] In this specification, an antibody with "high human homology" means an antibody that contains a heavy chain variable domain (VH) and a light chain variable domain (VL), and whose combined amino acid sequence matches at least 90% the best-matching human germline VH and VL sequences. Antibodies with high human homology may include antibodies that contain VH and VL domains of a naturally occurring non-human antibody that show a sufficiently large percentage sequence match with a human germline sequence. Examples include antibodies containing VH and VL domains of a typical camelid antibody, as well as engineered (particularly humanized or germlined) variants of such antibodies, and "fully human" antibodies.

[0078] In one embodiment, the VH domain of an antibody with high homology to humans may have a sequence match or sequence homology of 80% or more with one or more human VH domains in the framework regions FR1, FR2, FR3, and FR4. In another embodiment, the amino acid sequence match or homology between the VH domain of the polypeptide of the present invention and the best-matching human germline VH sequence may be 85% or more, or 90% or more, or 95% or more, or 97% or more, or 99% or more, or even 100%.

[0079] In one embodiment, the VH domain of an antibody with high homology to humans may contain one or more (e.g., 1 to 10) amino acid sequence mismatches in the framework regions FR1, FR2, FR3, and FR4 compared to the best-matching human VH sequence. In another embodiment, the VL domain of an antibody with high homology to humans may have a sequence match or sequence homology of 80% or more with one or more human VL domains in the framework regions FR1, FR2, FR3, and FR4. In yet another embodiment, the amino acid sequence match or homology between the VL domain of the polypeptide of the present invention and the best-matching human germline VL sequence may be 85% or more, or 90% or more, or 95% or more, or 97% or more, or 99% or more, or even 100%.

[0080] In one embodiment, the VL domain of an antibody with high homology to humans may contain one or more (e.g., 1 to 10) amino acid sequence mismatches in the framework regions FR1, FR2, FR3, and FR4 compared to the best-matching human VL sequence. Standard folding can be determined before analyzing the sequence agreement between an antibody with high homology to humans and the VH and VL of the human germline. This makes it possible to identify families of human germline compartments that have the same standard folding combinations with respect to H1 and H1, or L1 and L2 (and L3). Then, the member of the human germline family with the highest sequence homology to the variable region of the antibody of interest is selected and its sequence homology is scored. Procedures for determining the best-matching human germline and for determining % sequence agreement / homology are well known to those skilled in the art.

[0081] Antibodies with high homology to humans may contain hypervariable loops or CDRs having a human or human-like standard folding structure. In one embodiment, at least one hypervariable loop or CDR in the VH domain or VL domain of an antibody with high homology to humans may be obtained from or derived from the VH domain or VL domain of a non-human antibody (e.g., a typical antibody from a species of camelid), and the expected or actual standard folding structure can still be substantially the same as that which occurs in a human antibody. In one embodiment, both H1 and H2 within the VH domain of an antibody with high homology to humans may result in an expected or actual standard folding structure that is substantially the same as that which occurs in a human antibody.

[0082] Antibodies with high homology to humans may contain VH domains in which hypervariable loops H1 and H2 form the same combination of standard folding structures as those known to occur in at least one human germline VH domain. Only some combinations of standard folding structures in H1 and H2 have been observed to actually occur in VH domains encoded by the human germline. In one embodiment, H1 and H2 in the VH domain of an antibody with high homology to humans can be obtained from a VH domain of a non-human species (e.g., a species of camelid), and the expected or actual combination of standard folding structures will still be the same as those known to occur in human germline or somatic mutant VH domains. In non-limiting embodiments, the H1 and H2 within the VH domain of an antibody with high homology to humans can be obtained from the VH domain of a non-human species (e.g., a species of camelid), and the standard folding combination will be one of 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1, or 3-5. Antibodies with high homology to humans may contain a VH domain that exhibits sequence matching / high sequence homology to human VH, as well as a hypervariable loop that exhibits structural homology to human VH.

[0083] The standard folding and combinations present in H1 and H2 within the VH domain of antibodies with high homology to humans may be advantageous for being "correct" for the human VH germline sequence that best matches the VH domain of antibodies with high homology to humans in terms of overall primary amino acid sequence matching. For example, if the sequence best matches the human germline VH3 domain, it may be advantageous for H1 and H2 to form a standard folding combination that also naturally occurs in the human VH3 domain. This may be particularly important for antibodies derived from non-human species that have high homology to humans (e.g., antibodies containing VH and VL domains derived from typical camelid antibodies, especially antibodies containing humanized camelid VH and VL domains).

[0084] Therefore, in one embodiment, the VH domain of a MET antibody with high homology to humans may have a sequence match or sequence homology with the human VH domain of 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 99% or more, or even 100% in framework regions FR1, FR2, FR3, and FR4. In addition, H1 and H2 within the same antibody are derived from a non-human VH domain (e.g., from a species of camelid (preferably a llama)), but the expected or actual standard folding structure is the same as the combination of standard foldings known to occur within the same human VH domain.

[0085] In another embodiment, L1 and L2 within the VL domain of an antibody with high homology to humans are each derived from a VL domain of a non-human species (e.g., a VL domain derived from a camelid), and their respective expected or actual standard folding structures are substantially the same as those that occur in human antibodies. The combination of expected or actual standard folding structures of L1 and L2 within the VL domain of an antibody with high homology to humans can be the same as the combination of standard folding structures known to occur in human germline VL domains. In non-limiting embodiments, L1 and L2 within the V-lambda domain of an antibody with high homology to humans (e.g., an antibody containing a VL domain derived from camelids, or a humanized variant thereof) can form one of the standard folding combinations: 11-7, 13-7(A, B, C), 14-7(A, B), 12-11, 14-11, 12-12 (as defined by Williams et al., J. Mol. Biol. Vol. 264, pp. 220-232, 1996, and shown at http: / / www.bioc.uzh.ch / antibody / Sequences / Germlines / VBase_hVL.html). In non-limiting embodiments, L1 and L2 within the V-kappa domain can form one of the standard folding combinations 2-1, 3-1, 4-1, and 6-1 (defined by Tomlinson et al., EMBO J. Vol. 14, pp. 4628-4638, 1995, and shown at http: / / www.bioc.uzh.ch / antibody / Sequences / Germlines / VBase_hVK.html). In yet another embodiment, all three L1, L2, and L3 within the VL domain of an antibody with high human homology can exhibit substantially human structures. It is preferable for the VL domain of an antibody with high human homology to both exhibit sequence matching / sequence homology with human VL and for the hypervariable loop within the VL domain to exhibit structural homology with human VL.

[0086] In one embodiment, the VL domain of a MET antibody with high homology to humans can exhibit 80% or more, or 85% or more, 90% or more, 95% or more, 97% or more, or even 99%, or even 100% sequence matching with the human VL domain in framework regions FR1, FR2, FR3, and FR4. In addition, the hypervariable regions L1 and L2 can make the expected standard folding structure or the combination of the actual standard folding structure the same as the standard folding combination known to occur naturally within the same human VL domain.

[0087] Of course, by combining a VH domain that exhibits high sequence matching / homology with human VH and structural homology with the hypervariable loop of human VH with a VL domain that exhibits high sequence matching / homology with human VL and structural homology with the hypervariable loop of human VL, it is conceivable to provide an antibody with high homology to humans, containing a VH / VL pair (for example, a VH / VL pair derived from camelids) and exhibiting maximum sequence homology and structural homology with a VH / VL pair that codes for humans.

[0088] Procedures for evaluating the presence of human-like standard folding in CDR, VH, and VL domains from camelid species (e.g., llamas) are described in WO 2010 / 001251 and WO 2011 / 080350 (their entire contents are incorporated herein by reference).

[0089] In this specification, the terms “affinity” or “binding affinity” should be understood in the context of antibody binding as they normally do in this art, and reflect the strength and / or stability of binding between the antigen and the binding site on the antibody or its antigen-binding fragment.

[0090] The anti-MET antibodies presented herein are characterized by their high affinity for binding to human MET (hMET) and mouse MET (mMET). The binding affinity to hMET and mMET can be evaluated using standard techniques known to those skilled in the art.

[0091] In one embodiment, the binding affinity of a Fab clone containing a predetermined VH / VL pair can be evaluated using surface plasmon resonance (e.g., using the Biacore® system). A Fab clone containing the antibody-antigen binding fragment VH / VL pair of the present invention typically has a binding affinity of 1 × 10⁻¹⁶ to hMET. -3 / sec~1×10 -2 Range per second, and in some cases 1 x 10 -3 / sec~6×10 -3 The off-rate is in the range of / second (measured by Biacore®). An off-rate within this range can be considered one indicator that the Fab and its corresponding divalent mAb bind to the hMET with high affinity. Similarly, the Fab clone containing the VH / VL pair of the antibody and antigen-binding fragment of the present invention binds to the mMET with 1 × 10⁻¹⁶ affinity, as described in the appended examples. -3 / sec~1×10 -2 Range per second, and in some cases 1 x 10 -3 / sec~6×10 -3 The off-rate is shown in the range of / second (measured by Biacore®). An off-rate within this range can be considered an indicator that the Fab and its corresponding divalent mAb bind to mMET with high affinity. Therefore, a Fab that shows an off-rate within the above range for both human MET and mouse MET will bind to both hMET and mMET with high affinity. In other words, this Fab is cross-reactive between hMET and mMET. A divalent mAb containing two Fabs that (individually) show off-rates within the above range for human MET and mouse MET is also considered to bind to human MET and mouse MET with high affinity.

[0092] Binding affinity is the dissociation constant for a particular antibody, i.e., K D It can also be expressed as K D The smaller the value, the stronger the binding interaction between the antibody and its corresponding target antigen. D For example, K can be determined by SPR measurement.on and K off This can be determined by combining them. Typically, when the antibody and antigen-binding fragment of the present invention are measured as mAbs, the K of mMET and hMET is obtained. D This is 0.1 nanomoles / l.

[0093] The binding affinity of human MET to mouse MET can also be evaluated using cell-based systems, as described in the attached examples. In the examples, for example, ELISA or flow cytometry is used to examine the binding of mAbs to mammalian cell lines expressing MET. The high affinity for hMET or mMET can be evaluated using EC in ELISA, as described in Example 3, for example. 50 This is demonstrated by the fact that it is less than or equal to 0.5 nM.

[0094] As summarized above, the present invention relates, at least in part, to an antibody and its antigen-binding fragment that binds with high affinity to hMET and mMET. The characteristics and properties of the MET antibody and antibody fragment of the present invention will now be described in more detail.

[0095] Antibodies and antigen-binding fragments described herein that have high affinity and cross-react with hMET and mMET are MET agonists. Hereinafter, MET agonists induce MET signaling (partially or entirely) when bound to the MET receptor. The MET agonist antibodies and MET agonist antigen-binding fragments of the present invention are agonists of hMET and mMET. The agonist activity of the antibodies described herein when bound to hMET or mMET can be demonstrated by molecular and / or cellular responses that (at least partially) mimic the molecular and cellular responses induced upon homologous HGF-MET binding (i.e., binding of human HGF to hMET, or mouse HGF to mMET). Antibodies that mimic such responses are also referred to herein as “anti-MET agonists” or “agonist antibodies” (and their grammatical variations). Similarly, antibodies that partially mimic such a response are referred to herein as “partial MET agonists” or “partial agonists,” and antibodies that fully mimic such a response are referred to herein as “full MET agonists” or “full agonists.” It should be emphasized that the antibodies and antigen-binding fragments of the present invention induce MET signaling in both human and mouse systems. That is, the antibodies and antigen-binding fragments of the present invention are agonists of both hMET and mMET. Therefore, the following discussion applies to both the response induced by the binding of the antibodies and antigen-binding fragments of the present invention to hMET and the response induced by the binding of the antibodies and antigen-binding fragments of the present invention to mMET.

[0096] The MET-activating effect of the antibody and antigen-binding fragment of the present invention can be demonstrated by molecular responses (e.g., phosphorylation of the MET receptor) and / or cellular responses (e.g., cellular responses detectable by cell scattering assays and / or anti-apoptotic assays and / or branching morphogenesis assays). These molecular and cellular responses are described in more detail below: (i) Phosphorylation of the MET receptor. In this context, a MET agonist antibody or MET agonist antigen-binding fragment phosphorylates MET if autophosphorylation of MET occurs upon binding of this antibody or antigen-binding fragment in the absence of receptor-ligand binding. That is, this antibody or antigen-binding fragment binds to human hMET in the absence of hHGF and phosphorylates hMET, and this antibody or antigen-binding fragment binds to mMET in the absence of mHGF and phosphorylates mMET. Phosphorylation of MET can be elucidated by assays known in this art (e.g., Western blotting and phospho-MET ELISA (described in Example 6 and Basilico et al., J Clin Invest. Vol. 124, pp. 3172-3186, 2014)). The antibodies and antigen-binding fragments described herein may exhibit "high phosphorylation efficacy" or "low phosphorylation efficacy" for hMET and "high phosphorylation efficacy" or "low phosphorylation efficacy" for mMET. In this context, the antibody or antigen-binding fragment is similar to the EC that this antibody or fragment has against mMET, and against HGF. 50 (<1 nM) and / or at least 80% of E (as a percentage of the maximum activation induced by HGF) MAX This demonstrates the effect of EC on hMET, similar to that on HGF. 50 (<1 nM) and / or at least 80% of E (as a percentage of the maximum activation induced by HGF) MAX When an antibody or antigen-binding fragment exhibits this effect, it is considered to have "high phosphorylation efficacy." This antibody or fragment exhibits 1 nM to 5 nM EC relative to mMET. 50 and / or 60-80% E (as a percentage of the maximum activation induced by HGF) MAX This demonstrates an effect of 1 nM to 5 nM EC relative to hMET. 50 and / or 60-80% E (as a percentage of the maximum activation induced by HGF) MAXWhen it exhibits this effect, it is described as showing "small phosphorylation activity." (ii) Induction of HGF-like cellular response. MET activation can be measured using assays such as the cell scattering assay and / or anti-apoptotic assay and / or branching morphogenesis assay described in the examples herein. In this context, the MET agonist antibody or MET agonist antigen-binding fragment according to the present invention induces a response in cell assays that is (at least partially) similar to the response observed after exposure to homologous HGF, for example. For example, being a MET agonist means that there is an increase in cell scattering in response to this antibody compared to cells exposed to a control antibody (e.g., IgG1); and / or a protective efficacy against drug-induced apoptosis with an EC of less than 32 nM. 50 and / or more than 20% larger E compared to untreated cells MAX This can be indicated by increased cell viability; and / or by an increase in the number of branches per spheroid in a cell spheroid preparation exposed to this antibody or antigen-binding fragment.

[0097] The antibodies and antigen-binding fragments described herein can "fully" or "partially" induce an HGF-like cellular response when in contact with human cells, and can "fully" or "partially" induce an HGF-like cellular response when in contact with mouse cells, depending on the assay used.

[0098] In this context, the "overall induction" of an HGF-like cellular response by an antibody or fragment can be measured as follows: In a cell scattering assay, the antibody or antigen-binding fragment induces an increase in cell scattering at a concentration of 0.1–1 nM that is at least equivalent to that of 0.1 nM homologous HGF; and / or In anti-apoptotic assays, the antibody or antigen-binding fragment has an EC of 1.1 times or less the HGF value. 50 and / or E greater than 90% of the values ​​observed in HGF MAX Shows cell viability; and / or In branching morphogenesis assays, cells treated with antibodies or antigen-binding fragments exhibit more than 90% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF.

[0099] In this context, if the antibody or antigen-binding fragment does not "fully induce" the HGF-like cellular response described above, the "partial induction" of the HGF-like cellular response can be measured as follows: In a cell scattering assay, the antibody or antigen-binding fragment induces cell scattering at a concentration of 1 nM or less that is at least 25% of the level induced by 0.1 nM homologous HGF; In anti-apoptotic assays, the antibody or antigen-binding fragment has an EC of 7.0 times or less the HGF value. 50 and / or E at least 50% of the value observed in HGF MAX Shows cell viability; In branching morphogenesis assays, cells treated with antibodies or antigen-binding fragments exhibit at least 25% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF.

[0100] As already described, the antibody and antigen-binding fragment of the present invention are both hMET agonists and mMET agonists. Therefore, in embodiments in which this antibody induces an HGF-like cellular response (partially or completely), the HGF-like cellular response is induced (partially or completely) when the antibody or antigen-binding fragment is brought into contact with human cells, and is induced (partially or completely) when the antibody or antigen-binding fragment is brought into contact with mouse cells.

[0101] Binding region mapping (Example 4) reveals that the anti-MET antibody of the present invention recognizes epitopes of MET within the PSI domain or within the SEMA domain of MET. Thus, in some embodiments, the antibody or antigen-binding fragment of the present invention recognizes epitopes within the PSI domain of MET (human MET preferred). In some alternative embodiments, the antibody or antigen-binding fragment of the present invention recognizes epitopes within the SEMA domain of MET (human MET preferred).

[0102] In some embodiments, an antibody or antigen-binding fragment that recognizes an epitope within the SEMA domain recognizes an epitope located on the surface of one blade of the SEMAβ-propeller. In some embodiments, the epitope is located on the surface of blade 4 or blade 5 of the SEMAβ-propeller. In some such embodiments, the epitope is located between amino acids 314 and 372 of human MET. In some embodiments, the epitope is located on the surface of blades 1 to 4 or blades 1 to 3 of the SEMAβ-propeller. In some embodiments, the epitope is located between amino acids 27 and 313 of human MET, or between amino acids 27 and 225 of human MET.

[0103] In some embodiments, an antibody or antigen-binding fragment that recognizes an epitope within the PSI domain of MET recognizes an epitope located between amino acids 516 and 545 of MET (human MET preferred). In some embodiments, an antibody or antigen-binding fragment that recognizes an epitope within the PSI domain of MET recognizes an epitope located between amino acids 546 and 562 of MET (human MET preferred).

[0104] In some aspects, the antibodies described herein recognize epitopes within the extracellular domain of MET that contain one or more amino acid residues conserved between human MET and mouse MET. In preferred embodiments, the antibodies described herein recognize epitopes within the extracellular domain of MET that contain one or more amino acid residues conserved between human MET, mouse MET, rat MET, and monkey (e.g., cynomolgus monkey) MET.

[0105] In some embodiments, the antibody of the present invention recognizes an epitope of human MET located in the region of amino acid residues 123 to 223. In some embodiments, the antibody of the present invention recognizes an epitope of human MET located in the region of amino acid residues 224 to 311. In some embodiments, the antibody of the present invention recognizes an epitope of human MET located in the region of amino acid residues 314 to 372. In some embodiments, the antibody of the present invention recognizes an epitope of human MET located in the region of amino acid residues 546 to 562.

