Anti-met antibodies and uses thereof

Abstract

A monoclonal antibody is provided. The antibody comprises an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236. Also provided are uses thereof.

Description

[0001] ANTI-MET ANTIBODIES AND USES THEREOF

[0002] RELATED APPLICATIONS:

[0003] This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 770,461 filed March 12, 2025, which is hereby incorporated by reference in its entirety.

[0004]

[0005] The XML file, entitled 106724. xml, created on March 11, 2026, comprising 82,738 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to anti-MET antibodies and uses thereof.

[0008] The tumor microenvironment (TME) is populated by infiltrating immune cells, including B cells, and their presence has been related to a positive prognosis and better responsiveness to immune checkpoint blockade (ICB) (1, 2). Within tumors, B cells can reside in tertiary lymphoid structures (TLS), which are germinal center-like immune niches where they can take up and present antigens to T cells and differentiate into antibody- secreting cells (ASC)(3-5). In addition, studies have shown that anti-cancer B cells and antibodies circulate in cancer patients' blood (6-8). Recent studies have characterized intratumoral antibodies, revealing key features including high clonality, class switching, autoreactivity, and high frequencies of somatic hypermutation (SHM) - characteristics that suggest an antigen-directed immune response(4, 9).

[0009] B cells develop in the bone marrow (BM), where they undergo positive and negative selection. B cells expressing self-reactive B cell receptors (BCRs) are supposed to be removed during B cell development, to prevent autoimmune responses. Yet, a significant frequency of the mature repertoire express BCRs targeting self-proteins (10). Such B cells exist by escaping either central tolerance checkpoints during development or peripheral tolerance checkpoints following affinity maturation against non-self targets (11). Antibody production is typically initiated by a B cell's encounter with cognate antigen, followed by class switch recombination and affinity maturation (12). This process may also generate self-reactive B cells that produce autoantibodies, which in some cases can be detrimental (13, 14) . However, not all autoantibodies are harmful. As such, immune suppression and dysregulation occurring during cancer can result in activation of B cells resulting in the production of autoreactive autoantibody responses (15). Both the function of these B cells and the role of their immunoglobulins in tumor progression remain unclear.Studies have reported autoantibodies against cancer-associated antigens in patients with various types of cancer (16, 17). In the TME of ovarian cancer patients, ASC produce antibodies directed against matrix metalloprotease- 14 (MMP14), an enzyme that is highly expressed on the tumor cells surface (18). An appealing group of naturally elicited anti-cancer antibodies includes immunoglobulins targeting tumor-associated growth factor receptors. As such, pre-existing anti-HER2 and anti-EGFR serum antibodies were found in patients, and these antibodies correlate with successful therapy outcomes and positive prognosis (19, 20). Other studies describing B cell responses in the circulation during cancer development generate unclear conclusions regarding to the correlation of these antibodies with cancer progression. On one hand, persistent plasmablast responses in the circulation have been linked to anti-tumor responses in breast cancer patients, and the secreted antibodies were shown to induce anti-cancer effects and cancer regression in mice (7). Conversely, serum antibody reactivity has also been correlated with more advanced disease in melanoma patients (21). Overall, since defined tumor-related antigens are scarce, isolation of B cells and antibodies that target specific tumor-associated antigens and the functional characterization of these antibodies remains a major goal and a significant challenge.

[0010] MET is a tyrosine-kinase receptor activated by hepatocyte growth factor (HGF) (22). Similarly to other growth factor receptors, MET functions physiologically during embryogenesis and tissue repair (23). During cancer, aberrant MET activation promotes tumorigenesis and metastasis (24, 25). MET overexpression is observed in breast, lung, gastric, and colorectal cancers and correlates with bad prognosis (24, 26, 27). However, whether MET autoantibodies are produced in cancer patients, and what is their function, is currently unknown.

[0011] SUMMARY OF THE INVENTION

[0012] According to an aspect of some embodiments of the present invention there is provided an antibody comprising an antigen binding domain which binds an antigenic determinant of MET for use in preventing or treating cancer in a subject in need thereof, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0013] According to an aspect of some embodiments of the present invention there is provided a method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein the antigen binding domaincomprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236, thereby preventing or treating cancer in the subject.

