Method for promoting pancreatic islet cell proliferation

Abstract

The present invention relates to methods for promoting pancreatic islet cell proliferation, particularly islet cell proliferation. In particular, the present invention relates to methods for promoting pancreatic islet cell proliferation by administering an HGF-MET agonist, such as a MET agonist antibody or a fragment thereof. The present invention further relates to an HGF-MET agonist, such as a MET agonist antibody or a fragment thereof, and a pharmaceutical composition comprising the agonist, for use in the methods of the present invention. [Selection diagram] Figure 18

Description

[Technical Field]

[0001] The present invention relates to methods for promoting the proliferation of pancreatic islet cells, particularly β-islet cells. In particular, the present invention relates to methods for promoting the proliferation of pancreatic islet cells by administering an HGF-MET agonist, such as a MET agonist antibody or a fragment thereof. The present invention further relates to HGF-MET agonists, such as a MET agonist antibody or a fragment thereof, for use in the methods of the present invention, and pharmaceutical compositions comprising the agonists. [Background technology]

[0002] Pancreatic islets, or islets of Langerhans, are regions of endocrine tissue and cells located in the pancreas in the so-called "dense pathway." Pancreatic islets contain α, β, γ, δ, and ε cells, each of which plays a role in the endocrine function of the pancreas. In particular, α and β cells are particularly important for regulating blood glucose levels.

[0003] Type 1 diabetes is an autoimmune disease characterized by immune-mediated destruction of pancreatic cells in the islets of Langerhans, particularly β-islet cells. This progressive degeneration leads to impaired insulin production, thereby inducing hyperglycemia. Clinical symptoms typically occur with an 80–95% loss of β-cell mass (Klinke, PloS One 3: e1374, 2008). Regenerating β-cells and protecting them from progressive immune-mediated destruction is a critical unmet medical need in diabetic patients and a holy grail in diabetes research.

[0004] Although characterized by a different pathogenic mechanism, type 2 diabetes also causes islet degeneration. Indeed, type 2 diabetes is characterized by abnormal insulin production in the setting of insulin resistance, leading to hyperglycemia and the inability of beta cells to compensate for the increased insulin demand (Christoffersen et al., Am J Physiol Regul Integr Comp Physiol 297:1195-201, 2009). In type 2 diabetes, beta islet cells exhibit defective insulin production, and in the late stages of the disease, the cells themselves may be destroyed.

[0005] The current management of patients suffering from pancreatic islet cell degeneration, such as diabetic patients, involves dietary restriction with or without insulin administration. However, this approach does not address the underlying pathogenesis of the condition. Therefore, new therapies are needed. Summary of the Invention

[0006] Surprisingly, it has now been identified that MET agonists promote the proliferation of pancreatic islet cells, and furthermore, the resulting islet cells are functional, leading to the restoration of insulin production and normalization of blood glucose.

[0007] Pancreatic islet cell proliferation and regeneration is particularly important in the treatment of diabetes in that the underlying etiology can be treated by the methods described herein, which is a significant improvement over current disease management approaches that attempt to control symptoms.

[0008] Promoting islet cell proliferation is particularly important when treating patients with early-stage type 1 diabetes. Typically, type 1 diabetes symptoms become apparent during adolescence. However, by the time pathology is diagnosed, a large proportion of the patient's islet β cells have already been destroyed (more than 50%, e.g., 70% or 80% destroyed). Destruction of islet cells occurs rapidly, resulting in a very narrow time window for effective therapeutic intervention.

[0009] For example, immunosuppressants are being investigated as a treatment for newly diagnosed patients with type 1 diabetes in an effort to reduce autoimmune-mediated islet cell destruction. However, immunosuppressants require several months to demonstrate initial clinical benefit. When this occurs, pancreatic beta cells continue to be destroyed, often completely, for nearly half a year after treatment begins. As a result, the use of immunosuppressants is futile. Preserving islet (beta) cells during this critical time window represents a significant unmet medical need for patients with diabetes.

[0010] Surprisingly, as demonstrated herein, MET agonists (e.g., MET agonist antibodies) can not only maintain pancreatic islet cell populations but also promote their proliferation and regeneration. While animals transgenically overexpressing HGF have been described to exhibit altered β-cell proliferation, it was unknown whether exogenous, non-native MET-binding agonists would have any effect. Surprisingly, it has been demonstrated that administration of non-native MET agonists can not only maintain islet cell levels in diabetes, but also promote their proliferation and regeneration. The provision of clinical therapeutic agents capable of promoting islet cell proliferation is a long-felt need in diabetes therapy that is addressed for the first time by the present invention.

[0011] Accordingly, in a first aspect, a method of promoting pancreatic islet cell proliferation is provided, comprising administering to a subject an HGF-MET agonist.

[0012] In a further aspect, a method of promoting insulin production in a subject exhibiting suppressed insulin production is provided, comprising administering to the subject an HGF-MET agonist, which, in a preferred aspect, is characterized by inducing increased pancreatic islet cell proliferation.

[0013] In a further aspect, a method for treating diabetes is provided comprising administering to a subject an HGF-MET agonist, wherein the method is characterized in a preferred embodiment by inducing increased pancreatic islet cell proliferation.

[0014] In a further aspect, provided are HGF-MET agonists for use in the methods provided herein.

[0015] In a further aspect, provided is a pharmaceutical composition for use in the methods provided herein, comprising an HGF-MET agonist and a pharmaceutically acceptable excipient or carrier.

[0016] In preferred embodiments of all aspects, the HGF-MET agonist is an anti-MET agonist antibody. [Brief explanation of the drawings]

[0017] [Figure 1A] MET agonist antibody treatment does not alter basal metabolism in healthy mice. To evaluate the biological effects of MET agonist antibodies on islet cells in vivo, adult BALB / c mice of both sexes were treated systemically with purified 71D6 antibody at 0, 3, 10, or 30 mg / kg for a 3-month period (48 animals total, 6 mice / sex / group). Antibodies were administered by intraperitoneal (ip) injection twice weekly. Body weight and fasting blood glucose were measured monthly throughout the entire experimental period. (A) Time course of body weight. (B) Time course of basal blood glucose. [Figure 1B] MET agonist antibody treatment does not alter basal metabolism in healthy mice. To evaluate the biological effects of MET agonist antibodies on islet cells in vivo, adult BALB / c mice of both sexes were treated systemically with purified 71D6 antibody at 0, 3, 10, or 30 mg / kg for a 3-month period (48 animals total, 6 mice / sex / group). Antibodies were administered by intraperitoneal (ip) injection twice weekly. Body weight and fasting blood glucose were measured monthly throughout the entire experimental period. (A) Time course of body weight. (B) Time course of basal blood glucose. [Figure 2A-B]MET agonist antibody treatment promotes islet proliferation in healthy mice. Adult BALB-c mice were chronically treated with increasing concentrations of 71D6 MET agonist antibody, as described in the legend to Figure 1. At the end of the experiment, mice were sacrificed and subjected to necropsy. Pancreases were extracted, processed for histological analysis, and embedded in paraffin. Sections were stained with hematoxylin and eosin, examined microscopically, and photographed. Images were analyzed using Image J software to measure the number and size of islets. (A) Average islet density. (B) Average islet size. (C) Representative image of hematoxylin and eosin-stained pancreatic sections. Magnification: 400x. [Figure 2C] MET agonist antibody treatment promotes islet proliferation in healthy mice. Adult BALB-c mice were chronically treated with increasing concentrations of 71D6 MET agonist antibody, as described in the legend to Figure 1. At the end of the experiment, mice were sacrificed and subjected to necropsy. Pancreases were extracted, processed for histological analysis, and embedded in paraffin. Sections were stained with hematoxylin and eosin, examined microscopically, and photographed. Images were analyzed using Image J software to measure the number and size of islets. (A) Average islet density. (B) Average islet size. (C) Representative image of hematoxylin and eosin-stained pancreatic sections. Magnification: 400x. [Figure 3] MET agonist antibody treatment promotes islet cell proliferation in healthy mice. Adult BALB-c mice were chronically treated with increasing concentrations of the 71D6 MET agonist antibody as described above. Pancreatic sections were analyzed by immunohistochemistry using an anti-insulin antibody. Figures show representative images taken under a microscope at 100x magnification. [Figure 4]MET agonist antibodies normalize basal blood glucose in a mouse model of type 1 diabetes. Streptozotocin (STZ), a chemical agent that selectively kills β cells and a standard compound used to induce type 1 diabetes in laboratory animals, was injected intraperitoneally (ip) into female BALB-c mice at a dose of 40 mg / kg every 24 hours for 5 consecutive days. One week after the final injection, STZ-treated mice were randomly assigned based on basal blood glucose levels into four treatment groups of 7 mice each. Each group received treatment with (i) vehicle alone (STZ), (ii) purified 71D6 antibody (STZ + 71D6), (iii) purified 71G2 antibody (STZ + 71G2), or (iv) purified 71G3 antibody (STZ + 71G3). The antibodies were administered intraperitoneally (ip) at a dose of 1 mg / kg twice weekly for 8 weeks. An additional fifth treatment group included seven mice that did not receive any STZ or antibody and served as healthy controls (CTRL). Basal blood glucose was monitored throughout the experimental period. (A) Time course of basal blood glucose. (B) Basal blood glucose at 6 weeks of treatment. [Figure 5A-B] MET agonist antibodies promote islet regeneration in a mouse model of type 1 diabetes. STZ-injected BALB-c mice were treated with 1 mg / kg of 71D6, 71G2, or 71G3 as described in the legend to Figure 4. Eight weeks after antibody treatment, mice were sacrificed and subjected to necropsy. Pancreatic sections were stained with hematoxylin and eosin, examined microscopically, and photographed. Digital images of islets were analyzed using Image J software. The number, density, and size of islets were determined by digital data analysis. (A) Mean islet density. (B) Mean islet size. (C) Representative images of hematoxylin and eosin-stained pancreatic sections. Magnification: 200x. [Figure 5C]MET agonist antibodies promote islet regeneration in a mouse model of type 1 diabetes. STZ-injected BALB-c mice were treated with 1 mg / kg of 71D6, 71G2, or 71G3 as described in the legend to Figure 4. Eight weeks after antibody treatment, mice were sacrificed and subjected to necropsy. Pancreatic sections were stained with hematoxylin and eosin, examined microscopically, and photographed. Digital images of islets were analyzed using Image J software. The number, density, and size of islets were determined by digital data analysis. (A) Mean islet density. (B) Mean islet size. (C) Representative images of hematoxylin and eosin-stained pancreatic sections. Magnification: 200x. [Figure 6] MET agonist antibodies promote islet cell regeneration in a mouse model of type 1 diabetes. STZ-injected BALB-c mice were treated with 1 mg / kg of 71D6, 71G2, or 71G3 as described above. Pancreatic sections were analyzed by immunohistochemistry using an anti-insulin antibody. This figure represents images taken under a microscope at 200x magnification. [Figure 7] MET agonist antibodies normalize basal blood glucose in a mouse model of type 2 diabetes. Female db / db mice were randomly assigned to four treatment groups of five mice each. Each group received treatment with (i) vehicle alone (PBS), (ii) purified 71D6 antibody, (iii) purified 71G2 antibody, or (iv) purified 71G3 antibody. Antibodies were administered intraperitoneally (ip) at a dose of 1 mg / kg twice weekly for 8 weeks. C57BL6 / J mice served as nondiabetic control animals. Basal blood glucose was monitored throughout the entire experimental period. (A) Time course of basal blood glucose. (B) Basal blood glucose at 8 weeks of treatment. [Figure 8A-B]MET agonist antibodies promote islet regeneration in a mouse model of type 2 diabetes. Female db / db mice were treated with 71D6, 71G2, or 71G3 as described in the legend to Figure 7. After 8 weeks of treatment, mice were sacrificed and subjected to necropsy. Pancreases were harvested, processed for histology, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin, examined microscopically, and photographed. Islets were analyzed using Image J software to estimate islet number, density, and size. (A) Average islet density. (B) Average islet size. (C) Representative images of hematoxylin and eosin-stained pancreatic sections. Magnification: 200x. [Figure 8C] MET agonist antibodies promote islet regeneration in a mouse model of type 2 diabetes. Female db / db mice were treated with 71D6, 71G2, or 71G3 as described in the legend to Figure 7. After 8 weeks of treatment, mice were sacrificed and subjected to necropsy. Pancreases were harvested, processed for histology, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin, examined microscopically, and photographed. Islets were analyzed using Image J software to estimate islet number, density, and size. (A) Average islet density. (B) Average islet size. (C) Representative images of hematoxylin and eosin-stained pancreatic sections. Magnification: 200x. [Figure 9] MET agonist antibodies promote islet cell regeneration in a mouse model of type 2 diabetes. Female db / db mice were treated with 71D6, 71G2, or 71G3 as described above. Pancreatic sections were analyzed by immunohistochemistry using an anti-insulin antibody. Figures show representative images taken under a microscope at 100x magnification. [Figure 10]Blood glucose levels in NOD mice. Blood glucose was measured in randomly fed (i.e., non-fasting) animals using human test strips (multiCare® Biochemical Systems International). At 6 weeks, NOD mice were in a prediabetic state, i.e., exhibited an average blood glucose of approximately 110 mg / dL. Starting at 7 weeks, animals were treated as described herein. Blood glucose was monitored weekly throughout the duration of the experiment. Animals were considered diabetic if they exhibited blood glucose levels greater than 250 mg / dL (horizontal dotted line) for two consecutive weeks. [Figure 11] Analysis of diabetes onset. (A) Time course of the proportion of diabetic mice. The vertical dotted line indicates the start of treatment. (B) Kaplan-Meier analysis of diabetes onset. Statistical analyses were performed using Prism software (Graph Pad). The Mantel-Cox test, the log-rank trend test, and the Gehan-Breslow-Wilcoxon test all gave p values ​​less than 0.001, indicating that the differences between the curves were statistically significant. [Figure 12] Analysis of non-fasting blood glucose over time. Blood glucose was measured weekly in random-fed (i.e., non-fasting) animals as described above. Consistent with the diabetes incidence data, blood glucose levels followed the following precise order: CONTROL > CD3 > 71D6 > COMBO. [Figure 13] Glucose tolerance test (GTT). Before sacrifice, all animals underwent a glucose tolerance test (GTT). For this, animals were fasted overnight. The next morning, blood samples were taken for blood glucose and insulin measurements. A glucose solution (3 g / kg in 200 μL of PBS) was injected intraperitoneally, and a second blood sample was taken 3 minutes later. Blood glucose concentrations were measured using test strips as described above. Insulin concentrations were measured using an Ultra-Sensitive Mouse Insulin ELISA kit (Crystal Chem). (A) Blood glucose at zero. (B) Blood glucose at 3 minutes. (C) Insulin concentration at zero. (D) Insulin concentration at 3 minutes. [Figure 14]Body weight and liver weight relative to body weight at necropsy. (A) Body weight. Consistent with the attenuated diabetic phenotype, body weight was slightly (but not significantly) higher in the treatment group compared to the control group. (B) Liver weight relative to body weight. No significant differences in liver weight relative to body weight were observed in either group, suggesting that 71D6-induced liver proliferation (observed in other mouse strains) is strain specific. [Figure 15] Histological analysis of pancreatic sections. Pancreatic samples were paraffin-embedded and processed for histological analysis. Tissue sections were stained with hematoxylin and eosin (H&E) and examined microscopically. Representative images of each treatment group are shown. Magnification: 200x. [Figure 16] Immunohistochemical analysis of insulin expression. Pancreatic sections were stained with anti-insulin antibody and analyzed by microscopy. Representative images from each treatment group are shown. Magnification: 40x. [Figure 17] High-resolution microscopic analysis of insulin expression. Pancreatic sections were stained with anti-insulin antibody as described above. Representative microscopic images of each treatment group are shown. Magnification: 200x. [Figure 18] Anti-insulin autoantibodies in mouse plasma. Plasma samples collected at necropsy from all mice and from young, prediabetic female NOD mice (7 weeks of life) were analyzed using a mouse IAA (insulin autoantibody) ELISA kit (Fine Test). This analysis revealed that the majority of mice exhibited higher levels of anti-insulin antibodies compared with prediabetic mice (the rightmost group). No statistically significant differences were observed between the different populations. Mice in the COMBO treatment group showed a trend toward lower levels. Mice in the 71D6 treatment group could be clearly divided into two subpopulations, with low and high levels of autoantibodies, respectively. Although these results require further investigation, they collectively strongly support the hypothesis that neither anti-CD3 antibody treatment nor 71D6 treatment affects autoantibody production in this system but rather acts downstream to prevent or delay the onset of diabetes. [Detailed Description]

[0018] As used herein, "pancreatic islet cells" refers to pancreatic islet cells, also known as "islets of Langerhans," and includes alpha, beta, and delta islet cells as well as islet stroma. Means for identifying pancreatic islet cells are known to those skilled in the art, for example, histological examination of cell biopsies.