[0106] In some embodiments, the antibody or antigen-binding fragment of the present invention recognizes an epitope of human MET containing the amino acid residue Ile367. In some embodiments, the antibody or antigen-binding fragment of the present invention recognizes an epitope of human MET containing the amino acid residue Asp372. In some embodiments, the antibody or antigen-binding fragment of the present invention recognizes an epitope of human MET containing the amino acid residues Ile367 and Asp372.

[0107] In some such embodiments, the antibody or antigen-binding fragment of the present invention recognizes an epitope of human MET located in the region of amino acid residues 314-372 and containing the amino acid residue Ile367. In some such embodiments, the antibody or antigen-binding fragment of the present invention recognizes an epitope of human MET located in the region of amino acid residues 314-372 and containing the amino acid residue Asp371. In some such embodiments, the antibody or antigen-binding fragment of the present invention recognizes an epitope of human MET located in the region of amino acid residues 314-372 and containing the amino acid residues Ile367 and Asp372.

[0108] In some embodiments, the antibody or antigen-binding fragment of the present invention binds to an epitope of human MET containing the amino acid residue Thr555 of human MET.

[0109] In some such embodiments, the antibody or antigen-binding fragment of the present invention recognizes an epitope of human MET located in the region of amino acid residues 546-562 and containing amino acid residue Thr555.

[0110] An antibody or antigen-binding fragment can recognize an epitope consisting of multiple amino acid residues. This epitope may be linear, steric, or a combination thereof. If the epitope is identified as existing within a certain amino acid region, it may be formed by one or more amino acids within that region that the antibody or fragment contacts. Thus, in some embodiments of the present invention, it will be found that an antibody or fragment can recognize an epitope consisting of multiple (contiguous or discontinuous) amino acid residues (e.g., amino acids 314-372 or 546-562) within a specific region, provided that the recognized epitope contains specific amino acid residues (e.g., Ile367, Asp372, Thr555). Methods for identifying residues recognized as part of an antibody epitope are known to those skilled in the art, including, for example, the methods described in Examples 4 and 26.

[0111] The anti-MET antibody and antigen-binding fragment of the present invention bind to an epitope that overlaps with the binding domain recognized by HGF, or to an epitope near that binding domain, and can therefore compete (at least partially) with HGF for binding to homologous METs (i.e., they compete with human HGF for binding to hMETs and with mouse HGF for binding to mMets). In other words, this antibody or antigen-binding fragment directly or indirectly prevents HGF from binding to homologous METs in a binding assay (e.g., the ELISA described in Example 5). Therefore, in some embodiments, the MET antibody and antigen-binding fragment of the present invention compete with mouse HGF and human HGF for binding to homologous METs. Antibodies or antigen-binding fragments that thus compete with HGF are also referred to herein as "HGF competitors." Assays that determine whether an antibody or antigen-binding fragment competes with HGF for binding to METs are well known to those skilled in the art. For example, in a competitive ELISA, HGF competitors have an IC50 or lower IC50. 50 and / or at least 50% max(Maximum competition rate at saturation) is shown. The antibody and antigen-binding fragment of the present invention compete with mouse HGF for binding to mMET and with human HGF for binding to hMET.

[0112] The antibody or antigen-binding fragment of the present invention may "fully compete" or "partially compete" with HGF for binding to homologous MET. In this context, "fully competitive" means an IC in a competitive assay (e.g., ELISA). 50 less than 2 nM and / or I max An antibody or antigen-binding fragment having at least 90% IC50 is possible. In some embodiments, the "overall competitor" is less than 1 nM IC50. 50 and / or more than 90% max This indicates that, as a "partial competitor," it exhibits an IC50 of 2–5 nM in competitive assays (e.g., ELISA). 50 and / or 50-90% max Antibodies or antigen-binding fragments exhibiting this characteristic are possible. The values ​​listed apply to competition with mouse HGF and human HGF for binding to homologous METs.

[0113] As already described, the antibodies and antigen-binding fragments of the present invention are advantageous because they have the ability to recognize both human MET and mouse MET. The antibodies or antigen-binding fragments described herein are particularly advantageous if they exhibit equivalent properties when bound to mMET and when bound to hMET. This equivalence allows the antibodies to be analyzed in preclinical mouse models with the expectation that they will exhibit the same or similar properties in a human context.

[0114] Therefore, in some embodiments, the antibody and antigen-binding fragment of the present invention exhibit equivalent binding affinity to hMET and mMET. In this context, "equivalent binding affinity" means that the affinity of the antibody or antigen-binding fragment to hMET is 0.5 to 1.5 times that of the antibody to mMET. In some embodiments, the antibody and antigen-binding fragment of the present invention have an affinity for hMET that is 0.8 to 1.2 times that of mMET.

[0115] For the purpose of clarification and illustration, it should be noted that when an antibody or antigen-binding fragment has equivalent affinity for mMET and hMET, the off-rate for hMET may be 0.5 to 1.5 times that for mMET when measured as a Fab fragment. For example, an antibody or antigen-binding fragment with equivalent affinity for mMET and hMET may have an off-rate of 2.6 × 10⁻¹⁶ for hMET. -3 Antibodies showing an off-rate of 1.3–3.9 × 10⁶ / second against hMET -3 It is thought to exhibit an off-rate of / second. As a further example, antibodies or antigen-binding fragments with equivalent affinity for mMET and hMET will have an EC of hMET (determined by, for example, ELISA or flow cytometry). 50 However, the EC of this antibody or fragment related to mMET 50 This could be 0.5 to 1.5 times. For example, if it has equivalent affinity for mMET and hMET, and the EC for mMET is 0.1 nanomoles / l 50 Antibodies exhibiting this characteristic have an EC of 0.05-0.15 nanomoles / l with respect to hMET. 50 This is thought to indicate that.

[0116] In some embodiments, the antibody and antigen-binding fragment of the present invention are equivalent agonists for mMET and hMET. In this context, “equivalence” means that the level of MET activation induced upon binding to hMET is 0.5 to 1.5 times the level of signaling induced upon binding to mMET. In some embodiments, the antibody and antigen-binding fragment of the present invention induce MET signaling upon binding to hMET at a level 0.8 to 1.2 times that of signaling induced upon binding to mMET.

[0117] In some embodiments, the antibodies and antigen-binding fragments of the present invention are equivalent agonists of mMET and hMET when measured by at least one MET activation assay described herein. For example, the antibodies or antigen-binding fragments of the present invention induce equivalent phosphorylation of MET and / or exhibit equivalent protective effects against drug-induced apoptosis and / or induce equivalent levels of branching in branching morphogenesis assays. In some embodiments, this antibody or antigen-binding fragment exhibits equivalent MET activation when measured by all of the assays described herein.

[0118] To clarify, the equivalent phosphorylation of MET by the antibody of the present invention is equivalent to the EC of that antibody with respect to hMET. 50 However, regarding mMET 50 It can be measured as being 0.5 to 1.5 times that. For example, EC related to mMET 50 If the concentration is 2.9 nM, the antibody is related to EC2 of hMET. 50 When the value is in the range of 1.45 to 4.35 nM, it is thought to induce equivalent phosphorylation of hMET. Similarly, equivalent MET activation shown in anti-apoptotic assays is observed in human cells compared to mouse cells. max E is 0.5 to 1.5 times max It can be detected as becoming E in mouse cells. max If the percentage is 37.5%, then the antibody is related to human cells. maxIf the concentration is in the range of 18.75-56.25%, it is considered to be an equivalent hMET agonist. Equivalent MET activation, as demonstrated by branching morphogenesis assays, can be detected when the number of branches observed in human cell spheroids after exposure to this antibody is 0.5-1.5 times the number of branches observed in mouse cell spheroids after exposure to the same (non-zero) concentration of this antibody. For example, if the number of branches shown in mouse cells after exposure to 0.5 nM antibody is 14, then if the number of branches shown in human cells after exposure to 0.5 nM antibody is in the range of 7-21, then that antibody is considered to be an equivalent hMET agonist.

[0119] Similarly, the equivalent activating effect of hMET and mMET can be demonstrated by equivalent cell scattering. The nature of the results of such assays indicates that the application of a factor of 0.5–1.5 is inappropriate. In cell scattering assays, the equivalent activating effect of hMET and mMET can be demonstrated by the cell scattering score of human cells exposed to the antibody being ±1 of the cell scattering score of mouse cells exposed to the same (non-zero) concentration of antibody. For example, if mouse cells exposed to 0.33 nM of antibody show a cell scattering score of 2, then this antibody is considered an equivalent agonist of HGF if human cells exposed to the same 0.33 nM antibody show a cell scattering score of 1–3.

[0120] In some embodiments, the antibodies and antigen-binding fragments of the present invention compete with HGF to the same extent as mMET and hMET. In this context, "comparable competition with HGF" means that the level at which the antibody or antigen-binding fragment competes with human HGF to hMET is 0.5 to 1.5 times the level at which the antibody or antigen-binding fragment competes with mouse HGF to mMET. In some embodiments, the level at which the antibodies and antigen-binding fragments of the present invention compete with human HGF to hMET is 0.8 to 1.2 times the level at which the antibody or antigen-binding fragment competes with mouse HGF to mMET.

[0121] As an illustration, the ability of an antibody to compete equivalently with human HGF and mouse HGF may be detected as the IC 50 for an antibody competing with the binding of human HGF-hMET being 50 0.5 to 1.5 times that for an antibody competing with the binding of mouse HGF-mMET. For example, when the IC 50 for the binding of mHGF-mMET is 0.34 nM, the antibody competes equivalently with hHGF and mHGF if the IC 50 for the binding of hHGF-hMET is in the range of 0.17 to 0.51 nM.

[0122] In some embodiments, the antibodies and antigen-binding fragments of the invention cross-react with rat MET and / or cynomolgus monkey MET. Cross-reactivity with one or both of rat MET and cynomolgus monkey MET has the advantage that toxicity studies can be conducted in rat and / or cynomolgus monkey model systems. In this regard, whether an antibody exhibits cross-reactivity with cynomolgus monkey MET or rat MET can be determined by ELISA as described in Example 25 attached hereto.

[0123] The antibodies or antigen-binding fragments described herein can comprise at least one hypervariable loop or complementarity-determining region obtained from a VH domain or VL domain of a species of camelid. In particular, such antibodies or antigen-binding fragments can comprise a VH domain and / or VL domain, or CDRs thereof, obtained by actively immunizing a crossbred camelid (e.g., llama) with a human MET antigen.

[0124] The phrase "hypervariable loop or complementarity-determining region obtained from the VH or VL domain of one species of camelid" means that the hypervariable loop (HV) or CDR has the same or substantially the same amino acid sequence as the hypervariable loop or CDR encoded by the camelid immunoglobulin gene. In this context, "immunoglobulin gene" includes germline genes, rearranged immunoglobulin genes, and somatically mutated genes. Therefore, the amino acid sequence of the HV or CDR obtained from the VH or VL domain of one species of camelid may be the same as the amino acid sequence of the HV or CDR present in a typical antibody of a mature camelid. In this context, the expression "obtained from" implies a structural relationship, meaning that the HV or CDR of the MET antibody realizes the amino acid sequence (or a minor variant thereof) that was originally encoded by the camelid immunoglobulin gene. However, this does not necessarily imply a special relationship regarding the manufacturing process used to prepare the MET antibody.

[0125] MET antibodies derived from camelids can originate from any species of camelid, including, in particular, llamas, dromedaries, alpacas, vicuñas, guanacos, and camels.

[0126] MET antibodies containing the VH and VL domains or their CDRs derived from camelid species are typically recombinant polypeptides, and chimeric polypeptides are possible. The term "chimeric polypeptide" refers to an artificial (non-natural) polypeptide, which is created by juxtaposing two or more non-contiguous peptide fragments. This definition includes "species" chimeric polypeptides created by juxtaposing peptide fragments encoded by two or more species (e.g., camel and human).

[0127] CDRs derived from camelids may include one of the CDR sequences shown in Tables 3 and 4 below.

[0128] In one embodiment, the entire VH domain and / or the entire VL domain can be obtained from a single species of camelid. In a particular embodiment, the camelid-derived VH domain may contain any of the amino acid sequences shown as SEQ ID NOs: 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, while the camelid-derived VL domain may contain any of SEQ ID NOs: 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176. Next, the camelid-derived VH domain and / or camelid-derived VL domain can be subjected to protein manipulation to introduce one or more amino acid substitutions, insertions, or deletions into the camelid amino acid sequence. Preferably, these changes by manipulation include amino acid substitutions relative to the camelid. Such changes include "humanization" or "germlineization," in which one or more amino acid residues in the VH domain or VL domain encoded by camelids are replaced by equivalent residues in the homologous VH domain or VL domain encoded by humans.

[0129] The isolated camelid VH and VL domains obtained by active immunization of camelids (e.g., llamas) with human MET antigens can be used as a basis for manipulating MET antibodies according to the present invention. Starting from the complete camelid VH and VL domains, it is possible to introduce one or more amino acid substitutions, insertions, or deletions that deviate from the starting camelid sequence. In some embodiments, such substitutions, insertions, or deletions may be located in the framework region of the VH and / or VL domains. The purpose of such alteration of the primary amino acid sequence may be to reduce properties that are considered unfavorable (e.g., immunogenicity in the human host (so-called humanization)), or to reduce sites of potential product heterogeneity and / or instability (e.g., glycosylation, deamidation, isomerization, etc.), or to enhance several other favorable properties of the molecule (e.g., solubility, stability, bioavailability, etc.). In another embodiment, alterations to the primary amino acid sequence can be introduced into one or more hypervariable loops (or CDRs) of the VH domain and / or VL domain of camelids obtained by active immunization. Such alterations can be introduced to enhance antigen-binding affinity and / or specificity, or to reduce properties that are considered unfavorable (e.g., immunogenicity in the human host (so-called humanization)), or to reduce sites of potential product heterogeneity and / or instability (e.g., glycosylation, deamidation, isomerization, etc.), or to enhance several other favorable properties of the molecule (e.g., solubility, stability, bioavailability, etc.).

[0130] In one embodiment, the present invention provides a variant MET antibody containing at least one amino acid substitution in at least one framework region or CDR region of a VH domain or VL domain compared to a camelid VH domain or VL domain. Non-limiting examples of camelid VH domains or VL domains include camelid VH domains containing amino acid sequences shown as SEQ ID NOs: 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, and 175, and camelid VL domains containing SEQ ID NOs: 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, and 176.

[0131] In some embodiments, a “chimeric” antibody molecule is provided comprising a VH domain and a VL domain (or an engineered variant thereof) derived from a camelid antibody, and one or more constant domains from a non-camelid antibody (e.g., a constant domain encoding human (or an engineered variant thereof)). In such embodiments, it is preferable that both the VH and VL domains are obtained from the same species of camelid, for example, both VH and VL can be from a llama (before being engineered to introduce changes in the amino acid sequence). In such embodiments, both the VH and VL domains can be derived from a single animal, in particular a single animal actively immunized with the human MET antigen.

[0132] In some embodiments, the present invention encompasses chimeric camelid / human antibodies, and in particular, chimeric antibodies in which the VH and VL domains are entirely camelid (e.g., llama or alpaca) sequences and the remainder is entirely human. MET antibodies may include antibodies containing a "humanized" or "germline" variant of the camelid-derived VH and VL domains or their CDRs, and camelid / human chimeric antibodies containing one or more amino acid substitutions in the framework regions of the VH and VL domains compared to camelid VH and VL domains obtained by active immunization of camelids using human MET antigens. Such "humanization" increases the % sequence matching with the human germline VH or VL domain by replacing mismatched amino acid residues in the starting camelid VH or VL domain with equivalent residues found in the VH or VL domain encoding the human germline.

[0133] In some embodiments, the present invention includes chimeric camelid / mouse antibodies, and in particular, chimeric antibodies in which the VH and VL domains are entirely camelid (e.g., llama or alpaca) sequences and the remainder is entirely mouse sequences.

[0134] The present invention allows for CDR-grafted antibodies in which a CDR (or hypervariable loop) derived from a camelid antibody (e.g., a camelid MET antibody produced by active immunization using human MET protein), or a CDR (or hypervariable loop) encoded by a camelid gene in a different manner, is grafted onto a human VH and VL framework, with the remainder being entirely of human origin. Such CDR-grafted MET antibodies may contain CDRs having the amino acid sequences shown in Tables 3 and 4 below.

[0135] Camelid-derived MET antibodies include variants in which the hypervariable loops or CDRs of the VH and / or VL domains are obtained from a normal camelid antibody against human MET, but at least one of the (camelid-derived) hypervariable loops or CDRs is manipulated to contain one or more amino acid substitutions, additions, or deletions compared to the sequence encoded by camelid. Such modifications include "humanization" of the hypervariable loop / CDR. The camelid-derived HV / CDR thus manipulated can still exhibit an amino acid sequence that is "substantially identical" to the amino acid sequence of the camelid-encoded HV / CDR. "Substantially identical" in this context means that one or fewer, or two or fewer, amino acid mismatches are acceptable compared to the camelid-encoded HV / CDR. Special embodiments of MET antibodies may contain humanized variants of the CDR sequences shown in Tables 3 and 4.

[0136] Typical antibodies in camelids (e.g., llamas) are the following factors discussed in United States Patent Application Publication No. 12 / 497,239 (the contents of which are incorporated herein by reference): 1) Large % sequence homology with the VH and VL domains of camelids; 2) Significant structural homology between the CDRs of the VH and VL domains of camelids and their human counterparts (i.e., the human-like standard folding structure and the human-like combination of the standard folding). For this reason, it provides a favorable starting point for preparing antibodies useful as human therapeutic agents. A camelid (e.g., llama) platform can also offer significant advantages in terms of the functional diversity of the MET antibodies that can be obtained.

[0137] The usefulness of human therapeutic MET antibodies containing the VH and / or VL domains of camelids can be further improved by "humanizing" the natural camelid VH and VL domains to reduce their immunogenicity in the human host. The overall goal of humanization is to create molecules in which the VH and VL domains exhibit minimal immunogenicity when introduced into human subjects, while maintaining the specificity and affinity of the antigen-binding sites formed by the parent VH and VL domains.

[0138] One approach to humanization is called "germlineization," which involves altering the amino acid sequence of the VH or VL domain of a camelid to more closely resemble the germline sequence of a human VH or VL domain.

[0139] Determining homology between camelid VH (or VL) domains and human VH (or VL) domains is a crucial step in the humanization process, both in selecting which camelid amino acid residues to change (in a given VH or VL domain) and in selecting appropriate substitution amino acid residues.

[0140] One approach has been developed to germline-enable typical camelid antibodies, based on the alignment of numerous novel camelid VH(and VL) domain sequences (typically somatically mutant VH(or VL) domain sequences known to bind to target antigens) with human germline VH(or VL) domain sequences and human VH(or VL) consensus sequences, as well as germline sequence information available for alpacas (lama pacos).