[0014] According to an aspect of some embodiments of the present invention there is provided a method of monitoring treatment of cancer in a subject in need thereof, the method comprising: (a) treating the subject with an anti-cancer treatment, wherein the cancer is characterized by MET expression;

[0015] (b) determining a level of the MET prior and following the treating using an antibody, wherein a reduction in MET is indicative of an efficacious treatment, wherein at least one of the anticancer treatment and the antibody, comprises an antibody which comprises an antigen binding domain which binds an antigenic determinant of MET, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0016] According to an aspect of some embodiments of the present invention there is provided a method of diagnosing cancer in a subject in need thereof, the method comprising:

[0017] (a) contacting cells of the subject with an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236;

[0018] (b) detecting an immunocomplex formation between the antibody and the cells, wherein presence of the immunocomplex is indicative of cancer.

[0019] According to an aspect of some embodiments of the present invention there is provided a method of treating cancer in a subject in need thereof, the method comprising:

[0020] (a) diagnosing cancer according to the method as described herein;

[0021] (b) treating the cancer with an anti-cancer treatment, optionally wherein the anti-cancer treatment comprises the antibody.

[0022] According to an aspect of some embodiments of the present invention there is provided a method of selecting treatment to cancer in a subject diagnosed with cancer, the method comprising:(a) contacting cells of the subject with an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236;

[0023] (b) detecting an immunocomplex formation between the antibody and the cells, wherein presence of the immunocomplex is indicative of cancer cells which express MET; and

[0024] (c) selecting a treatment which is efficacious for MET expressing cancer, wherein the treatment is optionally with the antibody.

[0025] According to an aspect of some embodiments of the present invention there is provided a method of producing an antibody capable of binding an antigenic determinant of MET, the method comprising:

[0026] (a) expressing in a host cell a heterologous polynucleotide encoding an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236; and optionally

[0027] (b) recovering the antibody from the host cell.

[0028] According to an aspect of some embodiments of the present invention there is provided a vaccine comprising an effective amount of an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0029] According to an aspect of some embodiments of the present invention there is provided a monoclonal antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.According to an aspect of some embodiments of the present invention there is provided a polynucleotide comprising a nucleic acid sequence encoding the antibody (SEQ ID Nos: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86).

[0030] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the polynucleotide of claim 9.1 under a transcriptional control of a cis-acting regulatory element, said element being heterologous to said polynucleotide.

[0031] According to an aspect of some embodiments of the present invention there is provided an antibody comprising an antigen binding domain which binds an antigenic determinant of MET attached to a heterologous effector moiety or carrier, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0032] According to some embodiments of the invention, the antibody is a recombinant antibody. According to some embodiments of the invention, the at least 80 % is at least 85 %.

[0033] According to some embodiments of the invention, the at least 80 % is at least 90 %.

[0034] According to some embodiments of the invention, the at least 80 % is at least 95 %.

[0035] According to some embodiments of the invention, the at least 80 % is 100 %.

[0036] According to some embodiments of the invention, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 69B287 or 87B156.

[0037] According to some embodiments of the invention, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 69B287.

[0038] According to some embodiments of the invention, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 87B156.

[0039] According to some embodiments of the invention, the antibody inhibits cancer cells growth. According to some embodiments of the invention, the antibody binds semaphorin (SEMA) domain of MET.

[0040] According to some embodiments of the invention, the antibody competes with hepatocyte growth factor (HGF) binding to MET, as determined by ELISA.

[0041] According to some embodiments of the invention, the cancer is selected from the group consisting of breast cancer, lung cancer, gastric cancer and hepatocellular cancer.

[0042] According to some embodiments of the invention, the cancer is hormone receptor positive (HR+) breast cancer.According to some embodiments of the invention, the antibody comprises an antibody fragment.

[0043] According to some embodiments of the invention, the antibody fragment comprises Fab and / or scFv.

[0044] According to some embodiments of the invention, the antibody is a chimeric antibody.