[0019] As used herein, promoting islet cell proliferation refers to an increase in islet cell proliferation in a subject receiving an HGF-MET agonist compared to the subject prior to the intervention. Similarly, promoting islet cell proliferation can refer to an increase in islet cells in a subject receiving an HGF-MET agonist compared to a matched control subject not receiving an HGF-MET agonist. Islet cell proliferation can be measured by islet density (per mm 2 These are characterized by an increase in island density (number per island), an increase in island size (e.g., area), or an increase in both island density and island size.

[0020] As used herein, promoting beta islet cell proliferation refers to an increase in beta islet cell proliferation in a subject administered an HGF-MET agonist compared to that in that subject prior to the intervention. Similarly, promoting beta islet cell proliferation refers to an increase in islet cells in a subject administered an HGF-MET agonist compared to a matched control subject not administered an HGF-MET agonist. Islet cell proliferation is measured by islet density (1 mm 2 The increase in island density can be characterized by an increase in island density (number per island), an increase in island size (e.g., area), or an increase in both island density and island size.

[0021] As used herein, promotion of insulin production can refer to an increase in insulin production by (β) islet cells in a subject administered an HGF-MET agonist compared to that in the subject prior to intervention. Similarly, promotion of insulin production can refer to an increase in insulin production by (β) islet cells in a subject administered an HGF-MET agonist compared to a matched control subject not administered an HGF-MET agonist. Insulin production can be characterized by one or more of an increase in plasma insulin concentration, an increase in β-cell density, an increase in β-cell area, an increase in the density and / or number of insulin-positive islet cells, or any combination of these measures.

[0022] As used herein, pancreatic tissue transplantation refers to the transplantation of any pancreatic tissue into a subject. The transplantation can be a complete organ transplant, i.e., a complete pancreas transplant, or a partial pancreas transplant. The transplantation can also be a transplantation of pancreatic islets or islet cells, sometimes referred to as pancreatic islet transplantation.

[0023] As used herein, "HGF-MET agonist" and "MET agonist" are used interchangeably to refer to non-naturally occurring substances that promote signal transduction through the MET protein, i.e., substances other than HGF that bind to MET and increase MET signaling. Agonistic activity of a MET agonist in response to MET binding is indicated by molecular and / or cellular responses that mimic (at least partially) the molecular and cellular responses induced by HGF-MET binding. Suitable methods for measuring MET agonist activity are described herein, including in the Examples. A "full agonist" is a MET agonist that increases MET signaling in response to binding to an extent at least similar to, and possibly greater than, the extent to which MET signaling increases in response to binding of the native HGF ligand. Examples of the levels of MET signaling induced by "full agonists," as measured by various methods of measuring MET signaling, are provided herein.

[0024] As used herein, immunosuppressive substances, also referred to as immunosuppressants, refer to therapeutic agents intended to reduce or inhibit immune responses in subjects, such as anti-inflammatory agents and tolerizing agents. Examples of immunosuppressants include checkpoint inhibitors (e.g., PD-L1 molecules, CTLA4 molecules (e.g., abatecept)), TNF inhibitors (e.g., anti-TNF antibodies, etanercept), tolerizing dendritic cells, anti-CD3 antibodies, and anti-inflammatory cytokines (e.g., IL-10).

[0025] The HGF-MET agonist can be a small molecule, a binding protein, such as an antibody or antigen-binding fragment, an aptamer, or a fusion protein. A particular example of a MET agonist is an anti-MET agonist antibody.

[0026] As used herein, "treatment" or "treating" refers to the effective treatment of the relevant condition, i.e., an improvement in the health of the subject. Treatment can be therapeutic or prophylactic, i.e., therapeutic treatment of a subject suffering from the condition, or prophylactic treatment of a subject to reduce the risk of contracting the condition or reduce the severity of the condition once contracted. Therapeutic treatment can be characterized by an improvement in the health of the subject compared to before treatment. Therapeutic treatment can be characterized by an improvement in the health of the subject compared to a comparable control subject who did not receive the treatment. Therapeutic treatment can also be characterized by a stabilization of the subject's health compared to before treatment, i.e., an inhibition of the progression of the subject's disease state. Prophylactic treatment can be characterized by an improvement in the health of the subject compared to a control subject (or a population of control subjects) who was not treated.

[0027] As used herein, the term "antibody" includes immunoglobulins having a combination of two heavy chains and two light chains that have significant specific immunoreactive activity against an antigen of interest (e.g., human MET). The terms "anti-MET antibody" or "MET antibody" are used interchangeably herein to refer to antibodies that exhibit immunological specificity for the human MET protein. "Specificity" for human MET does not exclude cross-reactivity with species homologs of MET. In particular, "agomAbs," as used herein, refer to MET antibodies that bind to both human and mouse MET.

[0028] As used herein, "antibody" includes antibodies of any human class (e.g., IgG, IgM, IgA, IgD, IgE) and its subclass / isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1). Antibody, as used herein, also refers to engineered antibodies. Engineered antibodies include synthetic forms of antibodies that have been engineered so that they are not naturally occurring, such as antibodies that contain at least two heavy chain portions but not two complete heavy chains (e.g., domain-deleted antibodies or minibodies); multispecific forms of antibodies (e.g., bispecific, trispecific, etc.) that have been engineered to bind to two or more different antigens or to different epitopes on a single antigen; heavy chain molecules linked to scFv molecules; and the like. In addition, the term "engineered antibody" encompasses multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three or more copies of the same antigen).

[0029] The antibodies described herein can have one or more of the following antibody effector functions: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP). Alternatively, in some embodiments, antibodies for use according to the invention have an Fc region that has been modified to abrogate one or more effector functions, for example, all effector functions.

[0030] Antibodies comprise light and heavy chains, with or without interchain covalent bonds between them. Antigen-binding fragments of antibodies include peptide fragments that exhibit specific immunoreactive activity against the same antigen as the antibody (e.g., MET). Examples of antigen-binding fragments include scFv fragments, Fab fragments, and F(ab)2 fragments.

[0031] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are intended to have equivalent meanings. The term "variable" refers to the fact that some portions of the variable domains, VH and VL, differ extensively in sequence among antibodies and are responsible for the binding and specificity of each particular antibody to its corresponding target antigen. This refers to the fact that antibodies utilize different amino acid sequences for different purposes. However, the variability is not uniformly distributed throughout the variable domains of antibodies. The variability is concentrated in three sections, termed "hypervariable loops," within the VL and VH domains, respectively, that form the antigen-binding site. The first, second, and third hypervariable loops of the V lambda light chain domain are referred to herein as L1(λ), ​​L2(λ), and L3(λ), and can be defined as comprising residues 24-33 (the 9, 10, or 11 amino acid residues of L1(λ)), residues 49-53 (the 3 residues of L2(λ)), and residues 90-96 (the 5 residues of L3(λ)) in the VL domain (Morea et al., Methods 20:267-279, 2000). The first, second, and third hypervariable loops of the V kappa light chain domain are referred to herein as L1(κ), L2(κ), and L3(κ), and can be defined as comprising residues 25-33 (6, 7, 8, 11, 12, or 13 amino acid residues of L1(κ)), residues 49-53 (3 residues of L2(κ)), and residues 90-97 (6 residues of L3(κ)) in the VL domain (Morea et al., Methods 20:267-279, 2000). The first, second, and third hypervariable loops of the VH domain are referred to herein as H1, H2, and H3, and can be defined as comprising residues 25-33 (H1 consisting of 7, 8, or 9 residues), 52-56 (H2 consisting of 3 or 4 residues), and 91-105 (H3, which is hypervariable in length) in the VH domain (Morea et al., Methods 20:267-279, 2000).

[0032] Unless otherwise specified, the terms L1, L2, and L3 refer to the first, second, and third hypervariable loops of the VL domain, respectively, and include hypervariable loops from both Vkappa and Vlambda isotypes. The terms H1, H2, and H3 refer to the first, second, and third hypervariable loops of the VH domain, respectively, and include hypervariable loops from any known heavy chain isotype, including gamma, epsilon, delta, alpha, and mu.

[0033] The hypervariable loops L1, L2, L3, H1, H2, and H3 each comprise a portion of a "complementarity-determining region" or "CDR," as defined below. The terms "hypervariable loop" and "complementarity-determining region" are not strictly synonymous, because hypervariable loops (HVs) are structurally determined, whereas complementarity-determining regions (CDRs) are sequence-dependent (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health (NIH), Bethesda, MD, 1991), and the HV and CDR restrictions may differ between some VH and VL domains.

[0034] The CDRs of the VL and VH domains are typically defined as comprising the following amino acids: residues 24-34 (CDRL1), residues 50-56 (CDRL2), and residues 89-97 (CDRL3) of the light chain variable domain, and residues 31-35 or 31-35b (CDRH1), residues 50-65 (CDRH2), and residues 95-102 (CDRH3) of the heavy chain variable domain (Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health (NIH), Bethesda, MD, 1991). Thus, hypervariable loops (HVs) can be included within the corresponding CDRs, and references herein to "hypervariable loops" of the VH and VL domains should be construed to encompass the corresponding CDRs, and vice versa, unless otherwise specified.

[0035] The more highly conserved portions of variable domains are called framework regions (FRs), as defined below. Native heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4, respectively) that largely adopt a β-sheet configuration and are connected by three hypervariable loops. The hypervariable loops in each chain are held in close proximity by the FRs and, in combination with hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies. Structural analysis of antibodies has revealed a relationship between the sequence and shape of the binding site formed by the complementarity-determining regions (Chothia et al., J. Mol. Biol. 227:799-817, 1992; Tramontano et al., J. Mol. Biol. 215:175-182, 1990). Despite their high sequence variability, five of the six loops adopt a very limited repertoire of main-chain conformations, referred to as "canonical structures." These conformations are determined firstly by the length of the loop and secondly by the presence of key residues at defined positions within the loop and in the framework regions that determine the conformation through their ability to fold (pack), hydrogen bond, and adopt unusual main-chain conformations.

[0036] As used herein, the term "CDR" or "complementarity-determining region" refers to the discontinuous antigen-binding sites found in the variable regions of both heavy and light chain polypeptides. These specific regions are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health (NIH), Bethesda, MD, 1991; and Chothia et al., J. Mol. Biol. 196:901-917 (1987); MacCallum et al., J. Mol. Biol. 262:732-745 (1996). The definitions in these documents include overlapping or subsets of amino acid residues when compared with each other. The amino acid residues encompassing the CDRs defined by each of the above-cited documents are shown for comparison. The term "CDR" preferably refers to the CDRs defined by Kabat based on sequence comparisons.

[0037] [Table 1]

[0038] As used herein, the term "framework region" or "FR region" includes amino acid residues that are part of the variable region but not part of the CDRs (e.g., when using the Kabat definition of CDRs). Thus, the variable region framework is approximately 100-120 amino acid residues in length, but includes only amino acids outside the CDRs. For specific examples of heavy chain variable domains, and for the CDRs defined by Kabat et al., framework region 1 corresponds to the domain of the variable region encompassing amino acids 1-30, framework region 2 corresponds to the domain of the variable region encompassing amino acids 36-49, framework region 3 corresponds to the domain of the variable region encompassing amino acids 66-94, and framework region 4 corresponds to the domain of the variable region from amino acid 103 to the end of the variable region. The framework regions of the light chain are similarly separated by each of the light chain variable region CDRs. Similarly, when using the CDR definitions of Chothia et al. or MacCallum et al., the boundaries of the framework regions are separated by the ends of the respective CDRs, as described above. In a preferred embodiment, the CDRs are defined by Kabat.

[0039] In native 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 assumes a three-dimensional configuration in an aqueous environment. The remainder of the heavy and light chain variable domains, which exhibit less intermolecular amino acid sequence variability, are called framework regions. The framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. Thus, these framework regions act as a scaffold, providing the correct orientation and positioning of the six CDRs through interchain noncovalent interactions. The antigen-binding site formed by the positioned CDRs defines a surface complementary to an epitope on the surface of an immunoreactive antigen. This complementary surface promotes noncovalent binding of the antibody to the immunoreactive antigen epitope. The positions of the CDRs can be readily identified by those skilled in the art.

[0040] As used herein, 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 middle hinge, and the lower hinge. (Roux et al., J. Immunol. 161:4083-4090, 1998) MET antibodies containing a "fully human" hinge region can contain one of the hinge region sequences shown in Table 2 below.

[0041] [Table 2]

[0042] As used herein, the term "CH2 domain" includes the portion of an antibody heavy chain molecule spanning from approximately residue 244 to residue 360, using conventional numbering (Kabat numbering, residues 244-360; EU numbering, residues 231-340; Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health (NIH), Bethesda, MD (1991)). The CH2 domain is unique in that it is not tightly paired with other domains. Rather, two N-linked branched hydrocarbon chains are inserted between the two CH2 domains in intact, native IgG molecules. The literature also clearly describes the CH3 domain, which extends from the CH2 domain to the C-terminus of IgG molecules and contains approximately 108 residues.

[0043] As used herein, the term "fragment" refers to a portion or portion of an antibody or antibody chain that contains fewer amino acid residues than an intact or complete antibody or antibody chain. The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with the complete antibody (i.e., the complete antibody from which it was derived) for antigen binding (i.e., specific binding to MET). As used herein, the term "fragment" of an antibody molecule includes antigen-binding fragments of antibodies (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 an intact or complete antibody or antibody chain, or by recombinant means.

[0044] As used herein, "subject" and "patient" are used interchangeably to refer to a human individual. A "control subject" refers to a comparable subject who does not receive the intervention.

[0045] Throughout this application, the term "comprising" should be interpreted to encompass all specifically mentioned features as well as optional additional, unspecified features. As used herein, use of the term "comprising" also discloses embodiments in which no features are present (i.e., "consisting of") other than those specifically mentioned.

[0046] treatment method It is demonstrated herein that HGF-MET agonists (particularly MET agonist antibodies) promote the proliferation of pancreatic islet cells in healthy subjects. It is also demonstrated that MET agonists (particularly MET agonist antibodies) protect pancreatic islet cells from degeneration in subjects experiencing islet cell depletion or injury. Furthermore, HGF-MET agonists (particularly MET agonist antibodies) not only protect the islet cells in these subjects, but also promote the proliferation and regeneration of new islet cells in subjects whose islet cell populations are depleted or degenerating. Furthermore, the new islet cells induced by MET agonist administration are highly functional, as they restore insulin production.

[0047] Stimulating pancreatic islet cell proliferation is particularly advantageous because it treats the underlying pathophysiology of conditions such as diabetes (particularly type 1 diabetes, but also type 2 diabetes). Existing treatments rely on passive management of symptoms through diet and, often, insulin injections. These approaches do not address the underlying cause of the disease. Surprisingly, the present invention has identified that administration of an exogenous, non-naturally occurring HGF-MET agonist effectively promotes pancreatic islet cell proliferation and regeneration. Thus, administration of an HGF-MET agonist (particularly a MET agonist antibody) provides a solution to the long-felt medical need for a clinically meaningful therapy that addresses the problem of pancreatic cell degradation.

[0048] Thus, in one aspect, a method for promoting pancreatic islet cell proliferation is provided, comprising administering an HGF-MET agonist to a subject. Also provided is an HGF-MET agonist for use in promoting pancreatic islet cell proliferation in a subject, or use of an HGF-MET agonist for the manufacture of a medicament for promoting pancreatic islet cell proliferation in a subject.