[0141] This procedure is described in WO 2011 / 080350 (the contents of which are incorporated herein by reference) and can be applied to (i) the selection of “camelid” amino acid residues to be replaced in a camelid-derived VH domain or VL domain or CDR, and (ii) the selection of “human” amino acid residues to be substituted when humanizing any given camelid VH (or VL) domain. Using this approach, humanized variants of camelid-derived CDRs having the amino acid sequences shown in Tables 3 and 4 can be prepared, and further germline-chained camelid-derived VH and VL domains having the sequences shown in Table 5 can also be prepared.

[0142] MET antibodies can take on various different embodiments, each containing both a VH domain and a VL domain. In this specification, the term “antibody” is used in its broadest sense, and its non-limiting examples include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) that meet the condition of exhibiting appropriate immunological specificity to human and mouse MET proteins. In this specification, the term “monoclonal antibody” means an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in that population are identical except for any spontaneously occurring and potentially minute variations. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, unlike typical (polyclonal) antibody preparations, which generally contain different antibodies targeting different determinants (epitopes) on the antigen, each monoclonal antibody targets a single determinant, i.e., epitope, on the antigen.

[0143] An "antibody fragment" is a portion of a full-length antibody, generally containing its antigen-binding domain or variable domain. Examples of antibody fragments include Fab, Fab', F(ab')2, bispecific Fab, Fv fragments, dimeric antibodies, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from multiple antibody fragments (see Holliger and Hudson, Nature Biotechnol, Vol. 23: pp. 1126-1136, 2005 (the contents of which are incorporated herein by reference)).

[0144] In non-limiting embodiments, the MET antibodies presented herein may include a CH1 domain and / or a CL domain, and their amino acid sequence may be entirely or substantially human. When this MET antibody is intended for use in human therapy, it is typical that the entire constant region of the antibody, or at least a portion thereof, has an entirely or substantially human amino acid sequence. Therefore, the amino acid sequence of one or more of the CH1 domain, hinge region, CH2 domain, CH3 domain, CL domain (and, if present, the CH4 domain), or any combination thereof, may be entirely or substantially human. Such antibodies may consist of any human isotype (e.g., IgG1).

[0145] It is advantageous that all of the CH1 domain, hinge region, CH2 domain, CH3 domain, CL domain (and CH4 domain, if present) have a human amino acid sequence, either entirely or substantially. In the context of the constant region of a humanized antibody or chimeric antibody, or the constant region of an antibody fragment, the term “substantially human” means an amino acid sequence that is at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% identical to a human constant region. In this context, the term “human amino acid sequence” means the amino acid sequence encoded by a human immunoglobulin gene (including germline genes, rearranged genes, and somatically mutated genes). Such antibodies can consist of any human isotype (human IgG4 and IgG1 are particularly preferred).

[0146] MET antibodies containing constant domains of a “human” sequence modified by the addition, deletion, or substitution of one or more amino acids are also provided, except for embodiments where the presence of a “fully human” hinge region is clearly required.

[0147] The "fully human" hinge region within the MET antibody of the present invention may be advantageous in both minimizing the immunogenicity of the antibody and optimizing its stability.

[0148] Any isotype is possible for the MET antibodies presented herein. Antibodies intended for use in human therapy are typically of the IgA, IgD, IgE, IgG, and IgM types, and often of the IgG type. In the case of the IgG type, IgG can belong to any of the four subclasses IgG1, IgG2a or IgG2b, IgG3, or IgG4. Within each of these subclasses, it is permissible to introduce one or more amino acid substitutions, insertions, or deletions in the Fc portion, or to introduce other structural changes, for example, to enhance or reduce Fc-dependent function.

[0149] In non-limiting embodiments, we consider achieving substitution, insertion, or deletion of one or more amino acids within the constant regions of the heavy and / or light chains, particularly within the Fc region. Amino acid substitutions may involve replacing the substituted amino acid with a different native amino acid, a non-native amino acid, or a modified amino acid. Other structural changes are also permitted, such as changes in the glycosylation pattern (e.g., by the addition or deletion of an N-linked or O-linked glycosylation site). Depending on the intended use of the MET antibody, it may be desirable to modify the binding properties of the antibody of the present invention to the Fc receptor, for example, to modulate effector function.

[0150] In some embodiments, a MET antibody may include an Fc region of a given antibody isotype (e.g., human IgG1) that has been modified for the purpose of reducing or substantially eliminating one or more antibody effector functions naturally associated with that antibody isotype. In non-limiting embodiments, a MET antibody may substantially lack any antibody effector function. In this context, “antibody effector function” includes one or more, or all, of antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0151] The amino acid sequence of the Fc portion of a MET antibody may contain one or more mutations (e.g., amino acid substitutions, deletions, or insertions) that reduce the antibody effector function (compared to the corresponding wild-type antibody without the mutation). Several such mutations are known in the field of antibody engineering. Non-limiting examples suitable for inclusion in the MET antibodies described herein include mutations in the Fc domain of human IgG4 or human IgG1: N297A, N297Q, LALA (L234A, L235A), AAA (L234A, L235A, G237A), and D265A (amino acid residue numbers follow the EU numbering system in human IgG1).

[0152] The monoclonal antibodies or antigen-binding fragments that "cross-compete" with the MET antibodies disclosed herein are monoclonal antibodies or antigen-binding fragments that bind to human MET at the same or overlapping site as the MET antibody of the present invention, and also bind to mouse MET at the same or overlapping site as the MET antibody of the present invention. Competing monoclonal antibodies or antigen-binding fragments can be identified, for example, through an antibody competition assay. For example, a purified or partially purified sample of human MET can be bound to a solid support. Next, the antibody compound or antigen-binding fragment of the present invention and a monoclonal antibody or antigen-binding fragment that is thought to be able to compete with such an antibody compound of the present invention are added. One of the two molecules is labeled. If the labeled compound and the unlabeled compound bind to separate, distant sites on MET, the labeled compound will bind at the same level regardless of whether or not a competing compound is present. However, if the interaction sites are the same or overlap, the unlabeled compound will compete, and the amount of the labeled compound that binds to the antigen will be reduced. If there is an excess of the unlabeled compound, the labeled compound will bind, if at all, only in a small amount. For the purposes of the present invention, a competing monoclonal antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment that reduces the binding of the antibody compound of the present invention to MET by about 50%, or about 60%, or about 70%, or about 80%, or about 85%, or about 90%, or about 95%, or about 99%. Details of the procedure for performing such a competing assay are well known in the art and can be found, for example, in Harlow and Lane, "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988, pp. 567-569, 1988, ISBN 0-87969-314-2. Such assays can be made quantifiable by using purified antibodies.A standard curve is established by titrating one antibody against itself, i.e., by using the same antibody as both the label and the competitor. The ability of an unlabeled competing monoclonal antibody or its antigen-binding fragment to inhibit the binding of the labeled molecule to the plate is titrated. The results are plotted and compared to the concentrations required to achieve the desired degree of binding inhibition.

[0153] The present invention also provides a polynucleotide molecule encoding the MET antibody of the present invention, an expression vector containing a nucleotide sequence functionally linked to a regulatory sequence that encodes the MET antibody of the present invention and enables the expression of an antigen-binding polypeptide in a host cell or a cell-free expression system, and a host cell or cell-free expression system containing this expression vector.

[0154] The polynucleotide molecules encoding the MET antibody of the present invention include, for example, recombinant DNA molecules. In this specification, the terms “nucleic acid,” “polynucleotide,” and “polynucleotide molecule” are interchangeable and mean any single-stranded or double-stranded DNA or RNA molecule (in the case of a single-stranded molecule, a molecule with a complementary sequence). In discussions of nucleic acid molecules, the sequence or structure of a particular nucleic acid molecule can be described according to the usual convention of indicating the sequence in the 5' to 3' direction. In some embodiments of the present invention, the nucleic acid or polynucleotide is “isolated.” When applied to a nucleic acid molecule, this term means a nucleic acid molecule isolated from a contiguous sequence in the natural genome of the organism from which it originates. For example, “isolated nucleic acid” can include a DNA molecule inserted into a vector (e.g., a plasmid or viral vector), or a DNA molecule integrated with the genomic DNA of a prokaryotic or eukaryotic cell or a non-human host organism. When applied to RNA, “isolated polynucleotide” means the RNA molecule encoded by the above-mentioned isolated DNA molecule. Alternatively, this term can mean an RNA molecule purified / isolated from other nucleic acids that may be involved in their natural state (i.e., in cells or tissues). Isolated polynucleotides (DNA or RNA) can further be directly produced by biological or synthetic means, representing molecules separated from other compounds present during their production.

[0155] To recombinantly produce the MET antibody of the present invention, the recombinant polynucleotide encoding it can be prepared (using standard molecular biology techniques), inserted into a replicable vector, and expressed in a selected host cell or cell-free expression system. Suitable host cells include prokaryotic cells, yeast cells, or higher eukaryotic cells, particularly mammalian cells. Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension medium, Graham et al., J. Gen. Virol. Vol. 36: pp. 59-74, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA Vol. 77: pp. 4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod. Vol. 23: pp. 243-252, 1980); mouse myeloma cells SP2 / 0-AG14 (ATCC CRL 1581; ATCC CRL 8287) or NS0 (HPA Culture Collection No. (No. 85110503): Monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human liver cells (Hep G2, HB 8065); Mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. Vol. 383: pp. 44-68, 1982); MRC5 cells; FS4 cells; Human hepatoma cell line (Hep G2); DSM's PERC-6 cell line. Expression vectors suitable for use with each of these host cells are also generally known in this field.

[0156] It should be noted that the term "host cell" generally refers to a cultured cell line. The entire human body into which the expression vector encoding the antigen-binding polypeptide of the present invention has been introduced is clearly excluded from the definition of "host cell."

[0157] In one important aspect, the present invention also provides a method for producing the MET antibody of the present invention, comprising culturing host cells (or a cell-free expression system) containing a polynucleotide encoding the MET antibody (e.g., an expression vector) under conditions that allow the expression of the MET antibody, and then recovering the expressed MET antibody. This recombinant expression process can be used for the large-scale production of the MET antibody of the present invention (including monoclonal antibodies intended for use in human therapeutics). Appropriate vectors, cell systems, and manufacturing processes for the large-scale production of recombinant antibodies suitable for use in vivo therapeutics are generally available in the art and will be well known to those skilled in the art.

[0158] The MET antibodies presented herein are useful for therapeutic use, particularly in the treatment of diseases, especially those in which stimulating MET function is beneficial. Non-limiting examples of diseases include degenerative diseases, inflammatory diseases, autoimmune diseases, metabolic diseases, transplant-related disorders, and wound healing. In this regard, the MET antibodies presented herein are examples of a broader class of MET agonists (e.g., HGF) that are useful in the treatment of the aforementioned diseases.

[0159] Hepatocytes express MET and are therefore a primary target of HGF, which promotes hepatocyte proliferation and protects hepatocytes from apoptosis. This specification demonstrates that the MET antibody of the present invention, which induces MET signaling, protects hepatocytes in mouse models of liver injury (both acute and chronic liver injury) (Examples 16 and 17). Since the antibody of the present invention exhibits equivalent properties in human and mouse strains, as already described herein, it can be expected to provide similar protective effects in the context of human liver injury. Therefore, one aspect of the present invention provides a method for treating or preventing liver injury in human patients, comprising administering a therapeutically effective amount of a MET antibody that induces MET signaling to a patient in need. In some embodiments, this method is a method for treating or preventing acute liver injury. In some embodiments, this method is a method for treating or preventing chronic liver injury. In some embodiments, the antibody is one of the antibodies described herein.

[0160] Because renal epidermal cells express MET at considerable levels, they are sensitive to HGF stimulation. This specification demonstrates that MET antibodies that induce MET signaling provide protection in a mouse model of chronic kidney injury (Example 18). As already described herein, the antibodies of the present invention exhibit equivalent properties in human and mouse strains, and can therefore be expected to provide similar protective effects in the context of human kidney injury. Thus, one aspect of the present invention provides a method for treating or preventing kidney injury in human patients, comprising administering a therapeutically effective amount of a MET antibody that induces MET signaling to a patient in need. In some embodiments, this method is a method for treating or preventing acute kidney injury. In some embodiments, the antibody is one of the antibodies described herein.

[0161] This specification also reveals that administration of MET antibodies that induce MET signaling provides an effective treatment in a mouse model of inflammatory bowel disease (IBD) (e.g., ulcerative colitis) (Examples 19 and 20). Thus, one aspect of the present invention provides a method for treating or preventing IBD in human patients, comprising administering a therapeutically effective amount of MET antibodies that induce MET signaling to patients in need. In some embodiments, this method is a method for treating or preventing ulcerative colitis. In some embodiments, the antibody is one of the antibodies described herein.

[0162] This specification further demonstrates that metabolic function can be restored in diabetes (including both type 1 and type 2 diabetes) by administering MET antibodies that induce MET signaling (Examples 21 and 22). In particular, in a model of type 1 diabetes (Example 21), it is shown that the MET antibody promotes glucose uptake. Furthermore, administration of the MET antibody together with insulin resulted in a synergistic effect on glucose uptake. In a model of type 2 diabetes (Example 22), it is shown that the MET antibody normalizes glucose control and reduces insulin resistance. Thus, one aspect of the present invention provides a method for treating or preventing diabetes in human patients, comprising administering a therapeutically effective amount of a MET antibody that induces MET signaling to a patient in need. In some embodiments, this method is a method for treating or preventing type 1 diabetes. In some such embodiments, this method further includes administering insulin to the patient. In some embodiments, this method is a method for treating type 2 diabetes. In some embodiments, the antibody is one of the antibodies described herein.

[0163] This specification further demonstrates that the degree of fatty liver can be reduced in a mouse model of non-alcoholic steatohepatitis (NASH) by administering MET antibodies that induce MET signaling (Example 23). In particular, the MET antibodies were able to reduce the number of adipocytes and alleviate the degree of fibrosis. Thus, one aspect of the present invention provides a method for treating or preventing NASH in human patients, comprising administering a therapeutically effective amount of MET antibodies that induce MET signaling to patients in need. In some embodiments, the antibody is one of the antibodies described herein.

[0164] This specification further demonstrates that wound healing can be promoted by administering MET antibodies that induce MET signaling (Example 24). Furthermore, MET antibodies were able to promote wound healing in diabetic mice that had poor wound healing. Thus, one aspect of the present invention provides a method for promoting wound healing in human patients, comprising administering a therapeutically effective amount of MET antibodies that induce MET signaling to patients in need. In some embodiments, the human patients have diabetes, possibly type 1 diabetes. In some embodiments, the antibody is one of the antibodies described herein. [Examples]

[0165] The present invention will be better understood by referring to the following non-limiting examples relating to experiments.

[0166] Example 1: Immunization of llamas

[0167] Immunization of llamas and collection of peripheral blood lymphocytes (PBLs), as well as subsequent RNA extraction and amplification of antibody fragments, were carried out as described in the paper (de Haard et al., J. Bact. Vol. 187: pp. 4531-4541, 2005). Immunization of two adult llamas (Lama glama) was achieved by intramuscular injection of a chimeric protein (MET-Fc; R&D Systems) consisting of the extracellular domain (ECD) of human MET fused to the Fc portion of human IgG1. Each llama received a total of six injections, once a week for six weeks. Each injection consisted of a Freund's incomplete adjuvant containing 0.2 mg of protein, which was divided and administered to two locations in the neck.

[0168] Blood samples of 10 ml were collected before and after immunization to examine the immune response. Approximately one week after the final immunization, 400 ml of blood was collected, and PBLs were obtained using the Ficoll-Paque method. Total RNA was extracted using the phenol-guanidine thiocyanate method (Chomczynski et al., Analyst. Biochem. Vol. 162: pp. 156-159, 1987) and used as a template for random cDNA synthesis using the SuperScript™ III First-Strand Synthesis System kit (Life Technologies). As described in the paper (de Haard et al., J Biol Chem. Vol. 274: pp. 18218-18230, 1999), cDNA encoding the VH-CH1 region and VL-CL domains (κ and λ) of Lama IgG1 was amplified, and subcloning into the phagemide vector pCB3 was performed. Recombinant phagemids were used to transform E. coli strain TG1 (Netherland Culture Collection of Bacteria), and four different Fab-expressing phage libraries were created (one lambda library and one kappa library per immunized llama). The diversity was 10. 8 ~10 9 It was within the range.

[0169] The immune response to the antigen was investigated by ELISA. For this purpose, the ECD of human MET (UniProtKB # P08581; amino acids 1-932) and mouse MET (UniProtKB # P16056.1; amino acids 1-931) were obtained using standard protein engineering techniques. Recombinant human or mouse MET ECD proteins were immobilized on a solid phase (100 ng / well in a 96-well plate) and exposed to serial dilutions of serum from llamas before (day 0) or after (day 45) immunization. Binding was elucidated using mouse anti-llama IgG1 (Daley et al., Clin. Vaccine Immunol. Vol. 12, 2005) and HRP-labeled donkey anti-mouse antibody (Jackson Laboratories). As shown in Figure 1, both llamas showed an immune response to human MET ECD. In line with the 87% homology observed between the extracellular portion of human MET and the mouse ortholog, extremely high cross-reactivity was also observed in mouse MET ECD.

[0170] Example 2: Selection and screening of Fabs that bind to both human and mouse METs.

[0171] Fab-expressing phages were prepared from the above library according to a standard phage presentation protocol. For selection, phages were first adsorbed onto immobilized recombinant human MET ECDs, washed, and then eluted with trypsin. After two cycles of selection using human MET ECDs, two more cycles were performed in the same manner using mouse MET ECDs. In parallel, phage selection was also performed by alternating between human and mouse MET ECD cycles for a total of four cycles. The phages selected by these two approaches were pooled and used to infect TG1 E. coli. After isolating individual colonies, Fab secretion was induced using IPTG (Fermentas). Peripheral plasma fractions containing bacterial Fab were collected, and the ability of these fractions to bind to human and mouse MET ECDs was examined by surface plasmon resonance (SPR). Human or mouse MET ECDs were immobilized on CM-5 tips using amine coupling in sodium acetate buffer (GE Healthcare). Fab-containing peripheral plasma extract was loaded into a BIACORE 3000 instrument (GE Healthcare) at a flow rate of 30 μl / min. The off-rate of Fab (k off The ) was measured over a period of 2 minutes. The binding of Fab to human MET and mouse MET was further characterized by ELISA using MET ECD in solid phase and crude extract in solution. Since Fab was manipulated with the MYC flag, binding was elucidated using an HRP-labeled anti-MYC antibody (ImTec Diagnostics).