[0045] According to some embodiments of the invention, the antibody is a human antibody.

[0046] According to some embodiments of the invention, the antibody is labeled.

[0047] According to some embodiments of the invention, the method is effected in-vivo.

[0048] According to some embodiments of the invention, the method is effected ex-vivo.

[0049] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0050] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0051] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0052] In the drawings:

[0053] FIGs. 1A-B show MET and SEMA recombinant proteins. (A) Schematic illustration of soluble MET and SEMA recombinant proteins and tags. (B) Protein SDS-PAGE showing recombinant MET and SEMA proteins after expression in Expi293™ cells and affinity purification. The protein ladder is shown on the left.

[0054] FIGs. 2A-D show an anti-MET response in a cohort of 100 cancer patients (A) Heatmap representing cancer patient sera binding as detected by ELISA. Each column represents one donor (donor IDs are given on top). The left side of the heat map presents breast cancer donors, while the right side presents lung cancer donors. The rows are based on raw O.D.650 values at sera dilution 1:50 for MET, SEMA and gpl20. Color code is given on the right of the figure, where light yellow indicates binding, and dark purple indicates lack of binding. The 13 identified patients who respond to MET are marked with red asterisks. (B) Mean O.D. of MET and SEMA binding of cancer patients(black / purple) and non-cancer donors (green). The p-value is calculated using the Kolmogorov-Smirnov test. The dashed line indicates the 95th percentile of healthy donors' sera scores (was set as the threshold for defining anti-MET positive response). The 13 patients who respond to MET are colored in purple. (C) Mean O.D. of MET and SEMA binding for each patient, subdivided by cancer type, subtype (adenoCa = adenocarcinoma, SCC = Squamous cell carcinoma, TN = triple negative), and stage (E = Early, M = metastasis). The 13 patients who respond to MET are indicated in purple. (D) Pie charts showing the tumor hormonal receptor status (HR+ versus HR-) in 67 breast cancer patients who did not respond to MET (left pie) as opposed to the 8 breast cancer patients who respond to MET (right pie), p-value is calculated by Fisher exact test.

[0055] FIGs. 3A-E show correlations and validation of anti-MET serological response. (A) Correlation plot between MET O.D.650 values (y-axis) and the O.D.650 values of either SEMA (left) or gpl20 (right) of patients whose sera reacted to MET or SEMA. R and p-value are indicated, and were calculated with Spearman correlation. (B) MET, SEMA and gpl20 O.D.650 values of cancer patients (purple) and non-cancer donors (green). The p-value is calculated using the Kolmogorov-Smirnov test. (C) Slides of surgery or biopsy section from patients with serological MET response (Pl, P45, P72, P76), stained for MET expression by immunohistochemistry, and a summarizing table. (D) Kaplan-Meier survival plot of 32 HR+ metastatic breast cancer patients, comparing those who responded to MET (n=6) with those who did not respond to MET (n=26). (E) Bar graph representing the IgG binding, as the mean O.D. of MET and SEMA binding, for purified IgG based on ELISA. Samples IDs are indicated on the bottom part of the graph. The dashed line indicates the IgG score of the highest binding healthy donor.

[0056] FIGs. 4A-E show the isolation and sequencing of MET -binding B cells. (A) Gating strategy for isolation of MET-binding B cells directly from PBMCs: lymphocytes

[0057]

[0058] singlets

[0059]

[0060] B cells (CD19+)

[0061]

[0062] MET+ (double fluorophore staining). The last gate was single cell sorted. (B) Gating strategy for isolation of MET-binding B cells following B cell in vitro culturing with MET: lymphocytes CD38hlCD27hl. The last gate was single cell sorted. (C) Pie charts representing the VH and VL usage of MET-enriched B cells. Each pie chart represents one patient (indicated on top), the numbers in the middle of the pies represent the total sequences recovered, slices are proportional to the representation of specific VH or VL gene. (D) Pie charts representing the IgG subclass distribution of MET-enriched B cells. Each pie chart represents one patient (indicated on top), the numbers in the middle of the pies represent the total sequences recovered, slices are proportional to the representation of specific subclass. (E) IgG subclass prevalence of MET-binding B cells compared to RBD-binding B cells.FIG. 5 shows frequency of IgG+ B cells. Comparison between cancer patients and healthy donors (data taken from previously published studies from the lab (47, 48)) of IgG+ cell frequencies out of total CD 19 positive, measured by flow-cytometry. The p-value is calculated using the Mann-Whitney test.