[0049] In a further aspect, there is provided a method for promoting insulin production in a subject in need thereof, comprising administering to the subject an HGF-MET agonist. In a preferred embodiment of this aspect, the method is characterized by inducing increased proliferation of pancreatic islet cells. Also provided are HGF-MET agonists for use in promoting insulin production in a subject, or uses of HGF-MET agonists for the manufacture of a medicament for promoting insulin production in a subject.

[0050] In a further aspect, a method of treating diabetes is provided, comprising administering an HGF-MET agonist to a subject. In a preferred embodiment of this aspect, the method is characterized by inducing increased proliferation of pancreatic islet cells. Alternatively, or in addition, the method is further characterized by promoting insulin production. In a further aspect, an HGF-MET agonist (e.g., an MET agonist antibody) is provided for use in a method of treating diabetes, wherein the HGF-MET agonist promotes proliferation of pancreatic islet cells. In yet another aspect, an HGF-MET agonist is provided for use in a method of treating diabetes, wherein the HGF-MET agonist promotes insulin production. Also provided are HGF-MET agonists for use in treating diabetes in a subject, or uses of HGF-MET agonists for the manufacture of a medicament for treating diabetes in a subject.

[0051] As demonstrated herein, HGF-MET agonists (particularly MET agonist antibodies) promote pancreatic islet cell proliferation, which is characterized by both an increase in islet cell area and an increase in islet density in pancreatic tissue.

[0052] Thus, in preferred embodiments of all methods provided herein, the methods increase islet cell density. In preferred embodiments of all methods provided herein, the methods increase islet cell area.

[0053] It has been shown herein that HGF-MET agonists (e.g., MET agonist antibodies) promote proliferation of all pancreatic islet cells, i.e., alpha, beta, gamma, delta, and epsilon cells. Accordingly, in certain embodiments of all methods provided herein, the method promotes proliferation of any one or more of alpha cells, beta cells, gamma cells, delta cells, and epsilon cells. In certain embodiments, the method promotes proliferation of alpha cells. In certain embodiments, the method promotes proliferation of beta cells. In certain embodiments, the method promotes proliferation of gamma cells. In certain embodiments, the method promotes proliferation of delta cells. In certain embodiments, the method promotes proliferation of epsilon cells.

[0054] It is further demonstrated herein that HGF-MET agonists (e.g., MET agonist antibodies) are particularly effective in promoting beta islet cell proliferation. This is particularly advantageous because beta cells are essential for insulin production and effective glucose regulation and are degraded in conditions such as diabetes. Not only do HGF-MET agonists (e.g., MET agonist antibodies) promote beta cell proliferation, but the newly produced cells are highly functional and insulin-producing.

[0055] Thus, in preferred embodiments of all methods provided herein, the methods promote beta islet cell proliferation. In preferred embodiments, the methods increase beta islet cell density. In preferred embodiments, the methods increase beta islet cell area. In preferred embodiments, the methods promote insulin-producing beta cell proliferation.

[0056] The method described herein is also particularly advantageous in subjects who undergo pancreatic tissue transplantation.Pancreatic tissue transplantation is a possible treatment for subjects whose pancreatic islet cells have been destroyed (such as diabetic patients).Such transplants can be in the form of whole pancreas transplants, partial pancreas transplants, or isolated islet transplants.In any case, the method provided herein will be particularly advantageous in patients who undergo such transplants and transplants, because it promotes the survival of transplanted islets as well as the proliferation and expansion of their cells.

[0057] Thus, in all method embodiments provided herein, the method further comprises administering a transplant of pancreatic tissue to the subject. In certain embodiments, the method further comprises administering a partial pancreas transplant to the subject. In certain embodiments, the method further comprises administering a pancreatic islet graft to the subject. In all such embodiments, administration of the HGF-MET agonist (e.g., a MET agonist antibody) and administration of the graft can be performed in any order, or simultaneously.

[0058] In a further aspect, a method of improving pancreatic tissue transplantation in a subject in need thereof is provided, the method comprising administering to the subject an HGF-MET agonist. Also provided is an HGF-MET agonist for improving pancreatic tissue transplantation in a subject, or the use of an HGF-MET agonist for the manufacture of a medicament for improving pancreatic tissue transplantation in a subject. "Improved pancreatic tissue transplantation," as used herein, means improved survival of the graft following transplantation and expansion of the engrafted cells or tissue.

[0059] Administration of an HGF-MET agonist (e.g., a MET agonist antibody) is particularly advantageous in the context of type 1 diabetes. Type 1 diabetes is characterized by significant, often complete, degradation of a subject's beta islet cells. As a result, the subject is unable to produce insulin and therefore unable to adequately control blood glucose (blood glucose). As shown herein, administration of an HGF-MET agonist (e.g., a MET agonist antibody) can promote pancreatic islet cell (particularly beta cells) growth even in subjects whose islet cell populations are depleted. These new islet cells resulting from the methods provided herein are functional and produce insulin. Thus, type 1 diabetes patients will benefit from the methods provided herein.

[0060] Thus, in certain embodiments of all of the methods provided herein, the subject has type 1 diabetes.

[0061] Type 2 diabetes is characterized by a different pathogenic mechanism, but (like type 1) causes degeneration of the islets of Langerhans. For example, insulin resistance, characteristic of type 2 diabetes, requires a subject's beta cells to produce more insulin, ultimately leading to islet cell depletion and degeneration. Therefore, regeneration of islet cells, particularly beta cells, is an unmet medical need for patients with type 2 diabetes as well. As shown herein, HGF-MET agonists (e.g., MET agonist antibodies) promote islet cell proliferation in models of type 2 diabetes, resulting in increased beta cell numbers, increased insulin production, and therefore improved glycemic control.

[0062] Thus, in certain embodiments of all of the methods provided herein, the subject has type 2 diabetes.

[0063] In vitro methods It is demonstrated herein that pancreatic islet cell proliferation is promoted by HGF-MET agonists. HGF-MET agonists (e.g., MET agonist antibodies) not only have important effects in vivo, but are also advantageously used to promote in vitro expansion of pancreatic islet cells. Promoting in vitro expansion of pancreatic islet cells is important, for example, in the preparation of islet cell transplants. Pancreatic islets isolated in preparation for transplantation have limited in vitro viability. Contacting isolated pancreatic islet cells with an HGF-MET agonist (e.g., an anti-MET agonist antibody) prolongs the survival of isolated pancreatic islet cells in vitro. As a result, the window for effective transplantation is extended, and a greater proportion of transplanted islets will be viable. Similarly, isolated islets to be transplanted can be expanded using HGF-MET agonists according to the provided methods, thereby increasing the cell population available for transplantation.

[0064] Thus, in a further aspect, an in vitro method for promoting the growth of a cell population or tissue comprising pancreatic islet cells is provided, the method comprising contacting the cell population or tissue with an HGF-MET agonist. In a preferred embodiment, the HGF-MET agonist is a MET agonist antibody. The present invention also relates to an ex vivo method for preserving pancreatic islet cells or pancreatic grafts, comprising contacting the pancreatic islet cells or pancreatic grafts with an HGF-MET agonist, preferably a MET agonist antibody.

[0065] Subjects or patients As shown herein, administration of a MET agonist (e.g., a MET agonist antibody) promotes the proliferation of functional pancreatic islet cells, which is particularly important for patients recently diagnosed with diabetes, particularly type 1 diabetes, or those with so-called "pre-diabetes."

[0066] Symptoms of type 1 diabetes typically appear during adolescence. However, by the time the condition is diagnosed, a large proportion of the patient's pancreatic beta cells have already been destroyed (more than 50%, e.g., 70% or 80% destruction). Degeneration of the islet cells of Langerhans develops rapidly, especially when clinical symptoms become apparent and a diagnosis of diabetes is most commonly made. As a result, the time window for effective therapeutic intervention is narrow. This is evidenced by the fact that treatment with immunosuppressants (to limit islet cell degeneration) is most effective immediately after diagnosis, preferably within six weeks.

[0067] Thus, in certain embodiments of the methods provided herein, the subject has been diagnosed with diabetes and the first administration of a MET agonist (e.g., a MET agonist antibody) is within 6 weeks of diagnosis. Preferably, the first administration is within 5 weeks, 4 weeks, or 3 weeks of diagnosis.

[0068] In certain embodiments, the subject has "pre-diabetes." In such embodiments, "pre-diabetes" can be defined according to American Diabetes Association (ADA) thresholds for fasting plasma glucose (FPG), oral glucose tolerance test (OGTT), or both FPG and OGTT thresholds.

[0069] According to the ADA definition, "prediabetes" is characterized by impaired fasting glucose, i.e., an FPG of at least 100 mg / dL (5.6 mmol / L) but less than 126 mg / dL (7.0 mmol / L). Prediabetes can also be characterized by impaired glucose tolerance, i.e., an OGTT result of at least 140 mg / dL (7.8 mmol / L) but less than 200 mg / dL (11.1 mmol / L). Patients with fasting glucose levels of 126 mg / dL (7.0 mmol / L) or greater have fasting glucose levels that are sufficiently low to be diagnosed with diabetes. Patients with an OGTT of 200 mg / dL (11.1 mmol / L) or greater have impaired glucose tolerance that is sufficient to be diagnosed with diabetes.

[0070] Promoting islet cell proliferation in subjects who still exhibit partial glucose regulation (e.g., subjects in the early stages of diabetes, or "prediabetes") is particularly advantageous because these subjects still have a population of functioning islet cells. Thus, methods according to the present invention can extend the period during which such patients have functional islet cells.

[0071] Thus, in certain embodiments, the methods provided herein are methods of treating pre-diabetes.

[0072] In certain embodiments of the methods provided herein, the subject exhibits a fasting glucose (FPG) greater than 5.6 mmol / L. In certain embodiments, the subject exhibits a fasting glucose (FPG) greater than 6.1 mmol / L. In certain embodiments, the subject exhibits a fasting glucose (FPG) greater than 5.6 mmol / L and less than 7.0 mmol / L. In certain embodiments, the subject exhibits a fasting glucose equal to or greater than 7.0 mmol / L.

[0073] In certain embodiments of the methods provided herein, the subject exhibits fasting glucose greater than 100 mg / dL. In certain embodiments, the subject exhibits fasting glucose greater than 110 mg / dL. In certain embodiments, the subject exhibits fasting glucose (FPG) greater than 100 mg / dL and less than 126 mg / dL. In certain embodiments, the subject exhibits fasting glucose greater than 110 mg / dL and less than 126 mg / dL. In certain embodiments, the subject exhibits fasting glucose equal to or greater than 126 mg / dL.

[0074] In certain embodiments of the methods provided herein, the subject exhibits an OGTT greater than 7.8 mmol / L. In certain embodiments, the subject exhibits a fasting glucose level greater than 7.8 mmol / L and less than 11.1 mmol / L. In certain embodiments, the subject exhibits a fasting glucose level equal to or greater than 11.1 mmol / L.

[0075] In certain embodiments of the methods provided herein, the subject exhibits an OGTT greater than 140 mg / dL. In certain embodiments, the subject exhibits a fasting glucose level greater than 140 mg / dL and less than 200 mg / dL. In certain embodiments, the subject exhibits a fasting glucose level of 200 mg / dL or greater.

[0076] In certain embodiments of the methods provided herein, the subject is an adolescent, i.e., the subject is between 10 and 19 years of age, for example, between 12 and 18 years of age.

[0077] As previously mentioned, the methods provided herein are particularly advantageous for subjects whose islet cell levels have been depleted but who still have a functional islet cell population, as the methods can promote the viability of the remaining islet cells and simultaneously promote the proliferation and regeneration of new islet cells.

[0078] Thus, in certain embodiments of all of the methods provided herein, the subject is characterized by having a population of pancreatic islet cells that is at least 50% smaller than that of a healthy individual. In certain embodiments, the subject has a population of pancreatic islet cells that is at least 70%, optionally at least 80%, at least 90%, or at least 95% smaller than that of a healthy individual. In certain embodiments, the subject has a population of pancreatic islet cells that is about 70% to about 80% smaller than that of a healthy individual.

[0079] The destruction of pancreatic islet cells by autoantibodies can occur for some time before clinical symptoms become apparent and diabetes is diagnosed.During this period, autoantibodies against islet cell antigens are detected, indicating that the destruction of pancreatic islet cells is ongoing.The method provided herein is particularly advantageous for subjects in that such antibodies can be detected, especially when the subject is not yet symptomatic.This is because the subject still has a functional islet cell population that can be protected and regenerated using this method.

[0080] Thus, in certain embodiments, the subject has detectable autoantibodies to an islet cell antigen in their serum. In preferred such embodiments, the subject has not been diagnosed with diabetes. In some embodiments, the methods include measuring the level of autoantibodies to an islet cell antigen in the subject's serum, and administering a MET agonist (e.g., a MET agonist antibody) if the level is elevated compared to levels characteristic of healthy subjects.

[0081] Patients with latent autoimmune diabetes in adults (LADA) will particularly benefit from the methods provided herein. LADA is a form of diabetes whose progression is usually slower than that of diabetes diagnosed at a younger age. LADA can be characterized by impaired glycemic control (e.g., hyperglycemia) along with the detection of C-peptide. Subjects may also have detectable antibodies against pancreatic islet cells. Degeneration of pancreatic islet cells (especially β-islet cells) in LADA patients is more delayed. As a result, such patients are expected to retain a functional islet cell population for longer. The methods provided herein will be particularly beneficial for LADA patients because they can promote the survival of remaining islet cells while simultaneously promoting the proliferation and regeneration of new islet cells.

[0082] Thus, in certain embodiments, the subject has LADA. In certain embodiments, the method is a method of treating LADA.

[0083] The method described herein is particularly advantageous in subjects receiving pancreatic tissue transplantation.Pancreatic tissue transplantation is a possible treatment for subjects whose pancreatic islet cells have already been destroyed (such as diabetic patients).Such transplantation can be in the form of a complete pancreas transplant, a partial pancreas transplant, or an isolated islet transplant.In all cases, the method provided herein is particularly advantageous in patients receiving such transplants and grafts, because it promotes the survival of transplanted islets and the proliferation and expansion of their cells.

[0084] Thus, in certain embodiments of all of the methods provided herein, the subject has previously undergone a pancreatic tissue transplant. In certain embodiments, the subject has previously undergone a complete pancreas transplant. In certain embodiments, the subject has previously undergone a partial organ transplant. In certain embodiments, the subject has previously undergone a pancreatic islet transplant.

[0085] In preferred embodiments of all methods provided herein, the subject has type 1 diabetes. In preferred embodiments of all methods provided herein, the subject has type 2 diabetes.

[0086] As described elsewhere herein, the provided methods are particularly advantageous in the context of pancreatic tissue transplantation. In this context, the methods are particularly advantageous in that they promote the proliferation of transplanted islet cells. However, the methods are also advantageous when administered to healthy subjects from whom islet cells can be harvested, i.e., donor subjects. As demonstrated herein, administration of an HGF agonist (especially a MET agonist antibody) to a healthy subject promotes the proliferation of those islet cells without side effects. Thus, healthy subjects from whom pancreatic tissue is to be harvested for transplantation, i.e., donor subjects, will benefit from the administration of an HGF-MET agonist (e.g., a MET agonist antibody) according to the methods provided herein, because this will promote the proliferation of those islet cells, thereby providing more cells for transplantation. In addition, if the donor is a living donor, the remaining islet cell population will be larger after administration of the HGF-MET agonist.

[0087] Thus, in certain embodiments of the provided methods, the subject is a healthy donor subject.

[0088] In preferred embodiments of all aspects, the subject or patient is a mammal, particularly a human.