[0172] Fabs that bound to both human and mouse METs were selected using both SPR and ELISA, and the phages corresponding to these Fabs were sequenced (LGC Genomics). The cross-reactive Fab sequences were divided into multiple families based on the length and content of the VH CDR3 sequence. The VH families were assigned internal numbers not based on IMTG (International Immunogenetic Information System) nomenclature. Ultimately, 11 different human / mouse cross-reactive Fabs belonging to eight VH families were identified. The CDR and FR sequences of the heavy chain variable region are shown in Table 3. The CDR and FR sequences of the light chain variable region are shown in Table 4. The complete amino acid sequences of the heavy chain and light chain variable regions are shown in Table 5. The complete DNA sequences of the heavy chain and light chain variable regions are shown in Table 6.

[0173] [Table 3]

[0174] [Table 4]

[0175] [Table 5]

[0176] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0177] Table 7 shows various Fab families and their ability to bind to human MET and mouse MET.

[0178] [Table 7]

[0179] Example 3: Chimera Fab is placed in mAb.

[0180] The cDNA encoding the VH and VL(κ or λ) domains of the selected Fab fragment was manipulated and placed into two separate pUPE mammalian expression vectors (U-protein Express) containing cDNA encoding human IgG1 or human CL(κ or λ) CH1, CH2, and CH3, respectively. The complete amino acid sequences of the heavy and light chains of the llama-human chimeric antibody are shown in Table 8.

[0181] [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0182] The production of the obtained chimerama-human IgG1 molecule (by transient transfection of mammalian cells) and purification (by protein A affinity chromatography) were outsourced to U-protein Express. Binding of chimeric mAbs to MET was examined by ELISA using hMET ECD or mMET ECD in solid phase and increasing concentrations of antibody (0-20 nM) in solution. Binding was confirmed using HRP-labeled anti-human Fc antibody (Jackson Immuno Research Laboratories). This analysis showed that all chimerama-human antibodies bound to human MET and mouse MET with picomolar affinity, and to EC2 at concentrations of 0.06 nM-0.3 nM. 50was revealed. The binding ability (E MAX ) differed for each antibody, probably because the epitope exposure in the immobilized antigen was partial, but was the same in the human and mouse settings. The values of EC 50 and E MAX are shown in Table 9.

[0183] [Table 9]

[0184] It was also analyzed whether the chimeric anti-MET antibody binds to native human and mouse MET in living cells. For that purpose, increasing concentrations of the antibody (0 - 100 nM) were incubated with A549 human lung cancer cells (American Type Culture Collection) or MLP29 mouse liver progenitor cells (gift from Professor Enzo Medico, University of Torino, Strada Provinciale 142 km 3.95, Candiolo, Torino, Italy; Medico et al., Mol Biol Cell Vol. 7, pages 495 - 504, 1996) (both express MET at physiological levels). Binding of the antibody to the cells was analyzed by flow cytometry using a phycoerythrin-labeled anti-human IgG1 antibody (eBioscience) and a CyAn ADP analyzer (Beckman Coulter). As a positive control for binding to human MET, a commercially available mouse anti-human MET antibody (R&D Systems) and a phycoerythrin-labeled anti-mouse IgG1 antibody (eBioscience) were used. As a positive control for binding to mouse MET, a commercially available goat anti-mouse MET antibody (R&D Systems) and a phycoerythrin-labeled anti-goat IgG1 antibody (eBioscience) were used. All antibodies showed dose-dependent binding to both human and mouse cells, and EC 50 was 0.2 nM - 2.5 nM. Consistent with the data obtained by ELISA, the maximum binding (E MAXThe results differed depending on the antibody, but were similar in human and mouse cells. These results indicate that in both human and mouse cell lines, the chimerama-human antibody recognizes membrane-bound MET in its native configuration. 50 and E MAX The values ​​are shown in Table 10.

[0185] [Table 10]

[0186] Example 4: Receptor region important for antibody binding

[0187] To map the receptor regions recognized by antibodies that bind to both human and mouse MET (hereinafter referred to as human / mouse equivalent anti-MET antibodies), we measured the ability of these antibodies to bind to a group of manipulated proteins derived from human MET, prepared as described in the paper (Basilico et al., J Biol. Chem. Vol. 283, pp. 21267-21227, 2008). This group included (Figure 2) the entire MET ECD (decoy MET); MET ECD lacking IPT domains 3 and 4 (SEMA-PSI-IPT 1-2); MET ECD lacking IPT domains 1-4 (SEMA-PSI); isolated SEMA domain (SEMA); and fragments containing IPT domains 3 and 4 (IPT 3-4). The manipulated MET proteins were immobilized on a solid phase and exposed to solutions containing increasingly concentrated chimeric antibodies (0-50 nM). Binding was elucidated using HRP-labeled anti-human Fc antibodies (Jackson Immuno Research Laboratories). As shown in Table 11, this analysis revealed that seven mAbs recognized epitopes within the SEMA domain, while the other four recognized epitopes within the PSI domain.

[0188] [Table 11]

[0189] To more precisely map the regions of MET that are important for antibody binding, we examined the absence of cross-reactivity between our antibodies and llama MET (the organism used to generate these immunoglobulins). For this purpose, a series of llama-human chimeric MET proteins and human-llama chimeric MET proteins spanning the entire MET ECD were generated as described in the paper (Basilico et al., J Clin Invest. Vol. 124, pages 3172 - 3186, 2014). After immobilizing the chimeras (Figure 3) in the solid phase, they were exposed to solutions containing increasing concentrations of mAb (0 - 20 nM). Binding was revealed using HRP-labeled anti-human Fc antibody (Jackson Immuno Research Laboratories). This analysis revealed that five SEMA-binding mAbs (71D6, 71C3, 71D4, 71A3, 71G2) recognize epitopes located at amino acids 314 - 372 of human MET. This corresponds to the region corresponding to blades 4 - 5 of the β-propeller of SEMA with seven blades (Stamos et al., EMBO J. Vol. 23, pages 2325 - 2335, 2004). The other two SEMA-binding mAbs (74C8, 72F8) recognize epitopes located at 123 - 223 and 224 - 311, respectively. These correspond to blades 1 - 3 and 1 - 4 of the β-propeller of SEMA. The PSI-binding mAbs (76H10, 71G3, 76G7, 71G12) did not appear to show significant binding to either of the two PSI chimeras. Considering the results shown in Table 11, these antibodies probably recognize epitopes located at amino acids 546 - 562 of human MET. These results are summarized in Table 12.

[0190]

Table 12

[0191] Example 5: HGF Competition Assay

[0192] The above analysis suggests that epitopes recognized by some human / mouse equivalent anti-MET antibodies may overlap with epitopes in which HGF is involved when binding to MET (Stamos et al., EMBO J. Vol. 23, pp. 2325-2335, 2004; Merchant et al., Proc Natl Acad Sci USA Vol. 110, pp. E2987-E2996, 2013; Basilico et al., J Clin Invest. Vol. 124, pp. 3172-3186, 2014). To investigate along this line, competition between mAbs and HGF was examined by ELISA. The N-terminus of recombinant human and mouse HGF (R&D Systems) was biotinylated using NHS-LC-biotin (Thermo Scientific). Human or mouse MET-Fc protein (R&D Systems) was immobilized on a solid phase and then exposed to 0.3 nM human or mouse biotinylated HGF in the presence of increasing antibody concentrations (0-120 nM). Binding of HGF to MET was elucidated using HRP-labeled streptavidin (Sigma-Aldrich). As shown in Table 13, this analysis allowed for the classification of human / mouse equivalent anti-MET antibodies into two groups: full HGF competitors (71D6, 71C3, 71D4, 71A3, 71G2) and partial HGF competitors (76H10, 71G3, 76G7, 71G12, 74C8, 72F8).

[0193] [Table 13]

[0194] In general, SEMA conjugates replaced HGF more effectively than PSI conjugates. In particular, antibodies that recognized epitopes in the β-propeller blades 4 and 5 of SEMA were the most potent HGF competitors (71D6, 71C3, 71D4, 71A3, 71G2). This observation is consistent with the idea that SEMA blade 5 contains a binding site with high affinity for the α-chain of HGF (Merchant et al., Proc Natl Acad Sci USA, Vol. 110, pp. E2987-E2996, 2013). Although the PSI domain has not been shown to be directly involved in HGF, it has been suggested that it functions as a "hinge" to regulate the position of HGF between the SEMA domain and the IPT region (Basilico et al., J Clin Invest., Vol. 124, pp. 3172-3186, 2014). Therefore, it is highly probable that PSI (76H10, 71G3, 76G7, 71G12) prevents HGF from binding to MET not through direct competition with the ligand, but by intervening in this process or due to steric hindrance. Finally, it has been found that the β-propeller blades 1-3 of SEMA are important for the low affinity binding of the HGF β-chain, which plays a central role in MET activation but contributes only partially to the HGF-MET binding strength (Stamos et al., EMBO J. Vol. 23, pp. 2325-2335, 2004). This may explain why the mAb that binds to that region of MET (74C8, 72F8) is a partial competitor of HGF.

[0195] Example 6: MET Activation Assay

[0196] Immunoglobulins that target receptor tyrosine kinases are divalent and therefore may exhibit receptor agonist activity, potentially mimicking the effects of native ligands. To investigate this line of reasoning, the ability of human / mouse equivalent anti-MET antibodies to promote MET autophosphorylation was examined using a receptor activation assay. A549 human lung cancer cells and MLP29 mouse liver progenitor cells were deprived of serum growth factor for 48 hours and then stimulated with increasing concentrations (0-5 nM) of antibody or recombinant HGF (A549 cells, recombinant human HGF, R&D Systems; MLP29 cells, recombinant mouse HGF, R&D Systems). After stimulating the cells for 15 minutes, they were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed as described in the paper (Longati et al., Oncogene Vol. 9, pp. 49-57, 1994). After separating protein lysates by electrophoresis, they were analyzed by Western blotting using antibodies (Cell Signaling Technology) specific to phosphorylated MET (tyrosine 1234-1235), regardless of whether they were human or mouse. The same lysates were also analyzed by Western blotting using anti-whole-human MET antibodies (Invitrogen) or anti-whole-mouse MET antibodies (R&D Systems). This analysis revealed that all human / mouse equivalent anti-MET antibodies exhibited MET agonist activity. As shown in Figure 4, several antibodies (71G3, 71D6, 71C3, 71D4, 71A3, 71G2, 74C8) promoted MET autophosphorylation to the same extent as HGF. Several others (76H10, 76G7, 71G12, 72F8) were less potent, which was particularly evident at lower antibody concentrations. No clear correlation was observed between MET activation activity and HGF competitive activity.

[0197] To obtain more quantitative data, the agonist activity of the antibodies was also characterized by phospho-MET ELISA. For this purpose, A549 cells and MLP29 cells were subjected to serum starvation as described above, and then stimulated with mAbs at increasing concentrations (0-25 nM). Recombinant human (A549) HGF or recombinant mouse (MLP29) HGF was used as a control. The cells were lysed, and phospho-MET levels were determined by ELISA as described in the paper (Basilico et al., J Clin Invest. Vol. 124, pp. 3172-3186, 2014). Briefly, 96-well plates were coated with mouse anti-human MET antibody or rat anti-mouse MET antibody (both from R&D Systems), and then incubated with cell lysates. After washing, the captured proteins were incubated with biotin-labeled anti-phosphotyrosine antibody (Thermo Fishe), and binding was confirmed using HRP-labeled streptavidin (Sigma-Aldrich).

[0198] The results of this analysis are consistent with the data obtained by Western blotting. As shown in Table 14, 71G3, 71D6, 71C3, 71D4, 71A3, 71G2, and 74C8 potently activated MET, while 76H10, 76G7, 71G12, and 72F8 produced a much smaller effect. In any case, all antibodies showed comparable effects in human and mouse cells.

[0199] [Table 14]

[0200] Example 7: Scattering Assay

[0201] To evaluate whether the agonist activity of human / mouse equivalent anti-MET antibodies could be translated into biological activity, scattering assays were performed using both human and mouse epidermal cells. For this purpose, HPAF-II human pancreatic cancer cells (from the American Cell Culture Lineage Preservation Institute) and MLP29 mouse liver progenitor cells were stimulated with increasing concentrations of recombinant HGF (human or mouse; both from R&D Systems), and cell scattering was examined microscopically after 24 hours as described in the paper (Basilico et al., J Clin Invest. Vol. 124, pp. 3172-3186, 2014). This preliminary analysis revealed that HGF-induced cell scattering was linear in both cell lines until saturation was reached at approximately 0.1 nM. Based on these HGF standard curves, a scoring system was created ranging from 0 (no HGF and no cell scattering) to 4 (0.1 nM HGF present and maximum cell scattering). HPAF-II and MLP29 cells were stimulated with increasing concentrations of human / mouse equivalent anti-MET antibodies, and cell scattering was assessed after 24 hours using the scoring system described above. As shown in Table 15, this analysis revealed that all mAbs examined promoted cell scattering in both human and mouse cell lines, resulting in substantially overlapping results in both species. 71D6 and 71G2 showed exactly the same activity as HGF; 71G3 and 71A3 were only slightly less potent than HGF; 71C3 and 74C8 required substantially higher concentrations to achieve the same activity as HGF; and 71D4, 76G7, 71G12, and 72F8 did not reach saturation in this assay.

[0202] [Table 15] [Table 16]

[0203] Example 8: Protection from drug-induced apoptosis

[0204] Some experimental evidence suggests that HGF exhibits a strong protein anti-apoptotic effect on MET-expressing cells (review by Nakamura et al., J Gastroenterol Hepatol. Vol. 26, Suppl 1, pp. 188-202, 2011). To examine the potential anti-apoptotic activity of human / mouse equivalent anti-MET antibodies, a cell-based drug-induced survival assay was performed. MCF10A human breast epithelial cells (American Type Culture Collection) and MLP29 mouse liver progenitor cells were incubated with increasing concentrations of staurosporine (Sigma Aldrich). After 48 hours, cell viability was determined by measuring the total ATP concentration with a Victor X4 multilabel plate reader (Perkin Elmer) using the Cell Titer Glo kit (Promega). From this preliminary analysis, it was revealed that the concentration of the drug that induces approximately 50% cell death was 60 nM in MCF10A cells and 100 nM in MLP29 cells. Next, MCF10A cells and MLP29 cells were incubated with the drug at the concentration determined above in the presence of increasing concentrations (0 - 32 nM) of anti-MET mAb or recombinant HGF (human or mouse; both from R&D Systems). After 48 hours, cell viability was determined as described above. The results of this analysis shown in Table 16 suggest that human / mouse equivalent antibodies protect human and mouse cells equally from cell death induced by staurosporine. In both the human cell line and the mouse cell line, some mAbs (71G3, 71D6, 71G2) showed protective activity equal to or greater than that of HGF, while other molecules (76H10, 71C3, 71D4, 71A3, 76G7, 71G12, 74C8, 72F8) showed only partial protection.

[0205]

Table 17

[0206] Example 9: Branch Morphogenesis Assay

[0207] As discussed in the background technology section, HGF is a multifunctional cytokine that promotes the harmonious regulation of various independent biological activities (including cell proliferation, motility, invasion, differentiation, and survival). A cell-based assay that better summarizes all of these activities is the branching morphogenesis assay, which replicates the formation of tubular organs and glands during embryonic development (overview by Rosario and Birchmeier, Trends Cell Biol. Vol. 13, pp. 328-335, 2003). In this assay, spheroids of epidermal cells are seeded inside a 3D collagen matrix and stimulated with HGF to generate tubules, which eventually form branched structures. These branched tubules resemble the hollow structures of epidermal glands (e.g., mammary glands) in that they form lumens surrounded by polarized cells. This assay is the most complete HGF assay that can be performed in vitro.

[0208] To investigate whether human / mouse equivalent anti-MET antibodies exhibit agonist activity in this assay, LOC human kidney epidermal cells (Michieli et al., Nat Biotechnol., Vol. 20, pp. 488-495, 2002) and MLP29 mouse liver progenitor cells were seeded in a collagen layer as described in the paper (Hultberg et al., Cancer Res. Vol. 75, pp. 3373-3383, 2015), and then exposed to increasing concentrations of mAb or recombinant HGF (human or mouse; both from R&D Systems). The time course of branching morphogenesis was tracked microscopically, and colonies were photographed after 5 days. Representative images are shown in Figure 5. Quantitative results of branching morphogenesis activity were obtained by counting the number of branches in each spheroid. As shown in Table 17, all antibodies examined induced branched tubule formation in a dose-dependent manner. However, consistent with the data obtained from MET autophosphorylation assays and cell scattering assays, 71D6, 71A3, and 71G2 showed the most potent agonist activity, comparable to or even exceeding that of recombinant HGF.

[0209] [Table 18] [Table 19]

[0210] Example 10: Human-mouse equivalent agonist anti-MET antibodies offer a wide range of opportunities to alter MET activity.

[0211] A comprehensive analysis was conducted to compare antibody functions based on the biochemical and biological assays described below. Table 18 summarizes the performance of various mAbs measured in the assays performed. Analysis of this table reveals that human-mouse equivalent agonist anti-MET antibodies exhibit a broad range of biochemical and biological activity, providing ample opportunities for customized MET activity modification. Depending on whether translational or clinical applications are selected, antibody selection can be based on antibodies identified as competing entirely or partially with HGF, strongly or mildly inducing MET activation, strongly or weakly promoting cell entry, or aggressively or mildly antagonizing apoptosis. This outlook suggests that agonist antibodies are far more versatile and flexible than HGF, as they can induce a more stepwise response compared to the on-or-off nature of HGF.

[0212] From a pharmacological standpoint, the ability to selectively induce biological activity downstream of MET could be extremely useful. For example, in some applications in oncology, the benefit of ligands that separate the nutritional properties of HGF from its invasiveness-promoting activity is sought (Michieli et al., Nat Biotechnol. Vol. 20, pp. 488-495, 2002). Another application in hepatology requires a factor that, ideally, protects hepatocytes from apoptosis without promoting cell invasion (Takahara et al., Hepatology, Vol. 47, pp. 2010-2025, 2008). Yet another application in muscular dystrophy requires blocking HGF-induced proliferation during myoblast-to-muscle cell differentiation, while also protecting against differentiation-related apoptosis (Cassano et al., PLoS One Vol. 3, p. e3223, 2008). In all these applications and other similar cases, one can imagine using a partial agonist mAb that replaces endogenous HGF while inducing mild MET activation, thereby enhancing some of HGF's biological activities while reducing others.