[0063] FIGs. 6A-E show MET-binding monoclonal antibodies cloned from cancer patients. (A) Binding of anti-MET mAbs to MET (left panel) and to SEMA (right panel), as detected by ELISA. mGO.53 serves as an isotype control(lO). The legend on the right side indicates mAbs ID. (B) Competition ELISA of anti-MET mAbs (69B287, 87B165, mGO.53) with HGF. O.D. values at 650 nm represent the binding of mAbs to MET following incubation with varying HGF concentrations (100, 1.56, 0.02, and 0 pg / mL). A commercial anti-HGF mAb (5 pg / mL) was included to assess HGF binding to MET. (C) Representative images of tumor tissue (left) and healthy adjacent tissue (right) stained with phalloidin and 87B156 (top) or 69B287 (bottom) mAbs (D) Quantification and normalization of 87B156 (top) or 69B287 (bottom) mAbs to phalloidin between tumor and healthy tissues. See also Supplementary Figure 5. P p-value is calculated by Wilcoxon test. (E) Representative images of tumor tissue stained with DAPI, phalloidin conjugated to FITC, mAb conjugated to Alexa Fluor 647, and the combined image with all staining.

[0064] FIGs. 7A-E show the binding of 69B287 and 87B156 mAbs to cancer cell lines. (A) Flow cytometry plots (left) and the summarized frequencies (right) showing the binding of mGO.53, 69B287, and 87B156 to CAL-51 (top), MDA-MB-468 (middle) and HCC70 (bottom) breast cancer cell lines. (B) Confluence measured by Incucyte of CAL-51, MDA-MB-468 and HCC70 breast cancer cell lines over time, incubated with each mAb (87B156, 69B287, and mGO.53). (C) Quantification of chemotactic cell migration and invasion. A total of 5,000 CAL-51 cells were seeded in the upper chamber of each well in a 96-well chemotaxis microplate and treated with 100 pM of monoclonal antibodies (87B156, 69B287, and mGO.53). Epidermal growth factor (EGF, 1 pg / mL) was added to the lower chamber of all wells as a chemoattractant. Cell migration was monitored using live imaging, and the number of migrated CAL-51 cells was quantified. Migration counts were normalized to the initial cell number. (D) Left panel: Western blot analysis showing pERK and ERK levels in CAL-51 breast cancer cells treated with mAbs 69B287 and 87B156 at the indicated time points. Right panel: Graph depicting the fold change in pERK levels over time, normalized to total ERK levels. (E) Calculated AUC of the confluence measured by Incucyte of CAL-51, MDA-MB-468 and HCC70 breast cancer cell lines over time, incubated with each mAb (87B156, 69B287, and mGO.53). p-value is calculated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons post-test.DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0065] The present invention, in some embodiments thereof, relates to anti-MET antibodies and uses thereof.

[0066] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0067] The presence of B cells in tumors is correlated with favorable prognosis and efficient response to immunotherapy. While tumor-reactive antibodies have been detected in several cancer types, identifying antibodies that specifically target tumor-associated antigens remains a challenge.

[0068] Whilst conceiving embodiments of the invention, the present inventors investigated antibodies spontaneously elicited during breast and lung cancer that bind the cancer-associated antigen MET.