[0089] In a preferred embodiment of all aspects, the subject is a subject in need of the method, i.e. The method is administered to a subject in need thereof. Combination therapy

[0090] The HGF-MET agonists administered according to the methods provided herein are particularly advantageous when administered as a combination therapy with immunosuppressive therapy, because immunosuppressive drugs can reduce autoimmune-mediated islet cell destruction. However, repeated administration of immunosuppressive drugs over a period of weeks or months may be required for this protection to be effective. During this lag period, islet cells continue to degenerate, often to the point of complete destruction by the time the immunosuppressive drugs achieve their clinical effect. Administration of an HGF-MET agonist according to the present invention can prolong islet cell survival. Thus, the therapeutic window during which the immunosuppressive drugs are effective is extended, meaning that the combination therapy is more likely to be effective in protecting a subject's islet cells. Furthermore, in addition to prolonging islet cell survival, the methods provided herein promote their proliferation. Therefore, combination therapy is more effective as a result of a longer effective therapeutic window during which the immunosuppressive drugs reduce islet cell destruction and, in parallel, promote the proliferation and expansion of new islet cells as a result of MET agonist administration.

[0091] Accordingly, in certain embodiments of all of the methods and second medical indications provided herein, the subject is administered one or more immunosuppressive drugs. Accordingly, in certain embodiments, HGF-MET agonists are also provided for use in combination with one or more immunosuppressive drugs to promote pancreatic islet cell proliferation, promote insulin production, and / or treat diabetes in a subject. Also provided are HGF-MET agonists for use in promoting pancreatic islet cell proliferation, promote insulin production, and / or treat diabetes in a subject receiving treatment with one or more immunosuppressive drugs.

[0092] Immunosuppressants reduce autoimmune-mediated degradation of pancreatic islet cells. In certain embodiments, the one or more immunosuppressants are selected from the list consisting of: cyclosporin A; mycophenolic acid, vitamin D3, anti-CD3 antibodies, anti-IL-21 antibodies, anti-CD20 antibodies (e.g., rituximab), anti-CTLA4 antibodies, anti-TNFα antibodies (e.g., infliximab), anti-IL1α antibodies, anti-IL1β antibodies, anti-CD4 antibodies, anti-CD45 antibodies, CTLA4 molecules (e.g., abatacept), TNFα inhibitors (e.g., etane). These include PD-L1 molecules, IL-1 receptor antagonists (e.g., anakinra), pegylated granulocyte colony-stimulating factor (e.g., pegfilgrastim), human recombinant IFN-α, IL-10, glutamic acid decarboxylase (GAD)-65, tolerizing insulin peptides (e.g., insulin B:9-23, proinsulin peptide 19-A3), DiaPep277 for HSP60, regulatory T cells (Tregs), and tolerizing dendritic cells. For example, GAD-65 and IL-10 can be administered together as transgenic bacteria (e.g., lactococci) expressing both molecules.

[0093] The combination of administration of a MET agonist (e.g., a MET agonist antibody) and an immunosuppressant is particularly advantageous for subjects exhibiting early-stage diabetes or impaired glucose regulation. Particularly preferred patients or subjects are those described in the "Subjects or Patients" section herein.

[0094] For example, it may be particularly advantageous in subjects with fasting blood glucose levels greater than 5.6 mmol / L, e.g., greater than 5.6 mmol / L and less than 7.0 mmol / L. These patients have some islet cell depletion, but still retain a population of islet cells. The combined use of an immunosuppressant and a MET agonist according to the methods provided herein can protect the remaining islet cell population from degradation and promote the growth of new islet cells.

[0095] In certain embodiments, the methods and uses for the second medical indication provided herein are combined with an antidiabetic drug. Examples of diabetes treatments include insulin, dietary management, metformin, sulfonylureas, thiazolidinediones, dipeptidyl peptidase-4 inhibitors, SGLT2 inhibitors, and glucagon-like peptide 1 analogs. Thus, in certain embodiments, HGF-MET agonists are provided for use in combination with an antidiabetic drug to promote pancreatic islet cell proliferation, promote insulin production, and / or treat diabetes in a subject. Also provided are HGF-MET agonists for promoting pancreatic islet cell proliferation, promote insulin production, and / or treat diabetes in a subject receiving treatment with an antidiabetic drug.

[0096] The methods provided herein and their use for the second medical indication can further be advantageously combined with the administration of insulin, which can manage the symptoms of degraded islet cell mass while the methods provided herein are expanding the islet cell mass.

[0097] Thus, in certain embodiments of all aspects of the methods and second medical indications of use provided herein, insulin is administered to the subject at least daily, i.e., at least once a day, and optionally more frequently.

[0098] Administration As used herein, it will be understood that administration of an HGF-MET agonist (eg, an anti-MET agonist antibody) to a subject refers to administration of an effective amount of the agonist.

[0099] In certain embodiments, the HGF-MET agonist (e.g., an anti-MET agonist antibody or antigen-binding fragment thereof) is administered at a dose ranging from about 0.1 mg / kg to about 40 mg / kg per dose. In certain embodiments, the HGF-MET agonist (e.g., an anti-MET agonist antibody or antigen-binding fragment thereof) is administered at a dose ranging from 0.5 mg / kg to about 35 mg / kg, optionally about 1 mg / kg up to about 30 mg / kg. In certain preferred embodiments, the HGF-MET agonist (e.g., an anti-MET agonist antibody or antigen-binding fragment thereof) is administered at a dose ranging from about 1 mg / kg to about 10 mg / kg, i.e., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg. In certain preferred embodiments, the HGF-MET agonist (eg, an anti-MET agonist antibody or antigen-binding fragment thereof) is administered at a dose of 1 mg / kg, 3 mg / kg, 10 mg / kg, or 30 mg / kg.

[0100] Suitable routes for administering an HGF-MET agonist (e.g., an anti-MET agonist antibody) to a subject will be well known to those of skill in the art. Preferably, the MET agonist is administered parenterally. In certain preferred embodiments, the HGF-MET agonist is administered orally or by mouth (po), subcutaneously (sc), intravenously (iv), intradermally (id), intramuscularly (im), or intraperitoneally (ip). In certain preferred embodiments, the HGF-MET agonist is a MET agonist antibody and is administered intravenously.

[0101] HGF-MET agonist In all aspects of the present invention, an HGF-MET agonist is administered to a subject or patient. The terms "HGF-MET agonist" and "MET agonist" are used interchangeably to refer to a non-naturally occurring agent that promotes signaling through the MET protein, i.e., an agent other than HGF that binds to MET and increases MET signaling. Such agents can be small molecules, binding proteins such as antibodies or antigen-binding fragments, aptamers, or fusion proteins. A specific example of a MET agonist is an anti-MET agonist antibody.

[0102] Agonistic activity of the MET agonists described herein on MET binding is demonstrated by molecular and / or cellular responses that mimic (at least in part) the molecular and cellular responses induced by HGF-MET binding.

[0103] In accordance with the present invention, methods for determining MET agonism, e.g., by MET agonist antibodies and antigen-binding fragments, will be well known to those skilled in the art. For example, MET agonism can be indicated by phosphorylation of the MET receptor and / or cellular responses, e.g., molecular responses such as those detectable in cell scattering assays, anti-apoptotic assays, and / or branching morphology assays.

[0104] MET agonism (agonism) can be determined by the phosphorylation level of MET receptor upon binding. In this situation, for example, MET agonist antibody or antigen-binding fragment causes MET autophosphorylation in the absence of receptor-ligand binding. That is, when antibody or antigen-binding fragment binds to MET, MET phosphorylation occurs in the absence of HGF. MET phosphorylation can be determined by assays known in the art, such as Western blotting or phospho-MET ELISA (Basilico et al., J Clin Invest. vol. 124, pp. 3172-3186, 2014; which is incorporated herein by reference).

[0105] MET agonism can alternatively be measured by induction of an HGF-like cellular response. MET agonism can be measured using assays such as cell scattering assays, anti-apoptosis assays, and / or branching morphogenesis assays. In this regard, a MET agonist, e.g., an antibody or antigen-binding fragment, induces a response in a cellular assay that resembles (at least in part) the response observed after exposure to, e.g., HGF.

[0106] For example, a MET agonist (eg, a MET agonist antibody) can increase antibody-responsive cell scattering compared to cells exposed to a control antibody (eg, IgG1).

[0107] By way of further example, a MET agonist (e.g., a MET agonist antibody) may exhibit protection against drug-induced apoptosis with an EC50 of less than 32 nM. By way of further example, a MET agonist (e.g., a MET agonist antibody) may exhibit an Emax cell viability of greater than 20% compared to untreated cells.

[0108] As a further example, a MET agonist (eg, a MET agonist antibody) can increase the number of branches per spheroid in cell spheroid preparations exposed to the antibody or antigen-binding fragment.

[0109] MET agonists used in accordance with the present invention preferably stimulate MET signaling to an extent at least 70% greater than that of the natural ligand, HGF, i.e., the agonist is preferably a "full agonist." In certain embodiments, MET agonists stimulate signaling to an extent at least 80%, optionally at least 85%, at least 90%, at least 95%, or at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% greater than that of HGF.

[0110] In certain embodiments, when MET agonism (receptor activation) is determined using a phosphorylation assay, the MET agonist, e.g., a MET antibody, exhibits a potency for MET with an EC50 of less than 1 nM. In certain embodiments, the MET agonist, e.g., a MET antibody, exhibits a potency for MET agonism of at least 80% Emax (as a percentage of maximal HGF-induced activation).

[0111] In certain embodiments, when MET agonism is measured in a cell scattering assay, a MET agonist, e.g., a MET antibody or antigen-binding fragment, induces an increase in cell scattering equivalent to at least 0.1 nM homologous HGF at an antibody concentration of 0.1-1 nM.

[0112] In certain embodiments, when MET agonism is measured in an anti-apoptotic assay, the MET agonist (e.g., a MET antibody or fragment thereof) exhibits an EC50 that is 1.1-fold or less than that of HGF. In certain embodiments, when MET agonism is measured in an anti-apoptotic assay, the MET agonist (e.g., a MET antibody or fragment thereof) exhibits an Emax cell viability of greater than 90% compared to that observed for HGF.

[0113] In certain embodiments, when MET agonism is measured in a branching morphogenesis assay, cells treated with a MET agonist (e.g., a MET antibody or antigen-binding fragment) exhibit greater than 90% of the number of branches per spheroid induced by the same (non-zero) HGF concentration.

[0114] Particularly preferred HGF-MET agonists in all aspects of the invention are anti-MET agonist antibodies, which are also referred to herein as "MET agonist antibodies," "agonist antibodies," and grammatical variations thereof. In other words, MET agonist antibodies (or antigen-binding fragments thereof) for use in accordance with the invention bind to MET and promote cell signaling via MET.

[0115] As shown in the Examples, the MET agonist antibodies 71D6 and 71G2 effectively promote the proliferation of pancreatic islet cells, particularly islet β cells. 71D6 and 71G2 bind to epitopes in the SEMA domain of MET, particularly epitopes on blades 4 and 5 of the SEMA β-propeller. Thus, it has been demonstrated that MET agonist antibodies that bind to epitopes on the SEMA domain of MET, particularly blades 4-5 of the SEMA β-propeller, promote the proliferation of pancreatic islet cells, particularly β cells.

[0116] Thus, in certain embodiments, the methods described herein comprise administering a MET agonist antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to an epitope in the SEMA domain of MET. In certain preferred embodiments, the antibody or antigen-binding fragment thereof binds to an epitope located on a blade of the SEMA β-propeller. In certain embodiments, the epitope is located on blade 4 or 5 of the SEMA β-propeller. In certain preferred embodiments, the antibody or antigen-binding fragment thereof binds to an epitope located between amino acids 314 and 372 of MET.

[0117] As shown in the Examples, MET agonist antibodies that bind to the SEMA domain of MET, including 71D6, were found to bind to an epitope on MET that includes residues Ile367 and Asp371. Mutation at either of these residues attenuates binding of the antibody to MET, and mutation of both residues completely abolishes binding.

[0118] Thus, in certain preferred embodiments, the methods described herein comprise administering a MET agonist antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment recognizes an epitope comprising amino acid residue Ile367. In certain preferred embodiments, the methods described herein comprise administering a MET agonist antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment recognizes an epitope comprising amino acid residue Asp371.

[0119] In certain preferred embodiments, the antibody or antigen-binding fragment binds to an epitope comprising amino acid residues Ile367 and Asp372 of MET.

[0120] As well as the MET agonist antibody that binds to SEMA domain, the agonist antibody that binds to other MET domains is also described herein.For example, 71G3 binds to an epitope on the PSI domain of MET.As demonstrated in the examples, antibody 71G3 can also promote the proliferation of pancreatic islet cells in all models tested.

[0121] Thus, in certain embodiments, the methods described herein comprise administering a MET agonist antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds to an epitope in the PSI domain of MET. In certain preferred embodiments, the antibody or antigen-binding fragment binds to an epitope located between amino acids 546 and 562 of MET.

[0122] As shown in the Examples, MET agonist antibodies that bind to the PSI domain of MET, including 71G3, were found to bind to an epitope on MET containing residue Thr555. Mutation of this residue completely abolished binding of the PSI-binding agonist antibodies to MET.

[0123] Thus, in certain preferred embodiments, the methods described herein comprise administering a MET agonist antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment recognizes an epitope comprising amino acid residue Thr555.

[0124] Examples of MET agonist antibodies particularly suitable for use in the methods described herein are those that have CDR combinations that correspond to the CDRs of the anti-MET antibodies described herein. Thus, in certain embodiments, the antibody or antigen-binding fragment comprises a combination of VH and VL CDR sequences that corresponds to a VH CDR combination from a MET agonist antibody listed in Table 3, and a corresponding combination of VL CDRs of the same antibodies in Table 4.

[0125] In certain such embodiments, the antibody or antigen-binding fragment comprises a combination of CDRs corresponding to a combination of VH CDRs from a MET agonist antibody set forth in Table 3, and a corresponding combination of VL CDRs from the same antibody in Table 4, and further comprises VH and VL domains having at least 90%, optionally at least 95%, optionally at least 99%, and preferably 100% sequence identity to the corresponding VH and VL sequences of the antibodies set forth in Table 6. For clarity, in such embodiments, the allowed variation in percent identity of the VH and VL domain sequences is not in the CDR regions.

[0126] As demonstrated in the Examples, 71D6, 71G2, and 71G3 are MET agonist antibodies that are "full agonists" of MET. That is, upon binding of these antibodies to MET, the signaling response is similar to or exceeds the response to binding of the natural HGF ligand. Each of these antibodies has been demonstrated herein to effectively promote pancreatic islet cell proliferation. Thus, in certain preferred embodiments of all aspects and methods described herein, the method comprises administering an HGF-MET agonist that is a full agonist, i.e., an agonist that promotes MET signaling upon binding to an extent similar to or exceeding that of MET signaling upon binding of HGF. Examples for measuring MET agonism and examples of the effects of full agonists have been previously described herein.

[0127] As demonstrated in the Examples, examples of MET full agonists, such as anti-MET antibodies that are full agonists, include 71D6, 71G2, and 71G3. Thus, in particularly preferred embodiments of all methods described herein, the method comprises administering a MET agonist antibody or antigen-binding fragment thereof that is a full agonist of MET.

[0128] The MET agonist antibodies 71D6, 71G2, and 71G3 have each been demonstrated to effectively promote pancreatic islet cell proliferation. Accordingly, in preferred embodiments of all aspects and methods described herein, the antibody or fragment comprises a combination of CDRs with the corresponding CDR sequences of antibody 71D6 (SEQ ID NOs: 30, 32, 34, 107, 109, and 111), antibody 71G2 (SEQ ID NOs: 44, 46, 48, 121, 123, and 125), or antibody 71G3 (SEQ ID NOs: 9, 11, 13, 86, 88, and 90).

[0129] In a preferred embodiment of all aspects, the MET agonist is a [71D6] MET agonist antibody or antibody-binding fragment thereof having an HCDR1 of SEQ ID NO: 30, an HCDR2 of SEQ ID NO: 32, an HCDR3 of SEQ ID NO: 34, an LCDR1 of SEQ ID NO: 107, an LCDR2 of SEQ ID NO: 109, and an LCDR3 of SEQ ID NO: 111.