[0213] Conversely, various applications in the field of regenerative medicine require potent survival-promoting signals and rapid tissue repair to prevent irreversible cell damage or cytopathogenesis. This situation is found, for example, in cases of sudden liver failure, acute kidney injury, and severe pancreatitis (review by Nakamura et al., J Gastroenterol Hepatol. Vol. 26, Supplement 1, pp. 188-202, 2011). In all of these applications and other similar cases, it is preferable to use a total agonist mAb that promotes tissue healing and regeneration as powerfully as possible. Competition with HGF does not actually play a role in this case because total agonist mAbs are as potent as HGF, if not more so, and can reach pharmacological concentration logarithms higher than the physiological levels at which endogenous ligands are found.

[0214] In other pathological conditions involving non-standard and poorly characterized functions of HGF, such as those related to the immune system (inflammatory diseases, autoimmune diseases, transplant-related complications), the hematopoietic system (stem cell recruitment, hematopoiesis), and the nervous system (nerve growth, neurodegeneration), the role of the HGF / MET pathway has been largely unstudied. While some experimental evidence suggests that recombinant HGF or HGF gene therapy improves these diseases in preclinical models (review by Nakamura et al., J Gastroenterol Hepatol. Vol. 26, Suppl. 1, pp. 188-202, 2011), we do not have sufficient information to determine whether all functions of HGF are beneficial or only some functions of HGF are. For these therapeutic applications, the ability to finely tune MET activity using a very diverse population of MET agonist antibodies may be advantageous compared to HGF (even excluding the numerous pharmacological problems inherent in using recombinant HGF as a drug, as discussed in the summary of the invention).

[0215] In conclusion, we note that all identified human-mouse equivalent anti-MET antibodies may be useful for therapeutic purposes, regardless of whether they fully or partially compete with HGF, or whether they fully or partially activate the MET receptor.

[0216] [Table 20]

[0217] Example 11: The biochemical and biological characteristics of human / mouse equivalent antibodies do not change by exchanging the constant region.

[0218] The objective of this invention is to produce and identify agonist anti-MET antibodies that function similarly well in both human and mouse strains. Therefore, we attempted to investigate whether exchanging the human heavy chain constant region and light chain constant region with the corresponding mouse constant region affects the main biochemical and biological activities of a representative antibody population. For this purpose, three representative molecules were selected from a population of human / mouse equivalent antibodies (71G3, a partial competitor of HGF and a partial agonist in biological assays; 71G6 and 71G2, total competitors and total agonists in biological assays). The VH and VL regions of 71G3, 71D6, and 71G2 were conjugated to a mouse IgG1 / λ antibody frame. Sequences of any variant of mouse immunoglobulin are available from publicly accessible databases such as the ImMunoGeneTics information system (www.imgt.org). Fusion with the desired variable region can be achieved using standard genetic engineering procedures. Table 19 shows the complete amino acid sequences of the heavy and light chains of the llama-mouse chimeric antibody that was prepared.

[0219] [Table 21]

[0220] The production and purification of recombinant immunoglobulins can be achieved by transient transfection and affinity chromatography in mammalian cells, respectively, following well-established protocols. Subsequently, the biochemical and biological activities of mouse forms 71G3, 71D6, and 71G2 were compared with the activities of the same human forms of antibodies.

[0221] The ability of these antibodies to bind to purified human or mouse MET ECD was evaluated by ELISA; their ability to recognize native MET on human or mouse cells was evaluated by FACS; their ability to induce scattering in human and mouse epidermal cells was evaluated; and their ability to promote branching morphogenesis in collagen was evaluated. The results of this analysis, summarized in Table 20, clearly show that exchanging the human constant region with the mouse constant region has no substantial effect on any of the analyzed properties.

[0222] [Table 22]

[0223] Example 12: Comparison with prior art antibodies: Human-mouse cross-reactivity

[0224] As discussed in detail in the background technology section, a few other studies have already described agonist anti-MET antibodies that at least partially mimic HGF activity. As of the writing of this specification, such studies include: (i) 3D6 mouse anti-human MET antibody (United States Patent No. 6,099,841); (ii) 5D5 mouse anti-human MET antibody (United States Patent No. 5,686,292); (iii) NO-23 mouse anti-human MET antibody (United States Patent No. 7,556,804 B2); (iv) B7 human naive anti-human MET antibody (United States Patent Application Publication No. 2014 / 0193431 A1); (v) DO-24 mouse anti-human MET antibody (Prat et al., Mol Cell Biol. Vol. 11, pp. 5954-5962, 1991; Prat et al., J Cell Sci. Vol. 111, pp. 237-247, 1998); (vi) DN-30 mouse anti-human MET antibody (Prat et al., Mol Cell Biol. Volume 11, pp. 5954-5962, 1991; Prat et al., J Cell Sci. Volume 111, pp. 237-247, 1998).

[0225] All prior art agonist anti-MET antibodies were obtained as follows: 3D6 hybridomas were purchased from the American Cell Culture Lineage Preservation Service (catalog number ATCC-HB-12093). 3D6 antibodies were purified from hybridoma conditioning medium using a standard affinity chromatography protocol.

[0226] The cDNA encoding the variable region of the 5D5 antibody (a bivalent precursor of the antagonist anti-MET antibody onartuzumab (Merchant et al., Proc Natl Acad Sci USA Vol. 110, pp. E2987-E2996, 2013)) was synthesized based on the VH and VL sequences published in U.S. Patent No. 7,476,724 B2. The resulting cDNA fragment was fused with the mouse constant IgG1 / λ domain using a standard protein engineering protocol to produce a bivalent monoclonal antibody.

[0227] The NO-23 antibody was obtained from Professor Maria Prat (University of Novara, Italy; inventor of NO-23; U.S. Patent No. 7,556,804 B2). The NO-23 antibody can also be obtained by requesting the corresponding hybridoma from the Interlab Cell Line Collection (ICLC), an international depositary at the Advanced Biotechnology Center (ABC) in Genoa, Italy (clone number ICLC 03001).

[0228] The cDNA encoding the variable region of the B7 antibody was synthesized based on the VH and VL sequences published in U.S. Patent Application Publication No. 2014 / 0193431 A1. The resulting cDNA fragment was fused with the mouse constant IgG1 / λ domain as described above to produce a bivalent monoclonal antibody.

[0229] The DO-24 and DN-30 antibodies were obtained from Professor Maria Prat (University of Novara, Italy; the first person to identify and characterize DO-24 and DN-30; Prat et al., Mol Cell Biol. Vol. 11, pp. 5954-5962, 1991; Prat et al., J Cell Sci. Vol. 111, pp. 237-247, 1998). The DO-24 antibody was commercially available for many years from Upstate Biotechnology, although it is currently discontinued. The DN-30 antibody can also be obtained by requesting the corresponding hybridoma from the Interlab Cell Line Collection (ICLC), an international depositary at the Advanced Biotechnology Center (ABC) in Genoa, Italy (clone number ICLC PD 05006).

[0230] Since most animal models of human diseases use mice as hosts, cross-reactivity with mouse antigens is an essential prerequisite for antibodies whose biological activity needs to be validated in preclinical systems. Because all prior art antibodies were produced in mice (with the exception of B7, which was identified using a human naive phage library), the likelihood of these molecules cross-reacting with mouse METs is small. Even if slight cross-reactivity with autoantigens is possible in principle, the affinity of such interactions is usually very low.

[0231] As detailed in U.S. Patent No. 6,099,841, the 3D6 antibody does not bind to mouse MET, so the inventors had to prove its in vivo activity using ferrets and minks. It is clearly established that these animal models are not ideal systems for modeling human diseases or for use in preclinical medicine. Furthermore, the inventors have not presented any quantitative data regarding differences in antibody affinity and activity between human and ferret or mink strains.

[0232] It has been clearly shown that 5D5 antibodies and their derivatives do not bind to mouse MET (Merchant et al., Proc Natl Acad Sci USA, Vol. 110, pp. E2987-E2996, 2013). No information is available regarding cross-reactivity with other preclinical species.

[0233] Similarly, United States Patent Application Publication 2014 / 0193431 A1 also does not present any information regarding the cross-reactivity of the B7 antibody with mouse MET or other types of MET.

[0234] United States Patent No. 7,556,804 B2 claims that the NO-23 antibody cross-reacts with mouse MET, rat MET, and canine MET, but no quantitative experimental evidence has been presented to support this statement. The inventors immunoprecipitated MET from lysates of mouse cells, rat cells, human cells, or canine cells using a single saturated dose of NO-23, and then radioactively treated the immunoprecipitated protein. 32 It is incubated with P-ATP. After radiolabeling, it was incorporated. 32 P-ATP is visualized using autoradiography. This method is extremely sensitive and completely non-quantifiable. Therefore, it is impossible to determine the degree of cross-reactivity represented by the bands on the gel.

[0235] Similarly, DO-24 antibodies have been suggested to cross-react with mouse MET because DO-24-containing Matrigel pellets, when implanted into the peritoneal cavity of mice, promote vascular recruitment (Prat et al., J Cell Sci. Vol. 111, pp. 237-247, 1998). However, this may also be due to increased inflammation, and no direct evidence has been presented for DO-24 interacting with mouse MET. Another study showed that a single saturated dose of DO-24 (20 nM) induced autophosphorylation of MET in rat cardiomyocyte lineage H9c2 and mouse cardiomyocyte lineage HL-5 (Pietronave et al., Am J Physiol Heart Circ Physiol. Vol. 298, pp. H1155-H1165, 2010; Figure 1). In the same experiment, a much lower dose of recombinant HGF (0.5 nM) was shown to induce a similar level of MET phosphorylation. As the authors themselves acknowledge in the discussion section, these results suggest that DO-24 is dramatically less potent than HGF in these rodent cell lines. Since the same authors claim that DO-24 is a total agonist mAb comparable to HGF activity in human cell models (Prat et al., J Cell Sci. Vol. 111, pp. 237-247, 1998), it should be concluded that DO-24 does not induce the same efficacy or potency in human and mouse cells. Furthermore, it should be noted that the experiments by Pietronave et al. are not quantitative and therefore do not provide information on the degree of cross-reactivity between DO-24 and mouse or rat MET, and that measuring cross-reactivity would require direct comparative dose-response studies, such as those conducted by us (see below). In a third study, MET was immunoprecipitated from mouse mesenchymal stem cell lysates using a mixture of DO-24 and DN-30 antibodies (Forte et al., Stem Cells, Vol. 24, pp. 23-33, 2006). Both the presence of DN-30 and the type of assay (immunoprecipitation from cell lysates) prevented us from obtaining accurate information about DO-24's ability to interact with native mouse MET.In conclusion, there is absolutely no experimental evidence that the DO-24 antibody elicits equivalent biological responses in human and mouse cells.

[0236] Finally, it was clearly shown that the DN-30 antibody does not interact with mouse MET (Prat et al., J Cell Sci. Vol. 111, pp. 237-247, 1998; Petrelli et al., Proc Natl Acad Sci USA Vol. 103, pp. 5090-9095, 2006).

[0237] ELISA assays were performed to directly investigate whether prior art agonist anti-MET antibodies cross-react with mouse MET and to what extent, and to compare these antibodies with our human / mouse equivalent anti-MET antibodies. Since all prior art antibodies were obtained or designed in mouse IgG / λ form, we used mouse IgG / λ forms 71G3, 71D6, and 71G2. Human or mouse MET ECDs were immobilized in solid phase (100 ng / well in a 96-well plate) and exposed to increasing concentrations of antibody in solution (0–40 nM). Binding was elucidated using HRP-labeled anti-mouse Fc antibody (Jackson Immuno Research Laboratories). As shown in Table 21, this analysis revealed that prior art antibodies exhibited K2 binding in the range of 0.059 nM (B7) to 4.935 nM (3D6). D While the antibodies bound to human MET, none of them showed any affinity for mouse MET, even at a high concentration of 40 nM. Of the antibodies tested, only 71G3, 71D6, and 71G2 bound to both human and mouse MET, and with indistinguishable affinity and ability. The overall binding profiles of all antibodies are shown in Figure 6.

[0238] [Table 23]

[0239] Example 13: Comparison with prior art antibodies: MET autophosphorylation

[0240] To compare the agonist activity of prior art antibodies with that of human / mouse equivalent anti-MET antibodies, MET autophosphorylation experiments were performed using both human and mouse cells. A549 human lung cancer cells and MLP29 mouse liver progenitor cells were deprived of serum growth factor for 48 hours, and then stimulated with increasing antibody concentrations (0-25 nM). After stimulating the cells for 15 minutes, they were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed as described in the paper (Longati et al., Oncogene Vol. 9, pp. 49-57, 1994). Using an anti-MET antibody for capture (R&D Systems) and an anti-phosphotyrosine antibody for validation (R&D Systems), phospho-MET levels were determined by ELISA as described in the paper (Basilico et al., J Clin Invest. Vol. 124, pp. 3172-3186, 2014).

[0241] This analysis revealed two significant differences between the prior art antibodies and the human / mouse equivalent anti-MET antibodies described herein. First, consistent with the results obtained in binding experiments, only 71G3, 71D6, and 71G2 were able to promote MET autophosphorylation in both human and mouse cells. The prior art antibodies (including DO-24 and NO-23) induced MET activation only in human cells. We could not detect any activity against mouse cells in the systems we analyzed. Second, all of the prior art antibodies consistently showed lower agonist activity compared to 71G3, 71D6, and 71G2. The mAbs with the highest agonist activity in the prior art were 5D5 and B7, which showed slightly lower activity than 71G3, 71D6, and 71G2. The mAb with the lowest agonist activity in the prior art was 3D6. The other molecules showed intermediate activity. These analytical results are shown in Figure 7.

[0242] Example 14: Comparison with prior art antibodies: Branching morphogenesis

[0243] To compare the biological activity of prior art antibodies with that of human / mouse equivalent anti-MET antibodies, a branching morphogenesis assay was performed. This assay summarizes all important biological activities of HGF (including cell proliferation, scattering, differentiation, and survival). LOC human kidney epidermal cells and MLP29 mouse liver progenitor cells were seeded in a collagen layer as described above and incubated with increasing concentrations of mAb or recombinant HGF (human or mouse, both from R&D Systems). The time course of branching morphogenesis was tracked by microscopy, and colonies were photographed after 5 days. Quantification of branching morphogenesis activity was achieved by counting the number of branched tubules arising from each spheroid, which is shown in Table 22. Representative images of spheroids are shown in Figure 8 (LOC cells) and Figure 9 (MLP29 cells).

[0244] [Table 24] [Table 25]

[0245] The following findings were derived from the data presented above: In human cells, 71D6, 71G2, and 5D5 showed activity equivalent to human HGF. 71G3, 3D6, B7, and DO-24 functioned as partial agonists. NO-23 and DN-30 showed very slight agonist activity. In mouse cells, only 71G3, 71D6, and 71G2 effectively induced branched tubule formation. All other antibodies did not induce branched morphogenesis at all, consistent with their inability to bind to mouse MET in ELISA.

[0246] Our conclusion is that prior art antibodies, unlike human / mouse equivalent anti-MET antibodies, induce different biological activities in human and mouse strains.

[0247] Example 15: Plasma half-life of human / mouse equivalent anti-MET antibody

[0248] Next, we moved on to in vivo studies of selected human / mouse equivalent anti-MET antibodies. As a preliminary analysis, the peak and trough levels of these antibodies were determined in mice. For this purpose, affinity-purified 71G3, 71D6, and 71G2 (in mouse IgG / λ format) were intraperitoneally injected into 7-week-old female BALB / c mice (Charles River). A single bolus of 1 mg / kg or 10 mg / kg was injected, and blood samples were collected from the tail vein 3, 6, 12, and 24 hours after injection. Blood samples were processed, and the antibody concentration in plasma was determined by ELISA. Standard 96-well plates were covered with human MET ECD (100 ng / well) as described in Example 1, and then exposed to increasing dilutions of mouse plasma to capture anti-MET antibodies. After repeated washing with PBS, the presence of anti-MET antibodies was revealed using HRP-labeled donkey anti-mouse antibody (Jackson Laboratories). To quantify the bound antibodies, standard curves were established under the same conditions for purified 71G3, 71D6, and 71G2 antibodies.

[0249] The results of this analysis are shown in Figure 10. The antibody concentrations in plasma were similar for all antibodies examined and were directly proportional to the amount of protein injected. After 24 hours, the antibody concentrations in plasma were approximately 15 nM for a 1 mg / kg bolus and 250 nM for a 10 mg / kg bolus. Considering that the agonist activity of these antibodies reached saturation at concentrations of 5 nM or less in the most stringent assay (branched morphogenesis assay), it can be reasonably concluded that the plasma levels of antibodies obtained by intraperitoneal injection are related from a biological standpoint to the small bolus of 1 mg / kg.

[0250] Furthermore, the plasma half-life of the injected antibody was also calculated. This was achieved by converting the antibody concentration to the natural logarithm (1g), fitting the data to a straight line, and then calculating the slope of that line. From this analysis, it was estimated that the half-lives of 71G3, 71D6, and 71G2 were very similar, corresponding to approximately 3 days for a 1 mg / kg bolus and approximately 9 days for a 10 mg / kg bolus. This represents significantly greater stability compared to recombinant HGF (Ido et al., Hepatol Res. Vol. 30, pp. 175-181, 2004), which has been reported to have a half-life of 2.4 minutes in rodents. All plasma stability data is summarized in Table 23.

[0251] These data suggest that replacing recombinant HGF with human / mouse equivalent anti-MET antibodies may be advantageous in any clinical application requiring systemic HGF administration.

[0252] [Table 26]

[0253] Example 16: In vivo activity: Protection from acute liver injury

[0254] Hepatocytes express MET, making them a major target of HGF, which promotes their proliferation and protects them from apoptosis (review by Nakamura et al., J Gastroenterol Hepatol. Vol. 26, Supplement 1, pp. 188-202, 2011). Therefore, we investigated whether human / mouse equivalent agonist anti-MET antibodies exhibit protective activity in a mouse model of acute liver failure. For this purpose, a single dose of CCl4 (0.2 ml of a 10% solution in olive oil; both from Sigma-Aldrich) was injected into the subcutaneous compartment of 7-week-old female BALB / c mice (Charles River). Immediately after CCl4 injection, the mice were randomly divided into four groups of six, and each group received a single bolus of either purified 71G3, 71D6, 71G2, or vehicle only (PBS). The antibody was administered by intraperitoneal injection at a dose of 5 mg / kg. Blood samples were collected at different time points after injection (0, 12, 24, and 48 hours). An additional fifth control group included six mice that had not received either CCl4 or antibodies, and these mice were euthanized at the end of the experiment. Blood and liver were collected for analysis during necropsy. Plasma levels of the liver markers aspartate transaminase (AST), alanine aminotransferase (ALT), and bilirubin (BIL) were determined using standard clinical biochemistry methods. Liver was embedded in paraffin and processed for histological analysis using a standard protocol.

[0255] As shown in Figure 11, injection of CCl4 into control mice resulted in a rapid and dramatic increase in the levels of all three analyzed blood parameters, peaking 12–24 hours after poisoning. In the control group, CCl4 injection increased AST, ALT, and bilirubin levels 286-fold, 761-fold, and 13-fold, respectively. These increases were significantly less pronounced in all antibody groups (53%, 62%, and 46% respectively in 71G3; 37%, 34%, and 48% respectively in 71D6; and 50%, 39%, and 54% respectively in 71G2). The most potent antibody for liver protection was 71D6.

[0256] Histological examination of the liver at autopsy revealed that CCl4 causes significant tissue damage around the central veins of each liver module. This damage is characterized by eosinophilic staining and enlarged cytoplasm, typical of damaged hepatocytes. The apparent loosening of cell-cell interactions allows red blood cells to leak and infiltrate from the damaged vessels. In the antibody-treated group, the centrally located areas of such damage were smaller and the signs of injury were less pronounced. This is evidenced by less eosinophilic staining, normal cytoplasmic size, and less hematopoietic cell infiltration. Representative images of liver sections stained with hematoxylin and eosin are shown in Figure 12.

[0257] These results suggest that human / mouse equivalent agonist anti-MET antibodies can be used clinically to treat acute hepatic injury typically characterized by abnormal biochemical values ​​of the liver, jaundice, coagulation disorders, cerebral edema, and rapid progression of hepatic dysfunction leading to encephalopathy. Non-limiting examples of these pathological conditions include paracetamol overdose, idiopathic reactions to drug therapies (e.g., tetracycline), drug abuse (ecstasy, cocaine), and viral infections (hepatitis A, B, E).

[0258] Example 17: In vivo activity: Protection from chronic liver injury

[0259] We also investigated whether human / mouse equivalent agonist anti-MET antibodies would show therapeutic effects in a mouse model of chronic liver injury. In fact, HGF is known to have antifibrotic effects in the liver (overview by Matsumoto and Nakamura, Ciba Found Symp. Vol. 212, pp. 198-211; Discussion pp. 211-214, 1997). For this purpose, 7-week-old female BALB / c mice (Charles River) were chronically exposed to CCl4 for several weeks. In the first week, the mice were subcutaneously injected twice with 0.1 ml of a 5% CCl4 solution in olive oil (both from Sigma-Aldrich). From the following week, the dose of CCl4 was increased (0.1 ml of a 10% solution in olive oil) while maintaining the injection frequency (twice a week). Immediately after the initial injection, mice were randomly divided into four groups of seven, and each group was treated with either purified 71G3, 71D6, 71G2, or vehicle alone (PBS). Antibodies were administered three times a week by intraperitoneal injection at a dose of 1 mg / kg. An additional fifth control group consisted of seven healthy mice that were not given CCl4 or antibodies. Mice were euthanized after 6 weeks of chronic CCl4 poisoning. At necropsy, blood and liver were collected for analysis. Plasma levels of the liver markers aspartate transaminase (AST) and alanine aminotransferase (ALT) were determined by standard clinical biochemical methods. Liver was embedded in paraffin and processed for histological analysis using a standard protocol.

[0260] As shown in Figure 13, chronic exposure of control mice to CCl4 impaired liver function, as evidenced by higher plasma levels of AST and ALT. Unlike the acute model, which produced a rapid but transient increase in liver marker levels, chronic CCl4 toxicity induced a more gradual increase in AST and ALT levels, approximately five times higher than in untreated mice. Notably, antibody treatment completely prevented the increase in AST concentration, actually lowering it to below basal levels. Although not as dramatic as the effect observed with AST, the antibody also clearly blocked the rapid increase in ALT levels.

[0261] Liver sections were stained using various techniques aimed at detecting fibrous tissue (including Masson's trichrome staining, picrosilius red staining, and anti-α-smooth muscle actin (α-SMA) antibody staining). Hematoxylin and eosin staining were also performed to examine general histological structure. This analysis revealed that chronic CCl4 treatment leads to the formation of a large amount of fibrous tissue in the interlobular spaces. This tissue is particularly characterized by positivity for picrosilius red staining and anti-α-smooth muscle actin (α-SMA) antibody staining. The fibrous tissue formed a kind of "ribbon" connecting the portal vein triplets. Evidence of this is the hexagonal shape of the liver units. Notably, liver sections from mice given both CCl4 and agonist anti-MET antibodies showed much weaker fibrosis in terms of staining intensity, and the fibrous region appeared to be limited to the space around the portal vein. Representative images of liver sections stained with picrosilius red antibody and anti-α-SMA antibody are shown in Figures 14 and 15, respectively.

[0262] These data suggest that clinical use of human / mouse equivalent agonist anti-MET antibodies may be able to treat pathological conditions associated with chronic liver disease, characterized by progressive destruction and regeneration of liver parenchyma, leading to cirrhosis and fibrosis. Agonist anti-MET antibodies may also be able to alleviate or prevent fibrosis and promote the restoration of liver structure and function. Agonist anti-MET antibodies can also be used to suppress inflammation and immune responses that often exacerbate chronic liver disease.

[0263] Example 18: In vivo activity: Protection from acute kidney injury

[0264] Renal epidermal cells express MET at considerable levels and are therefore highly sensitive to HGF stimulation (Summary by Mizuno et al., Front Biosci. Vol. 13, pp. 7072-7086, 2008). Therefore, we investigated whether human / mouse equivalent agonist anti-MET antibodies would show protective effects in a mouse model of acute renal failure. To this end, tubular damage was induced in 7-week-old female BALB / c mice (Charles River) by a single intraperitoneal bolus of HgCl2 (3 mg / kg). Immediately after HgCl2 poisoning, the mice were randomly divided into four groups and treated with either 71G3, 71D6, 71G2, or vehicle only (PBS). The antibody was administered intraperitoneally at a dose of 10 mg / kg every 24 hours. Each group contained 6 mice, which were euthanized 72 hours after HgCl2 injection. During the autopsy, blood and kidney tissue were collected for analysis. Plasma levels of blood urea nitrogen (BUN) and creatinine (CRE) were determined using standard clinical biochemical methods. The kidney was processed for histological analysis using a standard protocol.

[0265] As shown in Figure 16, injection of HgCl2 into control mice resulted in a sharp increase in BUN and CRE levels. In the control group, BUN and CRE increased 6-fold and 12-fold, respectively. This increase was significantly less pronounced in all antibody groups (52% and 54% respectively in 71G3; 39% and 30% respectively in 71D6; and 45% and 44% respectively in 71G2). The most potent antibody for kidney protection was 71D6.

[0266] Histological examination of the kidneys revealed that HgCl2 caused widespread tubular damage characterized by proximal tubular hypertrophy, atrophy, and necrosis. The glomerular structure was disintegrated and separated from the surrounding stigma, and the space around the glomeruli was substantially increased. In the antibody-treated group, there was less necrosis of proximal tubular cells, and the glomerular histological structure appeared healthier. Representative images of kidney sections stained with hematoxylin and eosin are shown in Figure 17.

[0267] We suggest that clinical use of human / mouse equivalent agonist anti-MET antibodies may be possible in treating pathological conditions associated with acute renal failure that may be caused by, for example, ischemic injury or nephrotoxic injury, hypovolemic shock, obstruction of the urinary collection system, atherosclerosis, sepsis, diabetes, autoimmune diseases, and rhabdomyolysis. Agonist anti-MET antibodies may be useful in preventing or reversing acute renal failure, protecting tubular epithelial cells from apoptosis, accelerating epithelial cell regeneration, and restoring renal function.

[0268] Example 19: In vivo activity: Protection from acute colon injury, reduction of inflammation, and promotion of regeneration in a mouse model of ulcerative colitis.

[0269] It is well established that intestinal epithelial cells express MET and that HGF plays a central role in maintaining homeostasis and regeneration of the gastrointestinal tract (overview by Nakamura et al., J Gastroenterol Hepatol. Vol. 26, Supplement 1, pp. 188-202, 2011). Therefore, we investigated whether human / mouse equivalent agonist anti-MET antibodies can promote intestinal protection and regeneration in a mouse model of ulcerative colitis. For this purpose, 7-day-old female BALB / c mice (Charles River) were exposed to dextran sulfate sodium (DSS) in drinking water for 10 days. On day 10, treatment with DSS was discontinued and the mice were returned to normal water. From day 1, the mice were randomly divided into 7 groups of 7 mice each and treated with either 71G3, 71D6, 71G2 (dose of 1 mg / kg or 5 mg / kg), or vehicle alone (PBS). The antibody was administered three times a week by intraperitoneal injection. An additional eighth control group included seven healthy mice that were not given DSS or antibody. The mice were euthanized on day 12, two days after discontinuation of DSS administration. The colon was collected, washed, and its length measured using a ruler during necropsy. After measurement, the colon was embedded in paraffin for histological analysis.

[0270] Throughout this experiment, mouse body weight was regularly monitored, and clinical symptoms of ulcerative colitis were assessed by examining fecal occult blood, rectal bleeding, and fecal consistency. Quantification was achieved using a standard scoring method employed in preclinical models (Kim et al., J Vis Exp. Vol. 60, pii:3678, 2012), assigning a score from 0 (asymptomatic) to 3 (maximum symptom occurrence) for each parameter. The scores for each parameter were summed to obtain a disease activity index (DAI) ranging from 0 to 9.

[0271] As shown in Figure 18, exposure to DSS in the PBS group resulted in a maximum 25% decrease in body weight, an increase in DAI scores to 4 or higher, and a maximum 40% reduction in colon length. Notably, all antibodies analyzed reversed these effects in a dose-dependent manner and showed significant activity even at lower doses than those tested. 71D6 was the most potent antibody. 71D6 temporarily reduced body weight before returning it to normal levels comparable to those observed in the PBS group. 71D6 blocked the increase in DAI and substantially suppressed all clinical symptoms. 71D6 blocked colonic shortening, and shortening was limited to negligible fluctuations.

[0272] Colon sections were stained with hematoxylin and eosin and examined under a microscope. As shown in Figure 19, administration of DSS caused severe damage to the colonic mucosa. The epidermal layer was eroded, and lymphocyte infiltration appeared. Cryptal abscesses were scattered throughout the colonic mucosa, with large colonies of foamy macrophages, the main cause of tissue destruction. Lymph nodes around the viscera appeared enlarged. Mucus glands were characterized by atrophy and marked depletion of mucus, replaced by inflammatory infiltrates (containing foamy macrophages, lymphocytes, and neutrophils). In some ulcers, granulocyte or macrophage exudate had invaded, and the glandular components had completely disappeared. Notably, mice treated with both DSS and agonist anti-MET antibodies showed much milder symptoms of degeneration and inflammation. Specifically, macrophages and granulocytes, which are components of acute inflammation, were absent, the mucosa showed only minor damage, glandular distortion and density reduction appeared slight, mucin secretion was restored, and there were no erosions or ulcers at all. These protective effects were dose-dependent in all antibody groups, but the effect was already evident at 1 mg / kg. This indicates that the antibody concentration reached at this dose is very close to saturation (see plasma stability in Example 15). In this model as well, the most effective antibody appeared to be 71D6.

[0273] Example 20: In vivo activity: Protection from acute colon injury, reduction of inflammation, and immunosuppression in a mouse model of ulcerative colitis.

[0274] Inspired by the above results, we also investigated whether agonist anti-MET antibodies would be effective in more specific mouse models of inflammatory bowel disease. To this end, acute colonic injury was induced in 7-week-old female C57BL / 6 mice (Charles River) by intrarectal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in ethanol. The TNBS / ethanol combination is known to induce colorectal inflammation through both immunological and invasive processes (reviewed in Jones-Hall and Grisham, Pathophysiology Vol. 21, pp. 267-288, 2014). TNBS dissolved in 50% ethanol was administered enema at a dose of 5 mg / mouse. Immediately after TNBS administration, the mice were randomly divided into four groups of six and treated with either purified 71G3, 71D6, 71G2, or vehicle alone (PBS). The antibody was administered every two days by intraperitoneal injection at a dose of 1 mg / kg. An additional fifth control group included six healthy mice that were not given TNBS or antibody. The weight of the mice was monitored daily. The mice were euthanized five days after administration of TNBS. The colon was collected at necropsy and measured as described above. After measurement, the colon was embedded in paraffin and processed for histological analysis.

[0275] As shown in Figure 20, exposure to TNBS resulted in a body weight reduction of approximately 15% and a colonic length reduction of over 20%. These effects were milder than those produced by DSS, but significantly different from the effects observed in all antibody groups. In fact, treatment with 71G3, 71D6, and 71G2 almost completely suppressed the TNBS-induced body weight loss and colonic shortening, making it nearly impossible to distinguish antibody-treated mice from healthy control mice.

[0276] Colon sections were stained with hematoxylin and eosin and examined under a microscope. As shown in Figure 21, administration of TNBS induced the appearance of typical signs of lymphocytic colitis. These were characterized by hypertrophy of lymph nodes around the viscera, the appearance of lymphocyte aggregates in the submucosa and mucosa, and increased lymphocyte infiltration. Several full-thickness ulcers were observed, accompanied by stromal hyperplasia and lymphocyte and neutrophil infiltration. All of these pathological processes were strongly suppressed in the agonist anti-MET antibody group, with reduced anti-lymphocyte infiltration and decreased mucosal damage. Even where lymphocytes were present, they were not associated with mucus depletion or epidermal damage.

[0277] These results, along with the data reported in previous examples, suggest that clinical use of human / mouse equivalent agonist anti-MET antibodies may be possible in treating pathological conditions associated with ulcerative colitis, and more generally, inflammatory bowel disease. Treatment with agonist anti-MET antibodies may improve the clinical course of the disease by reducing intestinal lesions, promoting epidermal cell proliferation, and decreasing inflammatory cell infiltration.

[0278] Example 21: In vivo activity: Promotion of glucose uptake and cooperation with insulin in a mouse model of type 1 diabetes.

[0279] It has been reported that HGF promotes insulin-dependent glucose uptake in cultured mouse skeletal muscle cells (Perdomo et al., J Biol Chem. Vol. 283, pp. 13700-13706, 2008). Therefore, we investigated whether our agonist anti-MET antibody could reduce hyperglycemia in a mouse model of type 1 diabetes. To this end, we induced pancreatic β-cell degeneration in 7-week-old female BALB / c mice (Charles River) by intraperitoneal injection of streptozotocin (STZ; Sigma Aldrich). STZ was injected daily at a dose of 40 mg / kg for 5 consecutive days. One week after the last injection, fasting blood glucose levels were measured using a standard glucose strip (GIMA). At that time, STZ-treated mice showed a mean basal blood glucose level twice as high as untreated mice (240 mg / dl vs. 120 mg / dl). Mice were randomly divided into four groups of seven based on their basal blood glucose levels, and each group was treated with either purified 71G3, 71D6, 71G2, or vehicle alone (PBS). Antibodies were administered twice weekly by intraperitoneal injection at a dose of 1 mg / kg. An additional fifth control group consisted of seven healthy mice that received neither STZ nor antibodies. Fasting blood glucose levels were monitored for five weeks. At the end of week five, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed. The GTT consisted of measuring blood glucose levels at different time points after forced oral administration of glucose to fasted animals. The ITT consisted of measuring blood glucose levels at different time points after administering insulin to some fasted animals by intraperitoneal or intravenous injection.

[0280] As shown in Figure 22A, the basal blood glucose levels of mice treated with STZ remained elevated throughout the entire experiment. This was attributed to chronic inflammation of the pancreas, which progressively worsened organ damage. Conversely, the blood glucose levels of mice treated with the antibody steadily decreased, finally reaching a plateau two weeks after treatment. While antibody administration did not bring blood glucose levels completely normal, it reduced them by up to 25%, placing them roughly midway between the levels observed in STZ-treated mice and control mice. Given that hyperglycemia in this model is due to the absence of insulin derived from β-cells, the question arose as to whether the lower blood glucose levels in the antibody group were due to elevated insulin levels. However, ELISA assays of blood samples revealed that this was not the case (data not shown). In the glucose tolerance test (GTT), the antibody-treated mice started with lower blood glucose levels but did not develop a normal glucose uptake curve (Figure 22B). In contrast, the antibody-treated mice showed a more rapid response to insulin in the insulin-to-the-tip test (ITT) (Figure 22C). Fifteen minutes after insulin injection, blood glucose levels in mice treated with the antibody over a long period decreased to approximately 30–40% of the value at time zero, which was significantly lower than the values ​​observed in both STZ-treated mice and control mice (Figure 22D). These results suggest that agonist anti-MET antibodies promote glucose uptake in the absence of insulin. These results also suggest that agonist anti-MET antibodies and insulin may work together to mediate glucose uptake when both are present.

[0281] This hypothesis was investigated using mouse skeletal muscle cells in a cell-based assay. Differentiation from C2C12 mouse myoblasts (obtained from a US cell culture lineage preservation institution) into muscle cells was induced as recommended by the provider, and then incubated with human / mouse equivalent agonist anti-MET antibodies (71G3, 71D6, 71G2). After 24 hours, the antibody-treated cells were divided into three groups and acutely stimulated for 1 hour with 0 nM, 100 nM, or 1000 nM human recombinant insulin (Sigma Aldrich) in the presence of the fluorescent glucose analog 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG; Life Technologies). 2-NBDG uptake was measured by flow cytometry.

[0282] As shown in Figure 23, 71G3, 71D6, and 71G2 promoted glucose uptake in a dose-dependent manner. The combination of insulin and agonist anti-MET antibodies produced a synergistic effect, promoting glucose uptake more than either insulin alone or the antibody alone. These data are consistent with the finding that HGF and insulin cooperate to regulate glucose metabolism in cultured cells (Fafalios et al., Nat Med. Vol. 17, pp. 1577-1584, 2011), thus confirming our hypothesis that agonist anti-MET antibodies can enhance both insulin-independent and insulin-dependent glucose uptake.

[0283] Example 22: In vivo activity: Normalization of blood glucose levels and overcoming insulin resistance in a mouse model of type II diabetes.

[0284] Inspired by observations that human / mouse equivalent agonist anti-MET antibodies work in conjunction with insulin to promote glucose uptake, we investigated the potential of using these antibodies for treatment in a mouse model of type II diabetes. Type II diabetes is characterized by hyperglycemia, hyperinsulinemia, and insulin resistance. One of the best-known mouse models of type II diabetes is the db / db mouse, a C57BLKS / J strain with a point mutation in the leptin receptor gene lepr. This mutation leads to a loss of satiety and unrestrained eating, resulting in obesity and the clinical characteristics of type II diabetes described above (review by Wang et al., Curr Diabetes Rev. Vol. 10, pp. 131-145, 2014).