[0130] In preferred such embodiments, the antibody or antigen-binding fragment comprises: a VH domain comprising SEQ ID NO: 163, or a sequence at least 90% identical thereto, optionally at least 95%, at least 98%, or at least 99% identical thereto; and a VL domain comprising SEQ ID NO: 164, or a sequence at least 95%, optionally at least 98% or at least 99% identical thereto. For clarity, in such embodiments, the allowed variation in the percent identity of the VH and VL domain sequences is not in the CDR regions.

[0131] MET agonist antibodies for use herein can take a variety of different embodiments in which both VH and VL domains are present. The term "antibody" as used herein is used in the broadest sense and includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), so long as they exhibit immunological specificity for human MET protein and mouse MET protein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies; that is, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes) on an antigen, each monoclonal antibody is directed against a single determinant or epitope on the antigen.

[0132] An "antibody fragment" comprises a portion of a full-length antibody, generally the antigen-binding or variable domain. Examples of antibody fragments include Fab, Fab', F(ab')2, bispecific Fab's, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single-chain variable fragments (scFv), and multispecific antibodies formed from antibody fragments (see Holliger & Hudson, Nature Biotechnol, 23:1126-1136, 2005, the contents of which are incorporated herein by reference).

[0133] In preferred embodiments of all aspects provided herein, the MET agonist antibody or antigen-binding fragment thereof is bivalent.

[0134] In non-limiting embodiments, the MET antibodies provided herein can comprise a CH1 domain and / or a CL domain whose amino acid sequence is fully or substantially human. Thus, one or more, or any combination, of the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) can be fully or substantially human with respect to their amino acid sequence. Such antibodies can be of any human isotype, e.g., IgG1 or IgG4.

[0135] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may all have completely or substantially human amino acid sequences. In the context of the constant region of a humanized or chimeric antibody or antibody fragment, the term "substantially human" refers to at least 90%, or at least 92%, or at least 95%, or in the case of a human constant region, at least 97%, or at least 99% amino acid sequence identity. The term "human amino acid sequence" in this context refers to an amino acid sequence encoded by a human immunoglobulin gene, including germline genes, rearranged genes, and somatically mutated genes. Such antibodies may be of any human isotype, with human IgG4 and IgG1 being particularly preferred.

[0136] MET agonist antibodies may comprise constant domains of "human" sequence altered by the addition, deletion, or substitution of one or more amino acids relative to the human sequence, except in embodiments where the presence of a "fully human" hinge region is expressly required. The presence of a "fully human" hinge region in a MET antibody of the invention may be beneficial both to minimize immunogenicity and to optimize antibody stability.

[0137] The MET agonist antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, or IgM. In preferred embodiments, the antibody is of the IgG type, e.g., IgG1, IgG2a and b, IgG3, or IgG4. IgG1 and IgG4 are particularly preferred. Within each of these subclasses, one or more amino acid substitutions, insertions, or deletions can be made within the Fc portion, or other structural modifications can be made.

[0138] In non-limiting embodiments, it is contemplated that one or more amino acid substitutions, insertions, or deletions may be made within the heavy and / or light chain constant regions, particularly within the Fc region. Amino acid substitutions can replace the substituted amino acid with a different naturally occurring amino acid or a non-natural or modified amino acid. Other structural modifications are also possible, such as altered glycosylation patterns (e.g., by adding or deleting N- or O-linked glycosylation sites). Depending on the intended use of the MET antibody, it may be desirable to modify the antibody of the invention with respect to its binding properties to Fc receptors, e.g., to modulate effector function.

[0139] In certain embodiments, a MET antibody can comprise an Fc region of a given antibody isotype, e.g., human IgG1, that is modified to reduce or substantially eliminate one or more antibody effector functions naturally associated with that antibody isotype. In non-limiting embodiments, a MET antibody can substantially lack an antibody effector function. In this context, "antibody effector function" includes one or more or all of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0140] The amino acid sequence of the Fc portion of a MET antibody may include one or more mutations, such as amino acid substitutions, deletions, or insertions, that have the effect of reducing one or more antibody effector functions (compared to a wild-type counterpart antibody lacking the mutations). Some such mutations are known in the field of antibody engineering. Non-limiting examples suitable for inclusion in the MET antibodies described herein include the following mutations in the Fc domain of human IgG4 or human IgG1: N297A, N297Q, LALA (L234A, L235A), AAA (L234A, L235A, G237A), or D265A (amino acid residue numbering according to EU numbering for human IgG1).

[0141] Thus, in certain embodiments of all aspects of the invention, the anti-MET agonist antibody is an agonist antibody to both human MET and mouse MET.

[0142] Pharmaceutical Composition Also provided in accordance with the present invention are pharmaceutical compositions for use in the methods described herein. Accordingly, in a further aspect of the present invention, there is provided a pharmaceutical composition comprising an HGF-MET agonist, e.g., an anti-MET agonist antibody, and a pharmaceutically acceptable excipient or carrier for use in the methods of the present invention. Suitable pharmaceutically acceptable carriers and excipients will be well known to those skilled in the art. Examples of pharmaceutically acceptable carriers and excipients suitable for inclusion in pharmaceutical compositions of the present invention include sodium citrate, glycine, polysorbates (e.g., polysorbate 80), and saline.

[0143] In certain embodiments, the MET agonist, e.g., an anti-MET agonist antibody, is administered to a subject parenterally, preferably intravenously (iv). In certain embodiments, the MET agonist, e.g., an anti-MET agonist antibody, is administered as a continuous intravenous infusion. The infusion is continued until the desired dose is achieved.

[0144] In certain embodiments, the MET agonist, eg, an anti-MET agonist antibody, is administered to the subject parenterally, preferably intraperitoneally (ip). [Example]

[0145] The present invention will be better understood by reference to the following non-limiting examples.

[0146] Example 1: Generation of anti-MET agonist antibodies - llama immunization

[0147] Immunization of llamas and collection of peripheral blood lymphocytes (PBLs), as well as subsequent RNA extraction and amplification of antibody fragments, were performed as described (De Haard et al., J. Bact. 187:4531-4541, 2005). Two adult llamas (Lama glama) were immunized intramuscularly with a chimeric protein consisting of the extracellular domain (ECD) of human MET fused to the Fc portion of human IgG1 (MET-Fc; R&D Systems). Each llama received a total of six injections, one injection per week for six weeks. Each injection consisted of 0.2 mg of protein in Freund's incomplete adjuvant, split into two sites in the neck.

[0148] Ten mL blood samples 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 by the phenol-guanidine thiocyanate method (Chomczynski et al., Anal. Biochem. 162:156-159, 1987) and analyzed using SuperScript. TMThe cDNAs were used as templates for random cDNA synthesis using the llama IgG1 VH-CH1 region and VL-CL domains (κ and λ) of the llama IgG1 gene as described (de Haard et al., J Biol Chem. 274:18218-18230, 1999). The cDNAs encoding the VH-CH1 region and VL-CL domains (κ and λ) of llama IgG1 were amplified and subcloned into the phagemid vector pCB3. The recombinant phagemids were used to transform E. coli strain TG1 (Netherlands Culture Collection of Bacteria) to generate four different Fab-expressing phage libraries (one λ library and one κ library per immunized llama). The diversity was 10 8 ~10 9 The range was.

[0149] The immune response to the antigen was examined by ELISA. For this purpose, the human MET ECD (UniProtKB # P08581; amino acids 1–932) and mouse MET ECD (UniProtKB # P16056.1; amino acids 1–931) were obtained using standard protein engineering techniques. Human or mouse MET ECD recombinant proteins were immobilized on a solid phase (100 ng / well in a 96-well plate) and exposed to serial dilutions of llama-derived serum before (day 0) or after (day 45) immunization. Binding was revealed using mouse anti-llama IgG1 (Daley et al., Clin. Vaccine Immunol. Vol. 12, 2005) and HRP-conjugated donkey anti-mouse antibody (Jackson Laboratories). Both llamas demonstrated an immune response to the human MET ECD. Consistent with the notion that the extracellular portion of human MET is 87% identical to the mouse orthologue, excellent cross-reactivity was also observed with the mouse MET ECD.

[0150] Example 2: Selection and screening of Fabs that bind to both human and mouse MET

[0151] Fab-expressing phages were generated from the library using standard phage display protocols. For selection, phages were first adsorbed to immobilized recombinant human MET ECD, washed, and then eluted with trypsin. After two cycles of selection using human MET ECD, two additional cycles were performed using mouse MET ECD in the same manner. In parallel, phages were selected by alternating between human and mouse MET ECD cycles for a total of four cycles. Phages selected by these two approaches were pooled and used to infect TG1 E. coli. Individual colonies were isolated, and Fab secretion was induced with IPTG (Fermentas). The Fab-containing periplasmic fractions of the bacteria were collected, and their ability to bind to human and mouse MET ECD was examined by surface plasmon resonance (SPR). Human or mouse MET ECD was immobilized on a CM-5 chip using amine coupling in sodium acetate buffer (GE Healthcare). The Fab-containing periplasmic extract was loaded onto a BIACORE® 3000 instrument (GE Healthcare) at a flow rate of 30 μL / min. The dissociation rate of Fab (k off ) was measured over a 2-minute period. Binding of the Fab to human and mouse MET was further characterized by ELISA using MET ECD in the solid phase and crude periplasmic extracts in solution. Because the Fab was engineered with MYC flag, binding was revealed using an HRP-conjugated anti-MYC antibody (ImTec Diagnostics).

[0152] Fabs that bound to both human and mouse MET by both SPR and ELISA were selected, and the corresponding phages were sequenced (LGC Genomics). The cross-reactive Fab sequences were classified into families based on the length and content of the VH CDR3 sequences. VH families were assigned national numbers that were not based on the International Immunogenetics Information System (IMTG) nomenclature. Ultimately, 11 distinct 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 and light chain variable regions are shown in Table 5. The complete DNA sequences of the heavy and light chain variable regions are shown in Table 6.

[0153] [Table 3-1] [Table 3-2]

[0154] [Table 4-1]

[0155] [Table 4-2] [Table 5-1] [Table 5-2]

[0156] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0157] The different Fab families and their ability to bind to human and mouse MET are shown in Table 7.

[0158] [Table 7]

[0159] [Table 8]

[0160] Example 3: Chimerization of Fab into mAb

[0161] The cDNAs encoding the VH and VL (κ or λ) domains of the selected Fab fragments were engineered and introduced into two separate pUPE mammalian expression vectors (U-protein Express) containing cDNAs encoding the CH1, CH2, and CH3 domains of human IgG1 or human CL (κ or λ), respectively.

[0162] Production (by transient transfection of mammalian cells) and purification (by protein A affinity chromatography) of the resulting llama-human chimeric IgG1 molecules were outsourced to U-protein Express. Binding of the 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 revealed using an HRP-conjugated anti-human Fc antibody (Jackson Immuno Research Laboratories). This analysis revealed that all llama-human chimeric antibodies bound human and mouse MET with picomolar affinities, with EC values ​​ranging from 0.06 nM to 0.3 nM. 50 It was found that the binding ability (E max ) varied for each antibody, likely due to partial exposure of the epitope in the immobilized antigen, but were similar in human and mouse backgrounds. 50 and E max The values ​​are shown in Table 9.

[0163] [Table 9]

[0164] We also analyzed whether the chimeric anti-MET antibodies bind to native human and mouse MET in live cells. To this end, increasing concentrations of antibody (0–100 nM) were incubated with A549 human lung carcinoma cells (American Type Culture Collection (ATCC)) or MLP29 mouse hepatic progenitor cells (a gift from Professor Enzo Medico, University of Turin, Strada Provinciale 142 km 3.95, Candiolo, Turin, Italy; Medico et al., Mol Biol Cell 7, 495–504, 1996). Both cells express MET at physiological levels. Antibody binding to cells was analyzed by flow cytometry using a phycoerythrin-conjugated anti-human IgG1 antibody (eBioscience) and a CyAn ADP analyzer (Beckman Coulter). As positive controls for binding to human MET, commercially available mouse anti-human MET antibody (R&D Systems) and phycoerythrin-conjugated anti-mouse IgG1 antibody (eBioscience) were used. As positive controls for binding to mouse MET, commercially available goat anti-mouse MET antibody (R&D Systems) and phycoerythrin-conjugated anti-goat IgG1 antibody (eBioscience) were used. All antibodies showed dose-dependent binding to both human and mouse cells, with EC 50 The maximum binding (E MAX ) varied depending on the antibody but were similar in human and mouse cells. These results demonstrate that the llama-human chimeric antibodies recognize membrane-bound MET in its native conformation in both human and mouse cell lines. EC 50 and E MAX The values ​​are shown in Table 10.

[0165] [Table 10]

[0166] Example 4: Receptor domains responsible for antibody binding

[0167] To map the receptor region recognized by antibodies that bind to both human and mouse MET (hereafter referred to as human / mouse equivalent anti-MET antibodies), we measured their ability to bind to a panel of engineered proteins derived from human MET, generated as described (Basilico et al., J. Biol. Chem. 283:21267-21227, 2008). This panel included 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); the isolated SEMA domain (SEMA); and a fragment containing IPT domains 3 and 4 (IPT 3-4). The engineered MET proteins were immobilized on a solid phase and exposed to solutions containing increasing concentrations of the chimeric antibodies (0-50 nM). Binding was demonstrated using an HRP-conjugated anti-human Fc antibody (Jackson Immuno Research Laboratories). As shown in Table 11, this analysis revealed that seven mAbs recognize epitopes within the SEMA domain, while the other four mAbs recognize epitopes within the PSI domain.

[0168] [Table 11]

[0169] To more precisely map the region of MET responsible for antibody binding, we examined the absence of cross-reactivity between our antibodies and llama MET (the organism used to generate these immunoglobulins). To this end, we generated a series of llama-human and human-llama chimeric MET proteins spanning the entire MET ECD as described (Basilico et al., J Clin Invest. 124:3172–3186, 2014). The chimeras were immobilized on a solid phase and then exposed to solutions containing increasing concentrations of mAb (0–20 nM). Binding was revealed using an HRP-conjugated anti-human Fc antibody (Jackson Immuno Research Laboratories). This analysis revealed that five SEMA-binding mAbs (71D6, 71C3, 71D4, 71A3, and 71G2) recognized epitopes located between amino acids 314 and 372 of human MET. This region corresponds to blades 4–5 of the seven-bladed SEMA β-propeller (Stamos et al., EMBO J. 23:2325–2335, 2004). Two other SEMA-binding mAbs (74C8 and 72F8) recognize epitopes located between amino acids 123–223 and 224–311, respectively, which correspond to blades 1–3 and 1–4 of the SEMA β-propeller. PSI-binding mAbs (76H10, 71G3, 76G7, and 71G12) did not appear to show significant binding to either of the two PSI chimeras. Considering the results shown in Table 11, these antibodies likely recognize epitopes located between amino acids 546–562 of human MET. These results are summarized in Table 12.

[0170] [Table 12]

[0171] Example 5: HGF competition assay

[0172] The above analysis suggests that the epitopes recognized by some human / mouse equivalent anti-MET antibodies may overlap with the epitope embedded by HGF when binding to MET (Stamos et al., EMBO J. 23:2325-2335, 2004; Merchant et al., Proc Natl Acad Sci USA 110:E2987-E2996, 2013; Basilico et al., J Clin Invest. 124:3172-3186, 2014). To investigate this idea, we analyzed competition between mAbs and HGF by ELISA. Recombinant human and mouse HGF (R&D Systems) were biotinylated at the N-terminus using NHS-LC-biotin (Thermo Scientific). Either human or mouse MET-Fc protein (R&D Systems) was immobilized on a solid phase and exposed to increasing concentrations of antibody (0–120 nM) and 0.3 nM biotinylated human or mouse HGF. Binding of HGF to MET was revealed using HRP-conjugated streptavidin (Sigma-Aldrich). As shown in Table 13, this analysis allowed the human / mouse equivalent anti-MET mAbs to be separated into two groups: full HGF competitors (71D6, 71C3, 71D4, 71A3, 71G2) and partial HGF competitors (76H10, 71G3, 76G7, 71G12, 74C8, 72F8).