[0285] 7-week-old female db / db mouse (JAX(trademark) mouse strain BKS.Cg-Dock7) m + / +Lepr db Antibody J) was obtained from Charles River. One week later, the mice were randomly divided into four groups of five mice each, and each group was treated with either purified 71G3, 71D6, 71G2, or vehicle alone (PBS). The antibody was administered twice weekly by intraperitoneal injection at a dose of 1 mg / kg. Fasting blood glucose levels were monitored every 14 days for 8 weeks. After 7 weeks of treatment, i.e., when the mice were 15 weeks old, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed.

[0286] As shown in Figure 24, the mean basal blood glucose level in the PBS group at the time of randomization was approximately 230 mg / dl, which clearly corresponds to a diabetic level. This value tended to increase over time, and at the end of the experiment, i.e., after 8 weeks, the mean blood glucose level in the PBS group was approximately 330 mg / dl. In contrast, in the antibody-treated groups, the fasting basal blood glucose level steadily decreased over time. At the end of the experiment, the mean blood glucose levels for the 71G3, 71D6, and 71G2 groups were 173 mg / dl, 138 mg / dl, and 165 mg / dl, respectively.

[0287] After 7 weeks of treatment, i.e., when the mice were 15 weeks old, we examined the acute response of the mice to glucose and insulin challenges. It should be noted that these mice, unlike the STZ-treated mice (see Example 21), were insulin-resistant and therefore hyperinsulinemia, exhibiting hyperglycemia. Indeed, when challenged with glucose in a GTT, the mice in the PBS group were unable to show a normal glucose uptake profile. All mice showed a rapid increase in blood glucose levels, which remained elevated throughout the entire duration of the study. In contrast, when an ITT was performed, the same mice showed a paradoxical response to insulin, with a temporary, slight increase in blood glucose levels. This paradoxical response is a prominent feature of insulin resistance, at least in preclinical models.

[0288] The antibody-treated mice, starting at lower baseline levels, still did not exhibit a normal glucose uptake profile in the GTT, suggesting that the agonist anti-MET antibody could not counteract the rapid increase in blood glucose levels. Remarkably, however, antibody treatment dramatically improved the insulin response in the ITT, reversing the paradoxical effect observed in the PBS group and making the ITT profile more similar to that of non-diabetic mice (C57BLKS / J; Charles River). We conclude that long-term treatment with the agonist anti-MET antibody improves type II diabetes in db / db mice and partially overcomes insulin resistance.

[0289] Based on these results and those shown in previous examples, we propose that human / mouse equivalent agonist anti-MET antibodies may be clinically used to treat pathological conditions associated with hyperglycemia. These pathological conditions may include type 1 diabetes, type 2 diabetes, and other diabetic-like conditions characterized by hyperglycemia and / or insulin resistance (e.g., metabolic syndrome).

[0290] Example 23: In vivo activity: Improvement of fatty liver in a mouse model of non-alcoholic steatohepatitis.

[0291] A study has shown that gene deletion targeting MET in the liver leads to the progression of severe non-alcoholic steatohepatitis (NASH) in mice (Kroy et al., J Hepatol. Vol. 61, pp. 883-890, 2014). In an independent study (Kosone et al., Am J Physiol Gastrointest Liver Physiol. Vol. 293, pp. G204-G210, 2007), HGF improved high-fat diet-induced fatty liver in mice by activating microsomal triglyceride transport protein (MTP) and apolipoprotein B (ApoB) to minimize fatty acid storage.

[0292] Hyperinsulinemia-prone db / db mice are also widely used as a model for NASH, and more commonly, as a model for fatty liver disease. With a normal diet, these mice accumulate large amounts of lipids in their hepatocytes, leading to fatty liver, fibrosis, and chronic liver failure. Feeding these mice a high-fat diet can further exacerbate this condition (review by Anstee and Goldin, Int J Exp Pathol. Vol. 87, pp. 1-16, 2006).

[0293] Based on the observations and discussions described above, we investigated whether human / mouse equivalent agonist anti-MET antibodies could improve moderate fatty liver in db / db mice fed a normal diet. For this purpose, female db / db mice were obtained as described above. At 8 weeks of age, the mice were randomly divided into four groups of 6 mice each and treated with either purified 71G3, 71D6, 71G2, or vehicle only (PBS). The antibody was administered twice a week by intraperitoneal injection at a dose of 1 mg / kg. Eight weeks after treatment, the mice were euthanized and necropsy was performed. The liver was removed for histological examination, embedded in paraffin, and processed. Blood was collected for analysis of liver function markers.

[0294] Liver sections were stained with hematoxylin and eosin or with picrosilius red to highlight fibrosis. As shown in Figure 25, livers from the PBS group showed marked steatosis, typically concentrated around the central vein. Hepatocytes appeared dramatically hypertrophied and lipid-filled. Fatty hepatocytes were mixed with normal hepatocytes, with steatosis occupying up to 60% of the central periphery space. In contrast, livers from antibody-treated mice contained significantly fewer adipocytes and appeared entirely normal overall. As shown in Figure 26, picrosilius red staining demonstrated moderate periportal fibrosis in the PBS group. This fibrosis was characterized by thickening of the interstitial layer around the portal vein triad (portal vein, hepatic artery, and bile duct), sometimes extending into the interlobular spaces. Notably, fibrosis was much less, if present, in liver sections from all antibody groups. These observations were confirmed by analysis of AST and ALT, liver function markers, in plasma (see Figure 27). In fact, mice treated with agonist anti-MET antibodies had exceptionally low plasma concentrations of AST and ALT, approximately 1 / 2.5 of that in the PBS group and half the average levels of AST and ALT in normal mice.

[0295] These data suggest the potential for clinical use of human / mouse equivalent agonist anti-MET antibodies to treat NASH and other pathological conditions associated with fatty liver. By suppressing lipid accumulation in hepatocytes using agonist anti-MET antibodies, it may be possible to prevent or reverse hepatic steatopathies and break the vicious cycle between fatty acid accumulation and macrophage infiltration. Chronic inflammation inevitably leads to extracellular matrix deposition; therefore, agonist anti-MET antibodies may also be used to alleviate steatosis-associated fibrosis.

[0296] Example 24: In vivo activity: Wound healing in diabetic mice

[0297] One of the most significant clinical complications of diabetes is the increased formation of ulcers and slow wound healing. Since HGF is involved in wound healing (Nakamura et al., J Gastroenterol Hepatol, Vol. 26, Supplement 1, pp. 188-202, 2011), we attempted to determine whether human / mouse equivalent anti-MET antibodies could promote wound healing under diabetic conditions. For this purpose, db / db diabetic mice were obtained as described above. At 8 weeks of age, the mice were anesthetized, and a 0.8 cm wide circular wound was made in the right posterior flank using a circular punch blade for skin biopsy (GIMA). The entire epidermal layer was removed. The day after surgery, the mice were randomly divided into four groups and treated with either purified 71G3, 71D6, 71G2, or vehicle only (PBS). The antibody was delivered every two days by intraperitoneal injection at a dose of 5 mg / kg. The diameter of the wound was measured daily with calipers.

[0298] As shown in Figure 28, antibody treatment significantly accelerated wound closure and re-epidermalization. In the control group, experimentally induced wounds healed at an average rate of 5% per day, but this increased to 8% in the 71G3 group, 12% in the 71D6 group, and 11% in the 71G2 group.

[0299] We propose that clinical use of human / mouse equivalent agonist anti-MET antibodies may be possible in treating diabetes-related ulcers and wounds that generally heal slowly. Diabetes-related wounds are a prime example of conditions that need medical attention but are not being adequately addressed. In the United States, diabetes is the leading cause of non-traumatic lower limb amputations. Using agonist anti-MET antibodies may accelerate healing, improve re-epidermalization, and promote angiogenesis in hyperglycemia-induced wounds.

[0300] Example 25: Cross-reactivity between rat (Rattus norvegicus) MET and cynomolgus monkey (Macaca fascicularis) MET

[0301] Since most animal models of human diseases use mice as hosts, cross-reactivity with mouse antigens is a prerequisite for antibodies that require validation in preclinical studies. This led to the idea of ​​identifying human-mouse equivalent anti-MET antibodies. However, some preclinical procedures are preferable to be performed in larger rodents or primates (e.g., organ transplantation or other experimental procedures requiring complex surgical interventions). Furthermore, pharmacodynamic and pharmacokinetic studies are preferable to be performed in higher vertebrates, typically rats and monkeys. Finally, and most importantly, toxicological evaluation of therapeutic antibodies should ideally be performed in monkeys, or in two different rodent species if that is not possible. Therefore, cross-reactivity with rats and monkeys is also ideally desirable.

[0302] To that end, we investigated whether our human / mouse equivalent anti-MET antibody cross-reacts with MET from other species, including rat (Rattus norvegicus) and cynomolgus monkey (Macaca fascicularis). Rat MET ECD (NCBI # NP_113705.1; amino acids 1-931) and monkey MET ECD (NCBI # XP_005550635.2; amino acids 1-948) were obtained using standard protein engineering techniques. Human and mouse MET ECD were used as controls. A limited antibody population representative of both SEMA conjugates (71D6, 71C3, 71D4, 71A3, 71G2) and PSI conjugates (76H10, 71G3) was selected. Prior art 5D5 antibodies were used as controls. MET ECD proteins were immobilized on a solid phase (100 ng / well in a 96-well plate) and exposed to antibodies (with a human constant region) at increasing concentrations (0-40 nM) in solution. HRP-labeled anti-human Fc antibodies (Jackson Immuno Research) were used. Binding was clarified using (Laboratories Inc.). As shown in Figure 29, all human / mouse equivalent antibodies tested bound to human MET, mouse MET, rat MET, and monkey MET with similar affinity and ability, except for 5D5, which bound only to human MET and monkey MET. We conclude that antibodies 71D6, 71C3, 71D4, 71A3, 71G2, 76H10, and 71G3 bind to human MET, mouse MET, rat MET, and monkey MET with similar affinity and ability, at least as determined by ELISA.

[0303] Example 26: Fine-grained mapping of epitopes

[0304] To precisely map the MET epitopes recognized by human / mouse equivalent agonist anti-MET antibodies, the following strategy was employed: When antibodies produced by llamas targeting human MET cross-react with mouse MET, one (or several) residues likely to be recognized by this antibody are conserved between humans (H. sapiens) and mice (M. musculus), but not between humans (H. sapiens), mice (M. musculus), and llamas (L. glama). The same reasoning can be extended to rats (R. norvegicus) and cynomolgus monkeys (M. fascicularis).

[0305] To investigate along this line, the amino acid sequences of human MET (UniProtKB # P08581; amino acids 1-932), mouse MET (UniProtKB # P16056.1; amino acids 1-931), rat MET (NCBI # NP_113705.1; amino acids 1-931), cynomolgus monkey MET (NCBI # XP_005550635.2; amino acids 1-948), and llama MET (GenBank # KF042853.1; amino acids 1-931) were aligned and compared (Figure 30). Referring to Table 12, attention was focused on regions within MET that are important for binding to antibodies 71D6, 71C3, 71D4, 71A3, and 71G2 (amino acids 314-372 of human MET) and antibodies 76H10 and 71G3 (amino acids 546-562 of human MET). The former region of human MET (amino acids 314-372) contains five residues 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 shown in Figure 30 as black rectangles with increasing numbers 1-5. Four of these residues are also conserved in rat MET and cynomolgus monkey MET (Ala 327, Ser 336, Ile 367, Asp 372). The latter region of human MET (amino acids 546-562) contains 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 shown in Figure 30 as black rectangles with increasing numbers 6-8. Two of these residues are also conserved in rat MET and cynomolgus monkey MET (Ser 553 and Thr 555).

[0306] Using human MET as a template, a series of MET variants that were entirely human except for a specific residue being llama were created by mutating each of these residues in different combinations. Schematic diagrams of the variants are shown in Figure 31. Next, the affinity of selected SEMA-binding mAbs (71D6, 71C3, 71D4, 71A3, 71G2) and PSI-binding mAbs (76H10 and 71G3) for these MET variants was investigated by ELISA. For this purpose, various MET proteins were immobilized on a solid phase (100 ng / well in a 96-well plate) and then exposed to antibody solutions of increasing concentrations (0-50 nM). Since the antibodies used were in human constant-region form, binding was clarified using HRP-labeled anti-human Fc secondary antibody (Jackson Immuno Research Laboratories). Wild-type human MET was used as a positive control. The results of this analysis are shown in Table 24.

[0307] [Table 27]

[0308] The results shown above provide a clear picture of the residues that are important for binding to our agonist antibody.

[0309] All SEMA conjugates tested (71D6, 71C3, 71D4, 71A3, 71G2) appear to bind to the same epitope containing two key amino acids (Ile367 and Asp372) located within the SEMAβ-propeller blade 5, which are conserved in human MET, mouse MET, cynomolgus monkey MET, and rat MET, but absent in llama MET. Indeed, mutations in Ala327, Ser336, and Phe343 had no effect on binding, mutations in Ile367 partially weakened binding, and mutations in both Ile367 and Asp372 prevented binding altogether. We conclude that both Ile367 and Asp372 in human MET are critical for binding to the SEMA-bound antibodies tested. These two residues are indicated by "S" (meaning SEMA) in Figure 30.

[0310] The PSI conjugates tested (76H10, 71G3) also appear to bind to the same epitope. However, unlike the SEMA epitope, the PSI epitope contains only one important amino acid (Thr555) that is conserved in human MET, mouse MET, cynomolgus monkey MET, and rat MET, but not in llama MET. In fact, mutations in Arg547 or Ser553 had no effect on binding, but the Thr555 mutation prevented binding altogether. We conclude that Thr555 is a critical determinant for antibody binding to the tested PSI. This residue is indicated by "P" (meaning PSI) in Figure 30.

[0311] Example 27: Uniqueness of human / mouse equivalent agonist antibodies

[0312] The detailed epitope mapping results shown in Example 26 provide meticulous molecular-level evidence that the agonist antibody provided by the present invention possesses unique characteristics not shared by any prior art molecule. This uniqueness is best understood by performing the following analysis.

[0313] For most of the prior art antibodies discussed in Examples 12–14, there is no available information detailing the epitopes they recognize on MET. However, since none of the prior art molecules cross-react with mouse MET, we know that these epitopes must be different from those recognized by our antibody. A clear example of this diversity is provided by 5D5 / onartuzumab, the only prior art anti-MET antibody with detailed molecular annotations regarding its interaction with MET. 5D5 / onartuzumab recognizes four different residues in the SEMAβ-propeller blade 5, located very close to amino acids crucial for antibody-binding interaction with SEMA (Merchant et al., Proc Natl Acad Sci USA Vol. 110, pp. E2987–E2996, 2013). These residues, indicated by "O" (meaning onartuzumab) in Figure 30, correspond to Gln328, Arg331, Leu337, and Asn338.

[0314] It is interesting that none of these residues are conserved between humans (H. sapiens) and mice (M. musculus). This is perfectly consistent with the fact that 5D5 / onartuzumab was produced using mice as a host, and explains why it does not cross-react with mouse MET (Merchant et al., cited above, and the data shown in Figures 6 and 29). Furthermore, none of these residues are conserved between humans (H. sapiens) and rats (R. norvegicus), but all of them are conserved between humans (H. sapiens) and cynomolgus monkeys (M. fascicularis). This explains why 5D5 / onartuzumab does not bind to rat MET but does bind to cynomolgus monkey MET (Figure 29).

[0315] Unlike 5D5 / onartuzumab and all other prior art molecules discussed here, our human / mouse equivalent agonist antibodies are produced using llamas as a host and have been clearly screened for their ability to cross-react with both human and mouse METs. Most of the few amino acids that are conserved between humans (H. sapiens) and mice (M. musculus) but not between humans (H. sapiens), mice (M. musculus), and llamas (L. glama) (shown as black rectangles in Figure 30) are also conserved in rats (R. norvegicus) and cynomolgus monkeys (M. fascicularis), so the selected antibodies are considered likely to cross-react with rats and monkeys as well.

[0316] In conclusion, both the immunization strategy and the screening design make the antibodies of this invention unique. On the one hand, the species used for immunization (llama (L. glama)) is sufficiently distant from humans (H. sapiens), mice (M. musculus), rats (R. norvegicus), and cynomolgus monkeys (M. fascicularis), ensuring that there is sufficient mismatch between the amino acid sequences of llama METs when compared to METs from other species (see Figure 30). These mismatches are crucial because an immunized host cannot produce antibodies against epitopes that the host recognizes as "self." On the other hand, the human / mouse dual screening protocol forces the selection of antibodies that recognize epitopes conserved between these two species. This step is also essential because without panning between the two species, the most representative antibody—that is, an antibody with higher affinity but not necessarily cross-reacting—would simply be selected. By introducing both of these criteria (the fifth species and the "double immersion" protocol), it became possible to identify antibodies with new and unique characteristics.