[0173] [Table 13]

[0174] In principle, SEMA conjugates displaced HGF more effectively than PSI conjugates. In particular, antibodies recognizing epitopes within blades 4 and 5 of the SEMA β-propeller were the most potent HGF competitors (71D6, 71C3, 71D4, 71A3, and 71G2). This finding is consistent with the notion that blade 5 of SEMA contains a high-affinity binding site for the HGF α-chain (Merchant et al., Proc Natl Acad Sci USA 110:E2987-E2996, 2013). Although the PSI domain has not been shown to directly interact with HGF, it has been suggested that it functions as a "hinge" between the SEMA domain and the IPT region, regulating HGF capacity (Basilico et al., J Clin Invest. 124:3172-3186, 2014). Thus, mAbs that bind to PSI (76H10, 71G3, 76G7, and 71G12) appear to interfere with HGF binding to MET by interfering with this process or through steric hindrance, rather than by direct competition with the ligand. Finally, blades 1–3 of the SEMA β-propeller have been shown to be responsible for low-affinity binding of the HGF β-chain, which plays a central role in MET activation but only partially contributes to HGF-MET binding strength (Stamos et al., EMBO J. 23:2325–2335, 2004). This may explain why mAbs that bind to this region of MET (74C8 and 72F8) are partial competitors of HGF.

[0175] Example 6: MET activation assay Due to their bivalent nature, immunoglobulins directed against receptor tyrosine kinases may exhibit receptor agonist activity, mimicking the action of natural ligands. To address this issue, we tested the ability of human / mouse equivalent anti-MET antibodies to promote MET autophosphorylation in a receptor activation assay. A549 human lung carcinoma cells and MLP29 mouse hepatic progenitor cells were deprived of serum growth factors for 48 hours and then challenged 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 15 minutes of challenge, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed as described (Longati et al., Oncogene 9:49–57, 1994). Protein lysates were separated by electrophoresis and analyzed by Western blotting using antibodies specific for the phosphorylated form of MET (tyrosine 1234-1235), regardless of whether they were human or mouse (Cell Signaling Technology). The same lysates were also analyzed by Western blotting using anti-total human MET antibody (Invitrogen) or anti-total mouse MET antibody (R&D Systems). This analysis revealed that all human / mouse equivalent anti-MET antibodies exhibited MET agonist activity. Some antibodies (71G3, 71D6, 71C3, 71D4, 71A3, 71G2, and 74C8) promoted MET autophosphorylation to a degree comparable to that of HGF. Some other antibodies (76H10, 76G7, 71G12, and 72F8) were less potent, especially at low antibody concentrations. No clear correlation was observed between MET activating activity and HGF competitive activity.

[0176] To obtain more quantitative data, the agonist activity of the antibodies was also characterized by phospho-MET ELISA. To this end, A549 and MLP29 cells were serum-starved as described above and then sensitized with increasing concentrations (0–25 nM) of mAb. Recombinant human (A549) HGF or recombinant mouse (MLP29) HGF was used as a control. Cells were lysed, and phosphorylated MET levels were measured by ELISA as described (Basilico et al., J Clin Invest. 124:3172–3186, 2014). Briefly, 96-well plates were coated with mouse anti-human MET or rat anti-mouse MET antibodies (both R&D Systems) and then incubated with cell lysates. After washing, the captured proteins were incubated with biotin-conjugated anti-phosphotyrosine antibody (Thermo Fishe), and binding was revealed using HRP-conjugated streptavidin (Sigma-Aldrich).

[0177] 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, whereas 76H10, 76G7, 71G12, and 72F8 elicited a less pronounced effect. In all cases, all antibodies were equally effective in human and mouse cells.

[0178] [Table 14]

[0179] Example 7: Scattering Assay To assess whether the agonist activity of the human / mouse equivalent anti-MET antibody could be translated into biological activity, we performed scattering assays using both human and mouse epithelial cells. To this end, HPAF-II human pancreatic adenocarcinoma cells (American Type Culture Collection (ATCC)) and MLP29 mouse hepatic progenitor cells were sensitized with increasing concentrations of recombinant HGF (human or mouse; both R&D Systems) and then examined for cell scattering microscopically 24 hours later as described (Basilico et al., J Clin Invest. 124:3172–3186, 2014). This preliminary analysis revealed that HGF-induced cell scattering was linear in both cell lines, reaching saturation at approximately 0.1 nM. Based on these HGF standard curves, we created a scoring system ranging from 0 (no cell scattering in the absence of HGF) to 4 (maximal cell scattering in the presence of 0.1 nM HGF). HPAF-II and MLP29 cells were sensitized with increasing concentrations of human / mouse equivalent anti-MET antibodies, and cell scattering was measured 24 hours later using the scoring method described above. As shown in Table 15, this analysis revealed that all tested mAbs promoted cell scattering in both human and mouse cell lines, with virtually overlapping results in both species. 71D6 and 71G2 exhibited activity identical to HGF; 71G3 and 71A3 were only slightly less potent than HGF; 71C3 and 74C8 required significantly higher concentrations to achieve activity equivalent to HGF; and 71D4, 76G7, 71G12, and 72F8 did not reach saturation in this assay.

[0180] [Table 15]

[0181] Example 8: Protection from drug-induced apoptosis

[0182] Several lines of experimental evidence suggest that HGF exerts potent anti-apoptotic effects on MET-expressing cells (reviewed 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, we performed a cell-based drug-induced survival assay. MCF10A human breast epithelial cells (ATCC; American Type Culture Collection) and MLP29 mouse hepatic progenitor cells were incubated with increasing concentrations of staurosporine (Sigma-Aldrich). After 48 hours, cell viability was determined by measuring total ATP concentration using a CellTiter-Glo® kit (Promega) on a Victor X4 multilabel plate reader (Perkin Elmer). This preliminary analysis revealed that the drug concentration inducing approximately 50% cell death was 60 nM in MCF10A cells and 100 nM in MLP29 cells. MCF10A and MLP29 cells were then incubated with the drug concentrations 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, demonstrate that the human / mouse equivalent antibodies protected human and mouse cells to a similar extent from staurosporine-induced cell death. In either human or mouse cell lines, several mAbs (71G3, 71D6, 71G2) demonstrated protective activity comparable to or superior to HGF, whereas other molecules (76H10, 71C3, 71D4, 71A3, 76G7, 71G12, 74C8, 72F8) demonstrated only partial protection.

[0183] [Table 16]

[0184] Example 9: Branching morphogenesis assay

[0185] HGF is a pleiotropic cytokine that promotes the coordinated regulation of multiple independent biological activities, including cell proliferation, motility, invasion, differentiation, and survival. A cell-based assay that effectively recapitulates all of these activities is the branching morphogenesis assay, which recapitulates the formation of tubular organs and tubular glands during embryonic development (reviewed by Rosario & Birchmeier, Trends Cell Biol. 13, 328–335, 2003). In this assay, epithelial cell spheroids are seeded inside a three-dimensional collagen matrix and stimulated with HGF to sprout tubules that ultimately form branched structures. These branched tubules resemble the hollow structures of epithelial glands (e.g., mammary glands) in that they display a lumen surrounded by polarized cells. This assay is the most complete HGF assay available in vitro.

[0186] To determine whether human / mouse equivalent anti-MET antibodies exhibit agonist activity in this assay, LOC human renal epithelial cells (Michieli et al., Nat Biotechnol. 20:488-495, 2002) and MLP29 mouse hepatic progenitor cells were seeded into a collagen layer as described (Hultberg et al., Cancer Res. 75:3373-3383, 2015) and then exposed to increasing concentrations of mAb or recombinant HGF (human or mouse; both from R&D Systems). Branching morphogenesis was followed microscopically over time, and colonies were photographed after 5 days. Quantitation of branching morphogenesis activity was obtained by counting the number of branches for each spheroid. As shown in Table 17, all antibodies tested induced the formation of branched tubules in a dose-dependent manner. However, consistent with the data obtained from the MET autophosphorylation assay and cell scattering assay, 71D6, 71A3, and 71G2 exhibited the most potent agonistic activity, which was similar to or even superior to that of recombinant HGF.

[0187] [Table 17]

[0188] Example 10: Fine epitope mapping

[0189] To finely map the epitopes of MET recognized by human / mouse equivalent anti-MET antibodies, we employed the following strategy. We reasoned that if an antibody raised in a llama and directed against human MET cross-reacts with mouse MET, it likely recognizes a residue (or residues) that is 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; Rattus norvegicus) and cynomolgus monkeys (M. fascicularis).

[0190] To investigate this concept, we aligned and compared 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). Referring to Table 12, we focused on the region of MET responsible 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 anterior region of human MET (amino acids 314–372) contains five residues (Ala327, Ser336, Phe343, Ile367, and Asp372) that are conserved between human and mouse MET but not between llama MET. Four of these residues (Ala327, Ser336, Ile367, and Asp372) are also conserved in rat and cynomolgus monkey MET. The posterior region of human MET (amino acids 546–562) contains three residues (Arg547, Ser553, and Thr555) that are conserved between human and mouse MET but not between llama MET. Two of these residues (Ser553 and Thr555) are also conserved in rat and cynomolgus monkey MET.

[0191] Using human MET as a template, we mutated each of these residues in different permutations to generate a series of MET mutants that were entirely human except for specific residues that were llama-specific. We then examined the affinity of selected SEMA-binding mAbs (71D6, 71C3, 71D4, 71A3, and 71G2) and PSI-binding mAbs (76H10 and 71G3) for these MET mutants by ELISA. To this end, various MET proteins were immobilized on a solid phase (100 ng / well in a 96-well plate) and exposed to increasing concentrations of antibody solution (0–50 nM). Because the antibodies used were human constant region forms, binding was revealed using an HRP-conjugated anti-human Fc secondary antibody (Jackson Immuno Research Laboratories). Wild-type human MET served as a positive control. The results of this analysis are shown in Table 18.

[0192] [Table 18]

[0193] The results presented above provide a clear and sharp picture of the residues relevant for binding to the agonist antibodies of the invention.

[0194] All tested SEMA binders (71D6, 71C3, 71D4, 71A3, and 71G2) appear to bind to the same epitope within blade 5 of the SEMA β-propeller, which contains two critical amino acids (Ile367 and Asp372) that are conserved in human, mouse, cynomolgus, and rat MET but not in llama MET. Indeed, mutations of Ala327, Ser336, and Phe343 had no effect on binding. On the other hand, mutation of Ile367 partially attenuated binding, and mutations of Ile367 and Asp372 completely abolished binding. From this, we conclude that both Ile367 and Asp372 of human MET are crucial for binding to the tested antibodies directed against SEMA.

[0195] The PSI binders tested (76H10, 71G3) also appear to bind to similar or identical epitopes. However, unlike the SEMA epitope, the PSI epitope contains only one critical amino acid (Thr555) that is conserved in human, mouse, cynomolgus, and rat MET but not in llama MET. Indeed, mutation of Arg547 or Ser553 had no effect on binding, whereas mutation of Thr555 completely abolished binding. From this, we conclude that Thr555 is a crucial determinant for binding to the PSI-directed antibodies tested.

[0196] Example 11: MET agonist antibodies promote islet proliferation and pancreatic beta cell regeneration in healthy mice

[0197] To evaluate the biological effects of MET agonist antibodies on pancreatic β cells in vivo, adult BALB / c mice (Charles River) of both sexes were treated systemically with purified 71D6 antibody at 0, 3, 10, or 30 mg / kg for 3 months (48 animals total, 6 mice per sex per group). The antibody was administered by intraperitoneal (ip) injection twice weekly. Body weight and fasting blood glucose concentrations were measured monthly throughout the study period. At the end of the 3-month period, mice were sacrificed; pancreases were harvested, paraffin-embedded, and processed for histological analysis. Sections were stained with hematoxylin and eosin, examined microscopically, and photographed. Images were analyzed using ImageJ® software (National Institutes of Health), and the number and size of islets of Langerhans were measured.

[0198] Chronic treatment with 71D6 did not affect total body weight in either male or female animals (Fig. 1A). Similarly, baseline blood glucose measured in fasting animals was unchanged at either antibody dose (Fig. 1B). On the other hand, histological analysis of pancreatic sections showed that treatment with the 71D6 agonist antibody significantly increased the number of islets of Langerhans in a dose-dependent manner (Fig. 2A). In untreated control animals (0 mg / kg), 1 unit (mm ) of pancreatic sections was significantly increased. 2 At the highest dose tested (30 mg / kg), the number of islets per mm2 The number of islets per 1000 cells reached a value of 6; doses of 3 mg / kg and 10 mg / kg showed intermediate islet densities. Treatment with 71D6 also significantly increased islet size (Figure 2B). In control animals, the mean islet size was approximately 0.01 mm. 2 (expressed as the area of ​​the islet section, as measured by microscopic imaging of hematoxylin and eosin-stained tissue sections). At the 3 mg / kg dose, mean islet area increased two-fold compared to 0 mg / kg; at the 10 mg / kg dose, it increased three-fold compared to controls; and at the 30 mg / kg dose, islet size was four-fold greater compared to treated animals. Representative images of hematoxylin and eosin-stained pancreatic sections are shown in Figure 2C.

[0199] Interestingly, immunohistochemical analysis using an anti-insulin antibody revealed that 71D6 resulted in an expansion of the pancreatic β-cell population and enhanced insulin expression (Figure 3). This finding suggests that the 71D6-induced increase in islet size is due to hyperproliferation of pancreatic β-cells. Furthermore, enhanced insulin expression demonstrates that these β-cells are healthy and functional. Collectively, these results indicate that 71D6 acts as a mitogen and pro-regenerative factor for pancreatic β-cells.

[0200] Example 12: MET agonist antibodies promote islet proliferation and pancreatic beta cell regeneration in a mouse model of type 1 diabetes

[0201] Prompted by the observation that agonistic anti-MET antibodies act as β-cell mitogens, we tested their therapeutic potential in a mouse model of type 1 diabetes. Multiple administrations of low-dose streptozotocin (STZ; a chemical that selectively kills β-cells and is the standard compound used to induce type 1 diabetes in laboratory animals) resulted in ablation of pancreatic β-cells in mice.

[0202] STZ was injected intraperitoneally (ip) into female BALB-c mice (Charles River) at a dose of 40 mg / kg every 24 hours for five consecutive days. One week after the final injection, STZ-treated mice exhibited a two-fold higher mean basal blood glucose level compared to untreated mice (240 mg / dL vs. 120 mg / dL), suggesting that the chemical effectively killed β cells. At this point, mice were randomly assigned based on their basal blood glucose levels into four groups of seven mice each: (i) vehicle only (PBS), (ii) purified 71D6 antibody, (iii) purified 71G2 antibody, or (iv) purified 71G3 antibody. The antibodies were administered at a dose of 1 mg / kg twice weekly by ip injection. An additional fifth treatment group contained seven animals that received neither STZ nor antibody and served as healthy controls. The experiment continued for 8 weeks; blood glucose was monitored throughout the experiment. At the end of the 8 weeks, the mice were sacrificed and subjected to necropsy. Blood was collected for analysis, and the pancreas was extracted, processed for histology, and embedded in paraffin.

[0203] As shown in Figure 4A, basal blood glucose levels in STZ-treated mice continued to increase over time. This suggests that STZ-induced β-cell damage leads to chronic pancreatitis and progressive worsening of organ damage. Interestingly, antibody administration did not completely normalize blood glucose, but significantly reduced it toward more normal levels. Six weeks after the start of treatment (i.e., seven weeks after the final STZ injection), mice treated with STZ alone showed an average basal blood glucose level of approximately 250 mg / dL; mice treated with STZ and 71D6 showed an average basal blood glucose level of approximately 150 mg / dL; mice treated with STZ and 71G2 or 71G3 showed slightly elevated blood glucose levels, but still significantly lower than those in the STZ-only treatment group. Control, untreated mice showed an average basal blood glucose level of 96 mg / dL (Figure 4B).

[0204] To examine the effects of MET agonist antibodies on islets of Langerhans, pancreatic sections were stained with hematoxylin and eosin and analyzed microscopically. Digital images of islets were analyzed using ImageJ® software (National Institutes of Health). The number, density, and size of islets were determined by digital data analysis. As shown in Figure 5A, STZ administration significantly reduced the number of islets in the pancreas of mice treated with this compound alone. In contrast, mice treated with STZ and 71D6 exhibited a more normal islet density, closely resembling that observed in untreated control mice. STZ treatment also significantly affected islet size; it reduced it by more than sixfold (i.e., more than sixfold) (Figure 5B). Notably, 71D6 antagonized this reduction, limiting it to 1.5-fold. Similar results were obtained with 71G2 and 71G3, which were similar but slightly less potent (in the order 71G6 > 71G2 > 71G3). Representative images of hematoxylin and eosin-stained pancreatic sections are shown in Figure 5C.

[0205] Pancreatic sections were further analyzed by immunohistochemistry using anti-insulin antibodies. This analysis revealed that STZ not only reduced the number and size of islets of Langerhans, but also significantly reduced β-cells and, consequently, insulin production. Equally notably, MET agonist antibody treatment rescued β-cells from STZ-induced destruction and maintained high insulin production. This may explain the lower blood glucose levels observed in animals treated with both STZ and MET agonist antibodies compared with mice treated with STZ alone. Representative images of pancreatic sections stained with anti-insulin antibodies are shown in Figure 6.

[0206] Example 13: MET agonist antibodies promote islet proliferation and pancreatic beta cell regeneration in a mouse model of type 2 diabetes

[0207] The observation that anti-MET agonist antibodies induce pancreatic β-cell regeneration in healthy mice and in a type 1 diabetes model prompts further testing of their therapeutic potential in other relevant indications. Although type 2 diabetes is characterized by a different pathogenic mechanism, it also causes degeneration of the islets of Langerhans. In fact, type 2 diabetes is characterized by hyperinsulinemia in the presence of insulin resistance, leading to hyperglycemia and the inability of β cells to compensate for the increased demand for insulin (Christoffersen et al., Am J Physiol Regul Integr Comp Physiol 297: 1195-1201, 2009). Therefore, β cell regeneration is also an unmet medical need in type 2 diabetes patients.

[0208] To explore the therapeutic potential of agonist MET antibodies in type 2 diabetes, we selected the db / db obese mouse model. These animals are hyperphagic, obese, hyperinsulinemic, and hyperglycemic due to a mutation in the leptin gene. Obesity becomes evident from 3 to 4 weeks of age, hyperinsulinemia becomes evident around 2 weeks, and hyperglycemia develops between 4 and 8 weeks. Female db / db mice were obtained from Charles River at 7 weeks of age. One week later, the animals were randomly assigned to four treatment groups of five mice each. Each group received treatment with (i) vehicle alone (PBS), (ii) purified 71D6 antibody, (iii) purified 71G2 antibody, or (iv) purified 71G3 antibody. The antibodies were administered by i.p. injection at a dose of 1 mg / kg twice weekly. Given the C57BL6 / J background strain of db / db mice, these mice served as healthy controls. Blood glucose was monitored throughout the entire experimental period. After 8 weeks of treatment (at 16 weeks of age), mice were sacrificed and subjected to necropsy. Pancreases were extracted, processed for histology, and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin to visualize the islets of Langerhans. β-cells and insulin production were highlighted by immunohistochemistry using an anti-insulin antibody.

[0209] As shown in Figure 7A, untreated db / db mice already exhibited well-established hyperglycemia (approximately 240 mg / dL) at 7 weeks of age. Thereafter, blood glucose levels steadily increased until reaching a plateau of over 300 mg / dL. Interestingly, mice treated with 71D6, 71G2, and 71G3 exhibited significantly lower blood glucose levels throughout the experimental period, which was not consistent with that of control C57BL6 / J mice. At the end of the experiment, untreated db / db mice exhibited a basal blood glucose level of approximately 330 mg / dL; 71D6-treated db / db mice instead exhibited an average basal blood glucose level of approximately 140 mg / dL; 71G2- and 71G3-treated mice exhibited a basal blood glucose level of approximately 180 mg / dL (Figure 7B).

[0210] Pancreatic sections were stained with hematoxylin and eosin, analyzed microscopically, and photographed. Islets of Langerhans were analyzed using ImageJ® software to estimate islet number, density, and size. This analysis revealed that islets of Langerhans were severely degenerated in db / db mice at 16 weeks of age compared to age-matched C57BL6 / J controls, both in terms of number and size. Indeed, C57BL6 / J mice had 2.3 islets / mm 2 showed a mean islet density of 1.6 islets / mm in untreated db / db mice, whereas untreated db / db mice showed a mean islet density of 1.6 islets / mm in untreated db / db mice. 2 Notably, islet density was dramatically increased in db / db mice treated with 71D6, exceeding the density observed in healthy controls (4.4 islets / mm). 2 ) reached significantly higher values ​​than C57BL6 / J controls. Islet size was also significantly reduced in db / db mice compared to C57BL6 / J controls (Figure 8B). In the latter strain, islets of Langerhans were 0.3 mm 2The mean area of ​​islets in untreated db / db mice was approximately 10-fold reduced in untreated db / db mice. Remarkably, 71D6 treatment completely rescued the reduction in islet size, restoring it to values ​​similar to or even larger than those characteristic of C57BL6 / J healthy mice. Similar results regarding both islet number and size were obtained with 71G2 and 71G3 treatment, although their efficacy was slightly lower (71D6 > 71G2 > 71G3). Representative images of hematoxylin and eosin-stained pancreatic sections are shown in Figure 8C.

[0211] We characterized the biological effects of 71D6 by assessing its ability to specifically affect the β-cell population. To this end, pancreatic sections were analyzed by immunohistochemistry using an anti-insulin antibody. This analysis revealed that the small number of surviving islets in db / db mice was significantly reduced. db / db mice treated with 71D6, 71G2, or 71G3 contained significantly fewer insulin-expressing β-cells than healthy controls (Figure 9). In contrast, db / db mice treated with 71D6, 71G2, or 71G3 contained significantly more functional β-cells, and these cells expressed significantly higher levels of insulin. This was particularly evident in the 71D6-treated group, confirming that this antibody is more potent than 71G2 and 71G3.

[0212] These results, as well as those presented in the previous examples, demonstrate that the 71D6, 71G2, and 71G3 MET agonist antibodies promote the survival and regeneration of β-cells, which contribute to maintaining normal levels of insulin. Given that restoring functional β-cells significantly improves diabetic symptoms and the quality of life of diabetic patients, we propose that agonist anti-MET antibodies could be an innovative tool for treating diabetes in the clinic.

[0213] Importantly, a key requirement for transferring MET agonist antibodies to the clinic is their complete cross-reactivity with preclinical species, including rodents and nonhuman primates. Indeed, we were able to demonstrate the therapeutic activity of 71D6, 71G2, and 71G3 in mice because they maintain complete cross-reactivity between human and mouse MET. Furthermore, 71D6 elicits identical biological activity and efficacy in tissues of human, mouse, rat, and monkey origin. Without this interspecies equivalence, it would be impossible to advance the described MET agonist antibodies toward first-in-human trials. Primarily for this reason (i.e., lack of equivalence in preclinical species), none of the agonist MET antibodies known in the prior art could be tested in preclinical models and therefore lack the necessary proof of efficacy.

[0214] Further along this path, pancreas transplantation, either as a whole organ or using isolated islets of Langerhans or purified beta cells, represents another approach to treating both type 1 and type 2 diabetes (Kieffer et al., J Diabetes Investig. 2017, epub ahead of print; doi: 10.1111 / jdi.12758). This approach has several limitations, particularly related to insufficient transplantation techniques and poor survival of transplanted beta cells in recipients. Given the enhanced ability of the MET agonist antibodies described herein to promote beta cell regeneration and insulin secretion, they may improve the utility of pancreatic tissue transplantation and potentially expand the beta cell population in transplant recipients.

[0215] Example 14: MET agonist antibodies preserve pancreatic beta cell function, prevent diabetes onset, and synergize with immunosuppressants in a mouse model of autoimmune type 1 diabetes Type 1 diabetes is characterized by autoimmune-mediated destruction of pancreatic beta cells, leading to insufficient insulin secretion and an inability of the tissue to uptake glucose. Autoantibody-mediated beta cell destruction begins before the hyperglycemic phenotype appears. By the time insulin-dependent diabetes is diagnosed, typically during adolescence, beta cell destruction is already advanced, leaving only a small fraction of the original surviving beta cells. Furthermore, because beta cell destruction progresses so rapidly, the window for therapeutic intervention after diagnosis is very narrow.

[0216] Immunosuppressants continue to be investigated as a treatment for newly diagnosed patients with type 1 diabetes in an effort to reduce autoimmune-mediated islet cell destruction. However, immunosuppressants take several months to show initial clinical benefit. When this occurs, approximately six months after initiating treatment, pancreatic beta cells continue to be destroyed, often completely destroyed. As a result, the efficacy of immunosuppressants is severely blunted, if not rendered ineffective. Maintaining viability of islet beta cells or further regenerating them is a largely unmet medical need for patients with diabetes.

[0217] To test whether MET agonist antibodies can antagonize immune-mediated β-cell destruction and collaborate with immune-targeted drugs in the context of type 1 diabetes, we selected an appropriate mouse model. The NOD / ShiLtJ strain (commonly referred to as NOD) is a polygenic model of autoimmune type 1 diabetes. Diabetes in NOD mice is characterized by hyperglycemia and leukocyte infiltration of pancreatic islets. A significant decrease in pancreatic insulin content occurs at approximately 12 weeks of age in females and several weeks later in males. NOD mice are considered to be the animal model of type 1 diabetes that best recapitulates the pathology observed in humans. Several studies have been conducted in this strain together with immunosuppressants to investigate their potential for attenuating hyperglycemia and / or delaying the onset of diabetes. In particular, antibodies directed against the lymphocyte-specific surface marker CD3 have proven particularly effective in several studies (Chatenoud et al., Proc Natl Acad Sci USA 91: 123-127, 1994; Chatenoud et al., J Immunol 158: 2947-2954, 1997; Gill et al., Diabetes 65: 1310-1316, 2016; Kuhn et al., Immunotherapy 8: 889-906, 2016; Kuhn et al., J Autoimmun 76: 115-122, 2017). Interestingly, these studies demonstrated that oral administration of these immune-targeting antibodies resulted in fewer side effects than systemic administration. The most effective protocol consisted of treating mice for five consecutive days and then discontinuing treatment (Ochi et al., Nat Med. 12: 627-635, 2006). Notably, the therapeutic effect declined rapidly when the oral drug dose exceeded 5 μg per mouse (0.25 mg / kg).

[0218] To test whether our agonistic anti-MET antibodies exhibited therapeutic efficacy and to investigate their potential synergistic effects with immune-targeted drugs, 72 6-week-old female NOD mice were obtained from Charles River. Blood glucose was measured in randomly fed (i.e., non-fasted) animals using human-grade test strips (multiCare in; Biochemical Systems International). At this time point, NOD mice exhibited prediabetes with a mean blood glucose level of approximately 110 mg / dL (Figure 10A). Mice were randomly assigned to four different treatment groups, each consisting of 18 mice, to ensure that all groups were as homogeneous as possible with respect to blood glucose. Starting at week 7, the four treatment groups received different treatments as follows: no drug (CONTROL); 0.15 mg / kg anti-CD3 antibody (CD3); 3 mg / kg purified 71D6 antibody (71D6); and 0.15 mg / kg anti-CD3 antibody + 3 mg / kg purified 71D6 antibody (COMBO). Anti-CD3 antibody was administered orally by gavage in 100 μL of PBS once daily for 5 consecutive days, followed by discontinuation as per protocol. 71D6 was administered i.p. in 200 μL of PBS twice weekly for the entire experimental period. Mice were fed ad libitum using a standard diet. Blood glucose was measured weekly using strips in random-fed animals as described above. Animals were considered diabetic if they showed blood glucose levels above 250 mg / dL for two consecutive weeks.

[0219] Consistent with the literature, no diabetic animals were recorded by week 12 (Figure 10B). At week 13, diabetes began to appear in the control and CD3-treated groups. At week 18, 50% of the control animals were diabetic (Figure 10C), as described by the original strain supplier (The Jackson Lab - 001976 Mouse Strain Datasheet; https: / / www.jax.org / strain / 001976). At week 21, when the experiment was discontinued, 88% of the control mice were diabetic, while the other treatment groups showed significantly lower values: CD3, 47%; 71D6, 21%; and COMBO, 14% (Figure 10D). A time course analysis of diabetes onset is shown in Figure 11A. A Kaplan-Meier plot is shown in Figure 11B. Statistical analysis was performed using Prism software (Graph Pad). The Mantel-Cox test, the log-rank test, and the Gehan-Breslow-Wilcoxon test all gave p-values ​​less than 0.001, indicating that the differences between the curves were statistically significant.

[0220] Mean nonfasting blood glucose levels increased steadily in all groups, reaching extremely high levels (>450 mg / dL) only in the control (untreated) group (Figure 12). Consistent with the diabetes onset data, blood glucose levels followed the following precise order: CONTROL > CD3 > 71D6 > COMBO. During the course of the experiment (4 months), several mice died for reasons unrelated to treatment, primarily due to cage fights and bacterial infection in male mice (CONTROL, 1 / 18; CD3, 1 / 18; 71D6, 4 / 18; COMBO, 4 / 14). One diabetic mouse's blood glucose level rapidly reached an extreme value (>550 mg / dL), so the mouse was sacrificed 3 weeks after diabetes diagnosis. In these cases, the value of 550 mg / dL was used to calculate the mean blood glucose level for the group, even after death. All mice, regardless of whether they were diabetic or not, were sacrificed at the end of the 21st week.

[0221] Before sacrifice, all animals underwent a glucose tolerance test (GTT). For this, the animals were fasted overnight. The following morning, blood samples were collected for blood glucose and insulin measurements. A glucose solution (3 g / kg in 200 μL PBS) was injected intraperitoneally (ip), and a second blood sample was collected 3 minutes later. Mice were sacrificed immediately afterward, and major organs, including the liver and pancreas, were collected for analysis. Blood glucose concentrations (blood glucose levels) were measured using test strips as described above. Insulin concentrations were measured using a high-sensitivity mouse insulin ELISA kit (Crystal Chem).

[0222] Analysis of blood glucose levels revealed the following scenario. At time 0, blood glucose was lower in the treatment groups compared with the controls (CONTROL > CD3 > 71D6 > COMBO; Figure 13A). However, 3 minutes after glucose administration, blood glucose rose to similar levels in all groups (>350 mg / dL; Figure 13B). In contrast, blood insulin concentrations at time 0 were very low, except for a slightly higher level in the COMBO-treated group (Figure 13C). Notably, after glucose injection, insulin concentrations appeared very different among the treatment groups, following the reverse order (COMBO > 71D6 > CD3 > CONTROL; Figure 13D). Because NOD mice exhibit unique modulation of insulin during their prediabetic phase (Amrani et al., Endocrinology 139:1115–1124, 1998), it is difficult to directly compare these absolute values ​​with those of other nondiabetic mouse strains. In any case, it can be concluded that animals in the treatment group respond to glucose stimulation by secreting insulin, while control animals do not.

[0223] Consistent with the attenuated diabetic phenotype, body weight at necropsy was slightly (although not significantly) higher in the treatment group compared to the control group (Figure 14A). There was no significant difference in liver to total body weight ratios in either group (Figure 14B). This suggests that 71D6-induced liver proliferation (observed in other mouse strains) is strain-specific. No other biological or pathological signs or processes were detected in 71D6-treated mice during necropsy or histological analysis.

[0224] Pancreatic samples were paraffin-embedded and processed for histological analysis. Histological sections were stained with hematoxylin and eosin and analyzed microscopically. This analysis revealed that the pancreata of most animals in the CONTROL group contained very few islets, and those that were visible were abnormally small and heavily infiltrated with lymphocytes (Figure 15). In contrast, islets in the CD3-treated group, while still infiltrated with lymphocytes, were more abundant and showed little degeneration. Pancreatic sections from the 71D6-treated group contained more islets than both the CONTROL and CD3 groups, and islet size was larger than average; however, lymphocyte infiltration was still evident. Finally, islets in the COMBO group were abundant and large, but similarly infiltrated.