[0317] References Anstee QM and Goldin RD, Int J Exp Pathol. 87, 1–16, 2006 Basilico C et al, J Biol. Chem. 283:21267–21227, 2008 Basilico C et al., J Clin Invest. 124, 3172–3186, 2014 Cassano M et al., PLoS One 3, e3223, 2008 Chomczynski P et al., Anal. Biochem. 162:156–159, 1987 Daley LP et al., Clin. Vaccine Immunity. 12, 2005 de Haard H et al., J Biol Chem. 274:18218–18230, 1999 De Haard H et al., J. Bact. 187:4531–4541, 2005 Fafalios A et al. Nat Med. 17, 1577–1584, 2011 Forte G et al., Stem Cells. 24, 23–33, 2006 Hultberg A et al., Cancer Res. 75, 3373–3383, 2015 Ido A et al., Hepatol Res. 30, 175–181, 2004 Jones-Hall YL and Grisham MB, Pathophysiology 21, 267–288, 2014 Kim JJ et al., J Vis Exp. 60, No: 3678, 2012 Kosone T et al., Am J Physiol Gastrointest Liver Physiol. 293, G204-210, 2007 Kroy DC et al. J Hepatol. 61, 883-890, 2014 Longati P et al., Oncogene 9, 49-57, 1994 Matsumoto K and Nakamura T, Ciba Found Symp. 212, 198-214, 1997 Medico E et al., Mol Biol Cell 7, 495-504, 1996 Merchant M et al., Proc Natl Acad Sci USA 110, E2987-2996, 2013 Michieli P et al. Nat Biotechnol. 20, 488-495, 2002 Mizuno S et al. Front Biosci. 13, 7072-7086, 2008 Nakamura T et al., J Gastroenterol Hepatol. 26 Suppl 1, 188-202, 2011 Perdomo G et al., J Biol Chem. 283, 13700-13706, 2008 Petrelli A et al., Proc Natl Acad Sci USA 103, 5090-9095, 2006 Pietronave S et al., Am J Physiol Heart Circ Physiol. 298, H1155-65, 2010 Prat M et al., Mol Cell Biol. 11, 5954-5962, 1991 Prat M et al., J Cell Sci. 111, 237-247, 1998 Rosario M and Birchmeier W, Trends Cell Biol. 13, 328-335, 2003 Stamos J et al., EMBO J. 23, 2325-2335, 2004 Takahara et al., Hepatology, 47, 2010-2025, 2008 Wang B et al. Curr Diabetes Rev. 10, 131-145, 2014 The present invention provides, for example, the following items: [1] An antibody or its antigen-binding fragment that binds with high affinity to both human MET protein (hMET) and mouse MET protein (mMET), and is both an hMET agonist and an mMET agonist. [2] It contains at least one heavy chain variable domain (VH) and at least one light chain variable domain (VL), and the VH domain and VL domain are 1 × 10⁶ against hMET when tested as a Fab fragment. -3 / sec~1×10 -2 Range per second, and in some cases 1 x 10 -3 / sec~6×10 -3 Offrate in the range of / second (measured by Biacore) off ) and also show 1 × 10 for mMET -3 / sec~1×10 -2 Range per second, and in some cases 1 x 10 -3 / sec~6×10 -3 Offrate in the range of / second (measured by Biacore) off An antibody or antigen-binding fragment described in item 1, which exhibits the characteristic shown. [3] The antibody or antigen-binding fragment described in item 1 or 2, having equivalent affinity for hMET and mMET. [4] An antibody or antigen-binding fragment described in any one of items 1 to 4, which induces phosphorylation of hMET and also induces phosphorylation of mMET. [5] Phosphorylation of hMET less than 3.0 nM, and in some cases less than 2.0 nM (measured by phospho-MET ELISA) 50This induces phosphorylation of mMET to less than 3.0 nM, and in some cases less than 2.0 nM (measured by phospho-MET ELISA) 50 An antibody or antigen-binding fragment described in item 4, which is induced by [the specified method]. [6] The antibody or antigen-binding fragment described in item 4 or 5 that induces equivalent phosphorylation of hMET and phosphorylation of mMET. [7] An antibody or antigen-binding fragment described in any one of items 4 to 6, which exhibits high phosphorylation activity against hMET and high phosphorylation activity against mMET. [8] Phosphorylation of hMET to less than 1 nM EC 50 and / or at least 80% E (as the percentage of activation induced by HGC in phospho-MET ELISA) max Inducing this, as well as phosphorylation of mMET at EC levels less than 1 nM 50 and / or at least 80% E (as the percentage of activation induced by HGC in phospho-MET ELISA) max An antibody or its antigen-binding fragment as described in item 7, which is induced by [the specified method]. [9] An antibody or antigen-binding fragment described in any one of items 1 to 6, which exhibits low phosphorylation activity against hMET and low phosphorylation activity against mMET.

[10] Phosphorylation of hMET in EC13 1 nM to 5 nM 50 and / or 60-80% E (as the percentage of activation induced by HGC in phospho-MET ELISA) max This induces phosphorylation of mMET with 1 nM to 5 nM EC2. 50 and / or 60-80% E (as the percentage of activation induced by HGC in phospho-MET ELISA) max An antibody or its antigen-binding fragment as described in item 9, which is induced by [the specified method].

[11] An antibody or antigen-binding fragment described in any one of items 1 to 10, which induces an HGF-like cellular response when in contact with human cells and when in contact with mouse cells.

[12] An antibody or antigen-binding fragment described in item 11 that comprehensively induces an HGF-like cellular response upon contact with human cells and mouse cells.

[13] Comprehensive induction of HGF-like cellular response (i) In a cell scattering assay, induce cell scattering comparable to the maximum scattering induced by HGF at a concentration of 0.1–1.0 nM; and / or (ii) In the anti-apoptotic cell assay, the EC was less than 1.1 times the HGF value. 50 and / or E greater than 90% of the values ​​observed in HGF max (Measured as a percentage of the total ATP content of non-apoptotic control cells) and / or; (iii) In a branching morphogenesis assay, the antibody-treated cells exhibit more than 90% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF. An antibody or its antigen-binding fragment as described in item 11 or 12, measurable as one, any two, or all of the following.

[14] The antibody or antigen-binding fragment described in item 11, which partially induces an HGF-like cellular response upon contact with human cells and / or mouse cells.

[15] Partial induction of HGF-like cellular response (i) In a cell scattering assay, when the concentration is 1 nM or less, it induces cell scattering of at least 25% of the value induced by homologous HGF at 0.1 nM; and / or (ii) In the anti-apoptotic cell assay, EC values ​​less than 7.0 times the HGF value 50 and / or E at least 50% of the value observed in HGF max To show cell viability; and / or (iii) In a branching morphogenesis assay, the antibody-treated cells exhibit at least 25% of the number of branches per spheroid induced by the same (non-zero) concentration of HGF. An antibody or its antigen-binding fragment as described in item 14, which can be measured as such.

[16] An antibody or antigen-binding fragment described in any one of items 1 to 15, which is an HGF competitor.

[17] ICs with a minimum impedance of 5 nM 50 and / or at least 50% max Therefore, it competes with hHGF for coupling to hMET, and ICs below 5 nM 50 and / or at least 50% max An antibody or its antigen-binding fragment as described in item 16, which competes with mHGF for binding to mMET.

[18] The antibody or antigen-binding fragment described in item 16 or 17, which competes equally with hHGF and mHGF.

[19] An antibody or antigen-binding fragment described in any one of items 16-18, which is a full-fledged HGF competitor.

[20] ICs less than 2 nM 50 and / or more than 90% max It competes with hHGF, and ICs with a minimum mass of 2 nM. 50 and / or more than 90% max An antibody or its antigen-binding fragment, as described in item 19, that competes with mHGF.

[21] An antibody or antigen-binding fragment described in any one of items 16-18, which is a partial HGF competitor.

[22] ICs of 2-5 nM 50 and / or 50% to 90% max It competes with hHGF, and also with ICs of 2-5 nM. 50 and / or 50% to 90% max An antibody or its antigen-binding fragment as described in item 21, which competes with mHGF.

[23] An antibody or antigen-binding fragment of human MET that recognizes an epitope located within the region of human MET amino acid residues 123-223, or 224-311, or 314-372, or 546-562.

[24] An antibody or antigen-binding fragment thereof that recognizes an epitope containing one or more amino acids located in the extracellular domain of MET that is conserved between human MET and mouse MET.

[25] An antibody or antigen-binding fragment thereof that recognizes an epitope of human MET, comprising the amino acid residue Ile367 and / or Asp372 of human MET, and possibly located within the region of amino acid residues 314–372 of human MET.

[26] An antibody or antigen-binding fragment of human MET that contains the amino acid residue Thr555 and, in some cases, recognizes an epitope of human MET located within the region of amino acid residues 546 to 562.

[27] It contains a heavy chain variable domain including H-CDR1, H-CDR2, and H-CDR3, and a light chain variable domain including L-CDR1, L-CDR2, and L-CDR3, among which H-CDR1 contains amino acid sequences selected from SEQ ID NOs: 2, 9, 16, 23, 30, 37, 44, 51, 58, 65, and 72; H-CDR2 contains amino acid sequences selected from SEQ ID NOs: 4, 11, 18, 25, 32, 39, 46, 53, 60, 67, and 74; H-CDR3 contains amino acid sequences selected from SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 55, 62, 69, and 76; L-CDR1 contains amino acid sequences selected from sequence numbers 79, 86, 93, 100, 107, 114, 121, 128, 135, 142, and 149; L-CDR2 contains amino acid sequences selected from sequence numbers 81, 88, 95, 102, 109, 116, 123, 130, 137, 144, and 151; L-CDR3 is an antibody or its antigen-binding fragment containing an amino acid sequence selected from SEQ ID NOs: 83, 90, 97, 104, 111, 118, 125, 132, 139, 146, and 153.

[28] An antibody or antigen-binding fragment thereof as described in any one of items 1 to 22, as described in any one of items 23 to 27.

[29] [71G2] Contains a heavy chain variable domain including H-CDR1, H-CDR2, and H-CDR3, and a light chain variable domain including L-CDR1, L-CDR2, and L-CDR3, among which H-CDR1 contains the amino acid sequence shown as SEQ ID NO: 44; H-CDR2 contains the amino acid sequence shown as SEQ ID NO: 46; H-CDR3 contains the amino acid sequence shown as SEQ ID NO: 48; L-CDR1 contains the amino acid sequence shown as Sequence ID No. 121; L-CDR2 contains the amino acid sequence shown as Sequence ID No. 123; L-CDR3 is an antibody or its antigen-binding fragment containing the amino acid sequence shown as SEQ ID NO: 125.

[30] [71G2] The antibody or antigen-binding fragment according to item 23, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 167 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 168 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence.

[31] An antibody or antigen-binding fragment thereof as described in any one of items 1 to 22, as described in item 29 or 30.

[32] [71D6] It contains a heavy chain variable domain including H-CDR1, H-CDR2, and H-CDR3, and a light chain variable domain including L-CDR1, L-CDR2, and L-CDR3, among which H-CDR1 contains the amino acid sequence shown as SEQ ID NO: 30; H-CDR2 contains the amino acid sequence shown as SEQ ID NO: 32; H-CDR3 contains the amino acid sequence shown as SEQ ID NO: 34; L-CDR1 contains the amino acid sequence shown as Sequence ID No. 107; L-CDR2 contains the amino acid sequence shown as Sequence ID No. 109; L-CDR3 is an antibody or antigen-binding fragment containing the amino acid sequence shown as SEQ ID NO: 111.

[33] [71D6] The antibody or antigen-binding fragment according to item 25, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 163 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 164 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence.

[34] The antibody or antigen-binding fragment described in any one of items 1 to 22, as described in item 32 or 33.

[35] [71G3] Contains a heavy chain variable domain including H-CDR1, H-CDR2, and H-CDR3, and a light chain variable domain including L-CDR1, L-CDR2, and L-CDR3, H-CDR1 contains the amino acid sequence shown as SEQ ID NO: 9; H-CDR2 contains the amino acid sequence shown as SEQ ID NO: 11; H-CDR3 contains the amino acid sequence shown as SEQ ID NO: 13; L-CDR1 contains the amino acid sequence shown as SEQ ID NO: 86; L-CDR2 contains the amino acid sequence shown as Sequence ID No. 88; L-CDR3 is an antibody or antigen-binding fragment containing the amino acid sequence shown as SEQ ID NO: 90.

[36] [71G3] The antibody or antigen-binding fragment according to item 27, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 157 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 158 or a sequence that is at least 90%, 95%, 97%, or 99% identical to that sequence.

[37] The antibody or antigen-binding fragment described in any one of items 1 to 22, as described in item 35 or 36.

[38] Affinity variants or human germline variants of antibodies or antigen-binding fragments as described in any one of items 1 to 37.

[39] An antibody or antigen-binding fragment as described in any one of items 1 to 38, comprising the hinge region of human IgG, optionally the hinge region of IgG1, and / or the CH2 domain, and / or the CH3 domain.

[40] The antibody according to any one of items 1 to 39, comprising human IgG and, preferably, CH2 and / or CH3 domains having a sequence that is at least 90%, 95%, 97%, or 99% identical to IgG1, wherein the CH2 and / or CH3 domains are modified to reduce or substantially eliminate one or more antibody effector functions.

[41] An antibody or antigen-binding fragment according to any one of items 1 to 40, wherein one or more of the VH domain and / or VL domain, or CDR, is derived from a camelid animal.

[42] The antibody or antigen-binding fragment described in item 41, wherein the animal is a llama.

[43] An isolated polynucleotide encoding an antibody or antigen-binding fragment as described in any one of items 1 to 40.

[44] An expression vector comprising the polynucleotide described in item 43, functionally linked to a regulatory sequence that enables the expression of the antibody or antigen-binding fragment in a host cell or a cell-free expression system.

[45] Host cells or cell-free expression systems containing the expression vector described in item 44.

[46] A method for producing a recombinant antibody or an antigen-binding fragment thereof, comprising culturing host cells or a cell-free expression system of item 45 under conditions that enable the expression of the antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

[47] A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of items 1 to 42, and a pharmaceutically acceptable base or excipient.

[48] ​​An antibody or antigen-binding fragment as described in any one of items 1 to 42, or a pharmaceutical composition as described in item 47, for use in therapeutic purposes.

[49] A method for treating or preventing hepatic impairment, or in some cases acute hepatic impairment, in a human patient, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need thereof.

[50] The method according to item 49, wherein the MET agonist antibody is an antibody or antigen-binding fragment described in any one of items 1 to 42.

[51] A method for treating or preventing renal injury, and in some cases acute renal injury, in a human patient, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need thereof.

[52] The method according to item 51, wherein the MET agonist antibody is an antibody or antigen-binding fragment described in any one of items 1 to 42.

[53] A method for treating or preventing inflammatory bowel disease, and in some cases ulcerative colitis, in a human patient, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need thereof.

[54] The method according to item 53, wherein the MET agonist antibody is an antibody or antigen-binding fragment described in any one of items 1 to 42.

[55] A method for treating or preventing diabetes mellitus, in particular type 1 or type 2 diabetes mellitus, in a human patient, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need thereof.

[56] The method of item 55, wherein the MET agonist antibody is an antibody or antigen-binding fragment described in any one of items 1 to 42.

[57] A method for treating or preventing non-alcoholic steatohepatitis in a human patient, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need thereof.

[58] The method according to item 57, wherein the MET agonist antibody is an antibody or antigen-binding fragment described in any one of items 1 to 42.

[59] A method for treating or promoting wound healing in a human patient, possibly a patient with diabetes, comprising administering a therapeutically effective amount of a MET agonist antibody to a patient in need thereof.

[60] The method according to item 59, wherein the MET agonist antibody is an antibody or antigen-binding fragment described in any one of items 1 to 42.

Claims

1. It contains a heavy chain variable domain including H-CDR1, H-CDR2, and H-CDR3, and a light chain variable domain including L-CDR1, L-CDR2, and L-CDR3. H-CDR1 contains the amino acid sequence shown as SEQ ID NO: 58, H-CDR2 contains the amino acid sequence shown as SEQ ID NO: 60, H-CDR3 contains the amino acid sequence shown as SEQ ID NO: 62, L-CDR1 contains the amino acid sequence shown as Sequence ID No. 135, L-CDR2 contains the amino acid sequence shown as SEQ ID NO: 137, and L-CDR3 is an antibody or antigen-binding fragment comprising the amino acid sequence shown as SEQ ID NO: 139, The antibody or antigen-binding fragment binds to human MET protein (hMET) with high affinity and to mouse MET protein (mMET) with high affinity, and is an hMET agonist and an mMET agonist.

2. The heavy chain variable domain contains, or is composed of, the amino acid sequence of Sequence ID No. 171, or a sequence that is at least 90%, 95%, 97%, or 99% identical to said sequence. The antibody or antigen-binding fragment according to claim 1, wherein the light chain variable domain contains, or comprises, the amino acid sequence of SEQ ID NO: 172, or a sequence that is at least 90%, 95%, 97%, or 99% identical to said sequence.

3. Full-length monoclonal antibody or F(ab') 2 An antibody or antigen-binding fragment according to claim 1 or 2, which is a fragment.

4. An antibody or antigen-binding fragment according to any one of claims 1 to 3, wherein the antibody is an IgG class immunoglobulin.

5. The antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment is an affinity variant or a human germline variant.

6. The following conditions: • Includes the hinge region of human IgG, • Contains the CH2 domain of human IgG, • Contains the CH3 domain of human IgG, - Having a sequence that is at least 90%, 95%, 97%, or 99% identical to human IgG, - The VH domain or one or more CDRs are of camelid origin, - The VL domain or one or more CDRs are of camelid origin, - The VH domain or one or more CDRs are derived from a llama, a camelid animal. - The VL domain or one or more CDRs are derived from a llama, a camelid animal. An antibody or antigen-binding fragment according to any one of claims 1 to 5, which conforms to one or more of the following.

7. The antibody or antigen-binding fragment according to claim 6, wherein the human IgG is IgG1.

8. The antibody or antigen-binding fragment according to claim 6, wherein the CH2 domain is modified to reduce or substantially eliminate one or more antibody effector functions.

9. The antibody or antigen-binding fragment according to claim 6, wherein the CH3 domain is modified to reduce or substantially eliminate one or more antibody effector functions.

10. An antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein H-CDR1 of SEQ ID NO: 58, H-CDR2 of SEQ ID NO: 60, H-CDR3 of SEQ ID NO: 62, L-CDR1 of SEQ ID NO: 135, L-CDR2 of SEQ ID NO: 137, and L-CDR3 of SEQ ID NO: 139 are grafted onto a completely human-derived antibody or antigen-binding fragment.

11. The antibody according to any one of claims 1 to 10, wherein the heavy chain contains or consists of the amino acid sequence of SEQ ID NO: 215, or the heavy chain contains a sequence that is at least 90%, 95%, 97%, or 99% identical to the sequence, and the light chain contains or consists of the amino acid sequence of SEQ ID NO: 216, or the heavy chain contains a sequence that is at least 90%, 95%, 97%, or 99% identical to the sequence.

12. An isolated polynucleotide encoding an antibody or antigen-binding fragment according to any one of claims 1 to 11.

13. An expression vector comprising a polynucleotide according to claim 12, operably linked to a regulatory sequence that enables the expression of the antibody or its antigen-binding fragment in a host cell or a cell-free expression system.

14. A host cell or cell-free expression system comprising the expression vector according to claim 13.

15. A method for producing a recombinant antibody or an antigen-binding fragment, comprising culturing the host cells or cell-free expression system described in claim 13 under conditions that enable the expression of an antibody or antigen-binding fragment, and recovering the expressed antibody or antigen-binding fragment.

16. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 11 and at least one pharmaceutically acceptable carrier or excipient.

17. An antibody or antigen-binding fragment according to any one of claims 1 to 11, or a pharmaceutical composition according to claim 16, for the manufacture of a pharmaceutical used for therapeutic purposes.

18. In human patients - To treat or prevent liver damage, - To treat or prevent kidney damage, - To treat or prevent inflammatory bowel disease, • To treat or prevent diabetes, - For the treatment or prevention of non-alcoholic steatohepatitis, - To promote wound healing A pharmaceutical composition for use in the manufacture of pharmaceuticals, comprising an antibody or antigen-binding fragment according to any one of claims 1 to 11.

19. The aforementioned liver injury is either acute or chronic; The aforementioned kidney injury is acute kidney injury; The aforementioned inflammatory bowel disease is ulcerative colitis; The aforementioned diabetes is type 1 or type 2 diabetes; Alternatively, the pharmaceutical composition according to claim 18, wherein the promotion of wound healing is performed in a patient having diabetes.

Images