[0225] Significant treatment-dependent differences were observed in pancreatic sections stained with anti-insulin antibodies (Figure 16). In the CONTROL group, very little, if any, staining was observed among the few visible islets. In the CD3-treated group, the insulin signal was higher, but not as intense as that observed in 71D6-treated animals. Islets observed in the COMBO-treated group showed the highest and most uniform insulin signal compared to all other treatment groups. At higher magnification, these features could be seen in more detail (Figure 17). Islets from untreated animals contained few, if any, insulin-producing cells. In contrast, the majority of islet cells in the CD3-treated group were insulin-positive. In the 71D6-treated group, islets were large and strongly stained. Pancreata from the COMBO group contained the largest and most insulin-producing islets of all other groups.

[0226] As mentioned above, the number of insulin-producing β cells in the islets of Langerhans was significantly higher in the treatment groups (COMBO > 71D6 > CD3 > CONTROL). However, the cellular infiltration was highly heterogeneous, and no significant differences were observed in the number of lymphocytes recruited to the islet periphery among the various treatment groups. This can be explained by two mechanisms that differ depending on the treatment. It is well established that oral delivery of anti-CD3 antibodies induces immunogenic tolerance rather than abolishing the immune response (Chatenoud et al., J Immunol 158:2947-2954, 1997). The tolerogenic process involves the activation and proliferation of T regulatory cells, which inhibit autoantibody-mediated β cell destruction (Chatenoud Novartis Found Symp 252:279-220, 2003). This explains why pancreatic β cells are not destroyed in the CD3 group despite immune cell infiltration. On the other hand, the data presented in the previous example suggest that 71D6 promotes β-cell survival and regeneration. Therefore, it can be hypothesized that 71D6 antagonizes immune-mediated β-cell death and promotes β-cell proliferation, thereby preserving β-cell mass despite severe immune cell infiltration.

[0227] To further explore the role of immune mechanisms in the response to anti-CD3 and anti-MET antibodies, we measured anti-insulin antibodies in mouse plasma. For this purpose, plasma samples were collected from all mice at necropsy and from young, prediabetic female NOD mice (7 weeks old) and analyzed using a mouse IAA (insulin autoantibody) ELISA kit (Fine Test). This analysis revealed that the majority of mice exhibited higher levels of anti-insulin antibodies compared with prediabetic mice (Figure 18). No statistically significant differences were observed between the different populations, although the COMBO-treated mice showed a trend toward lower levels. The 71D6-treated mice could be clearly separated into two subpopulations, with low and high autoantibody levels, respectively. While these results clearly require further study, the overall hypothesis is that neither anti-CD3 nor 71D6 treatment affects autoantibody production in this system but rather acts downstream to prevent or delay the onset of diabetes.

[0228] In summary, the data obtained in this study suggest that 71D6 treatment is highly effective in maintaining pancreatic β-cell integrity in the setting of type 1 diabetes. Systemic 71D6 treatment was not only more effective than established immunosuppressive therapy, but also increased the efficacy of the latter when administered concomitantly. The mechanism of action underlying 71D6's therapeutic activity appears to be related to its ability to promote β-cell survival and / or proliferation rather than interfering with autoantibody production or immune cell infiltration into pancreatic islets. These data provide experimental evidence that MET agonist antibodies, alone or in combination with immunotherapy, can be used to treat type 1 diabetes. [1] A method for promoting the proliferation of pancreatic islet cells, comprising administering an HGF-MET agonist to a subject. [2] A method of promoting insulin production in a subject in need thereof, comprising administering to the subject an HGF-MET agonist. [3] A method for treating diabetes, comprising administering an HGF-MET agonist to a subject. [4] The method of claim 2 or 3, wherein the method is characterized by inducing increased pancreatic islet cell proliferation. [5] The method according to any of the preceding items, wherein the subject exhibits a fasting blood glucose level greater than 5.6 mmol / L. [6] The method according to any of the preceding items, wherein the subject is characterized by having a population of pancreatic islet cells that is at least 50% smaller, optionally at least 70% smaller, and optionally about 70% to about 80% smaller than the cell population in a healthy individual. [7] The method according to any of the preceding items, wherein the subject has type 1 diabetes or type 2 diabetes. [8] The method according to any one of the above items 1, wherein the subject has previously received a pancreatic tissue transplant. [9] The method according to any one of items 1 to 7, further comprising administering a pancreatic tissue transplant to the subject.

[10] The method according to any of the preceding items, further comprising administering one or more immunosuppressive drugs to the subject.

[11] The method of item 10, wherein the one or more immunosuppressants are selected from the group consisting of an anti-CD3 antibody, an anti-IL-21 antibody, a CTLA4 molecule, a PD-L1 molecule, IL-10, and glutamic acid decarboxylase (GAD)-65.

[12] The method of item 1, wherein the subject is a healthy donor subject.

[13] The method of any of the preceding items, wherein administration of the HGF-MET agonist promotes proliferation of pancreatic islet β cells.

[14] The method of any one of items 1 to 13, wherein the HG-MET agonist is administered at a dose ranging from 0.1 to 40 mg / kg per administration.

[15] The method of any one of items 1 to 14, wherein the HGF-MET agonist is administered at a dose ranging from 0.5 to 35 mg / kg, optionally 1 to 30 mg / kg, and optionally 1 to 10 mg / kg.

[16] The method of any one of items 1 to 15, wherein the HGF-MET agonist is administered at a dose of 1 mg / kg, 3 mg / kg, 10 mg / kg, or 30 mg / kg.

[17] The method of any one of paragraphs 1 to 16, wherein the HGF-MET agonist is administered once a week, optionally 1 to 3 times a week, or optionally 2 times a week.

[18] The method according to any of the preceding items, wherein the method further comprises administering a diabetes drug, preferably insulin, to the subject.

[19] An HGF-MET agonist for use in the method according to any one of items 1 to 18.

[20] A pharmaceutical composition for use in the method of any one of items 1 to 18, comprising an HGF-MET agonist and a pharmaceutically acceptable excipient or carrier.

[21] An in vitro method for promoting proliferation of a cell population or tissue comprising pancreatic islet cells, the method comprising contacting the cell population with an HGF-MET agonist.

[22] An ex vivo method for preserving islet cells or pancreatic grafts, comprising contacting the islet cells or pancreatic grafts with an HGF-MET agonist.

[23] The method according to any one of the preceding items, an HGF-MET agonist for use therein, or a pharmaceutical composition for use therein, wherein the HGF-MET agonist is a full agonist of MET.

[24] The method according to any one of the preceding items, an HGF-MET agonist for use therein, or a pharmaceutical composition for use therein, wherein the HGF-MET agonist is an anti-MET agonist antibody or antigen-binding fragment.

[25] The method of Item 24, the antibody for use therein, or the pharmaceutical composition for use therein, wherein the anti-MET antibody or antigen-binding fragment thereof binds to the SEMA domain of MET, optionally to blades 4 to 5 of the SEMA β propeller.

[26] The method of Item 24, the antibody for use therein, or the pharmaceutical composition for use therein, wherein the anti-MET antibody or antigen-binding fragment thereof binds to an epitope comprising MET residue Ile367 and / or Asp372, optionally comprising both MET residues Ile367 and Asp372.

[27] The method, antibody for use therein, or pharmaceutical composition for use therein according to Item 24, wherein the anti-MET antibody or antigen-binding fragment thereof binds to the PSI domain of MET and optionally binds to an epitope between residues 546 and 562 of MET.

[28] The method, antibody for use therein, or pharmaceutical composition for use therein according to claim 24 or 27, wherein the anti-MET antibody or antigen-binding fragment thereof binds to an epitope comprising residue Thr555 of MET.

[29] The method, antibody for use therein, or pharmaceutical composition for use therein according to any one of Aspects 23 to 26, wherein the anti-MET agonist antibody or antigen-binding fragment thereof comprises a combination of the VH CDR1, CDR2, and CDR3 sequences and the VL CDR1, CDR2, and CDR3 sequences of 71D6. 30. The method of claim 29, the antibody for use therein, and the pharmaceutical composition for use therein, wherein the anti-MET agonist antibody or antigen-binding fragment thereof comprises a VH domain at least 90% identical to SEQ ID NO: 163 and / or a VL domain at least 90% identical to SEQ ID NO: 164.

[31] The method according to any one of Aspects 24 to 27, the antibody used therein, and the pharmaceutical composition used therein, wherein the anti-MET agonist antibody is 71D6.

[32] The method according to any one of Aspects 24 to 31, the antibody for use therein, and the pharmaceutical composition for use therein, wherein the anti-MET agonist antibody is an IgG4 antibody.

[33] A method of treating diabetes in a subject, comprising administering to the subject an effective amount of anti-MET antibody 71D6, and optionally further comprising administering to the subject insulin at least daily.

Claims

1. A pharmaceutical composition for treating diabetes comprising an anti-MET agonist antibody or antigen-binding fragment thereof, wherein diabetes is treated by promoting pancreatic islet cell proliferation in a subject.

2. The pharmaceutical composition of claim 1, wherein diabetes is treated by promoting insulin production.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition is administered to a subject exhibiting a fasting blood glucose level greater than 5.6 mmol / L.

4. 4. The pharmaceutical composition of claim 3, wherein the subject is characterized by having a population of pancreatic islet cells that is at least 50% smaller, optionally at least 70% smaller, and optionally about 70% to about 80% smaller than the population of cells in a healthy individual.

5. The pharmaceutical composition of claim 3 or 4, wherein the subject has type 1 diabetes or type 2 diabetes.

6. The pharmaceutical composition of any one of claims 3 to 5, wherein the subject has previously undergone a pancreatic tissue transplant.

7. A pharmaceutical composition according to any one of claims 1 to 6; a) one or more immunosuppressive agents; or b) the following: one or more immunosuppressants selected from the group consisting of anti-CD3 antibody, anti-IL-21 antibody, CTLA4 molecule, PD-L1 molecule, IL-10, and glutamic acid decarboxylase (GAD)-65 A combination pharmaceutical for treating diabetes comprising:

8. 8. The pharmaceutical combination of claim 7, further comprising an antidiabetic drug, preferably insulin.

9. The pharmaceutical composition according to any one of claims 1 to 7, which promotes pancreatic islet cell proliferation in a healthy donor of pancreatic islet cells.

10. 1. An in vitro method for promoting the growth of a cell population or tissue comprising pancreatic islet cells, the method comprising contacting the cell population with an anti-MET agonist antibody or antigen-binding fragment thereof.

11. 1. An ex vivo method for preserving islet cells or a pancreatic graft, comprising contacting the pancreatic islet cells or pancreatic graft with an anti-MET agonist antibody or antigen-binding fragment thereof.

12. The pharmaceutical composition of any one of claims 1 to 6 and 9, the pharmaceutical combination of claim 7 or 8, or the method of claim 10 or 11, wherein the anti-MET agonist antibody or antigen-binding fragment thereof is a full agonist of MET.

13. The anti-MET antibody or antigen-binding fragment thereof is selected from the group consisting of: a) binds to the SEMA domain of MET, optionally to blades 4-5 of the SEMA β propeller; b) an epitope comprising residues Ile367 and / or Asp372 of MET, optionally comprising both residues Ile367 and Asp372 of MET The pharmaceutical composition according to any one of claims 1 to 6 and 9, the combined pharmaceutical composition according to claim 7 or 8, or the method according to claim 10 or 11, wherein the

14. 12. The pharmaceutical composition of any one of claims 1 to 6 and 9, the pharmaceutical combination of claim 7 or 8, or the method of claim 10 or 11, wherein the anti-MET antibody or antigen-binding fragment thereof binds to the PSI domain of MET and optionally binds to an epitope at residues 546 to 562 of MET.

15. 12. The pharmaceutical composition of any one of claims 1 to 6 and 9, the pharmaceutical combination of claim 7 or 8, or the method of claim 10 or 11, wherein the anti-MET antibody or antigen-binding fragment thereof binds to an epitope comprising residue Thr555 of MET.

16. the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO: 30, an HCDR2 consisting of SEQ ID NO: 32, an HCDR3 consisting of SEQ ID NO: 34, an LCDR1 consisting of SEQ ID NO: 107, an LCDR2 consisting of SEQ ID NO: 109, and an LCDR3 consisting of SEQ ID NO: 111; or the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO: 44, an HCDR2 consisting of SEQ ID NO: 46, an HCDR3 consisting of SEQ ID NO: 48, an LCDR1 consisting of SEQ ID NO: 121, an LCDR2 consisting of SEQ ID NO: 123, and an LCDR3 consisting of SEQ ID NO: 125; or the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO:9, an HCDR2 consisting of SEQ ID NO:11, an HCDR3 consisting of SEQ ID NO:13, an LCDR1 consisting of SEQ ID NO:86, an LCDR2 consisting of SEQ ID NO:88, and an LCDR3 consisting of SEQ ID NO:90; or the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO:2, an HCDR2 consisting of SEQ ID NO:4, an HCDR3 consisting of SEQ ID NO:6, an LCDR1 consisting of SEQ ID NO:79, an LCDR2 consisting of SEQ ID NO:81, and an LCDR3 consisting of SEQ ID NO:83; or the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO: 65, an HCDR2 consisting of SEQ ID NO: 67, an HCDR3 consisting of SEQ ID NO: 69, an LCDR1 consisting of SEQ ID NO: 142, an LCDR2 consisting of SEQ ID NO: 144, and an LCDR3 consisting of SEQ ID NO: 146; or the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO: 72, an HCDR2 consisting of SEQ ID NO: 74, an HCDR3 consisting of SEQ ID NO: 76, an LCDR1 consisting of SEQ ID NO: 149, an LCDR2 consisting of SEQ ID NO: 151, and an LCDR3 consisting of SEQ ID NO: 153; or the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO: 58, an HCDR2 consisting of SEQ ID NO: 60, an HCDR3 consisting of SEQ ID NO: 62, an LCDR1 consisting of SEQ ID NO: 135, an LCDR2 consisting of SEQ ID NO: 137, and an LCDR3 consisting of SEQ ID NO: 139; or the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO: 16, an HCDR2 consisting of SEQ ID NO: 18, an HCDR3 consisting of SEQ ID NO: 20, an LCDR1 consisting of SEQ ID NO: 93, an LCDR2 consisting of SEQ ID NO: 95, and an LCDR3 consisting of SEQ ID NO: 97; or 12. The pharmaceutical composition of any one of claims 1 to 6 and 9, the pharmaceutical combination of claim 7 or 8, or the method of claim 10 or 11, wherein the anti-MET agonist antibody or antigen-binding fragment comprises a combination of an HCDR1 consisting of SEQ ID NO: 37, an HCDR2 consisting of SEQ ID NO: 39, an HCDR3 consisting of SEQ ID NO: 41, an LCDR1 consisting of SEQ ID NO: 114, an LCDR2 consisting of SEQ ID NO: 116, and an LCDR3 consisting of SEQ ID NO:

118.

17. the anti-MET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 163 and / or a VL domain consisting of SEQ ID NO: 164; or the anti-MET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 167 and / or a VL domain consisting of SEQ ID NO: 168; or the antiMET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 155 and / or a VL domain consisting of SEQ ID NO: 156; or the antiMET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 173 and / or a VL domain consisting of SEQ ID NO: 174; or the antiMET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 175 and / or SEQ ID NO: 176; or the antiMET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 157 and / or a VL domain consisting of SEQ ID NO: 158; or the antiMET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 171 and / or a VL domain consisting of SEQ ID NO: 172; or the antiMET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 159 and / or a VL domain consisting of SEQ ID NO: 160; or 12. The pharmaceutical composition of any one of claims 1 to 6 and 9, the pharmaceutical combination of claim 7 or 8, or the method of claim 10 or 11, wherein the antiMET agonist antibody or antigen-binding fragment comprises a VH domain consisting of SEQ ID NO: 165 and / or a VL domain consisting of SEQ ID NO:

166.

18. The pharmaceutical composition according to any one of claims 1 to 6 and 9, the combined pharmaceutical composition according to claim 7 or 8, or the method according to claim 10 or 11, wherein the anti-MET agonist antibody is an IgG4 antibody.

Images