[0069] Patients diagnosed with cancer were found positive to antibodies binding to both the recombinant ectodomain of MET and the ligand binding part of MET, SEMA. MET binding in the breast cancer cohort was significantly correlated with hormone receptor-positive status. The present inventors conducted immunoglobulin sequencing of peripheral MET-enriched B cells from MET-reactive patients. The MET-enriched B cell repertoire was found to be polyclonal and prone to non-IgGl subclass. Monoclonal antibodies were cloned and analyzed and these exhibited MET binding, low thermostability and high polyreactivity. Among these, antibodies which effectively bind to tumor cells and inhibit MET-expressing cancer cell lines were found. These antibodies were also found to compete with HGF binding to MET, inhibit cancer cell migration and inhibit MET signaling. Overall, it is demonstrated that some cancer patients develop polyreactive antibodies that cross-react with MET. The presence of MET autoantibodies may be harnessed towards patient diagnosis and prognosis, treatment selection, treatment monitoring. CDRs of MET autoantibodies or homologs thereof can be used in clinical applications which involve diagnosis prognosis and treatment.

[0070] As used herein “MET” also known as “hepatocyte growth factor receptor (HGFR)” is a protein that in humans is encoded by the MET gene. The protein possesses tyrosine kinase activity. Overexpression of MET, as well as its autocrine activation by co-expression of its hepatocyte growth factor ligand, are implicated in oncogenesis. UniProt P08581 provides the sequence of human MET where the semaphorin (SEMA) domain is located in amino acid coordinates 27-515. According to some embodiments, the SEMA domain is as set forth in SEQ ID NO: 91.

[0071] According to an aspect of the invention, there is provided an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domaincomprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0072] According to some embodiments, the heavy chain and / or light chain of the antibodies listed in Table 1 below. In the table, each row represents a specific antibody. Also contemplated are homologs of these antibodies as described below.

[0073] The CDRs of the heavy chain variable domain and the light chain variable domain are designated sequentially from the N-terminus to the C-terminus as CDRH1, CDRH2, and CDRH3 (heavy chain), and CDRL1, CDRL2, and CDRL3 (light chain), respectively. In the Table below, the CDR sequences are indicated by underlining within each variable domain sequence set forth herein. Each CDR may be characterized as an integral component of its respective heavy chain variable domain or light chain variable domain (or a homolog thereof) or alternatively as a discrete individual sequence. The six CDRs collectively, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, define the antigen-binding specificity of each antibody described herein.

[0074] Table 1

[0075]

[0076]

[0077] * (CDRs are underlined)Table la

[0078]

[0079]

[0080]

[0081] According to an aspect of the invention there is provided a monoclonal antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0082] According to an aspect of the invention there is provided an antibody comprising an antigen binding domain which binds an antigenic determinant of MET attached to a heterologous effector moiety or carrier, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0083] The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VE) that are capable of binding to an epitope of an antigen, in this case PstS.

[0084] According to specific embodiments, the antibody is a whole or intact antibody.

[0085] According to specific embodiments, the antibody is an antibody fragment.

[0086] Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv Fv (scFv), a disulfide- stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.

[0087] As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDRH1 or Hl; CDRH2 or H2; and CDRH3 or H3) and three in each of the VL (CDRE1 or El; CDRL2 or L2; and CDR L3 or L3).

[0088] CDRs shown in Table 1 were determined using the IMGT numbering system as implemented in IgBLAST.As used herein, the “variable regions” and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.

[0089] Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:

[0090] (i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;

[0091] (ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

[0092] (iii) disulfide-stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond.

[0093] (iv) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CH domains thereof;

[0094] (v) Fab’ , a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);

[0095] (vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and

[0096] (vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.

[0097] According to specific embodiments the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgA2, IgD, and IgE.

[0098] As shown in the Examples section, antibodies were recombinantly produced as IgGl, though their naturally occurring counterparts isolated from cancer patients may belong to other IgG subclasses.

[0099] The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.

[0100] According to specific embodiments, the antibody comprises an Fc domain.

[0101] According to specific embodiments, the antibody is a naked antibody.As used herein, the tern "naked antibody" refers to an antibody which does not comprise a heterologous effector moiety e.g., therapeutic moiety, detectable moiety.

[0102] As used herein “heterologous” means not occurring in nature in conjunction with the antibody. According to specific embodiments, the antibody comprises a heterologous effector moiety e.g. e.g. therapeutic moiety, detectable moiety. The effector moiety can be proteinaceous or non-proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner. The effector moiety may be any molecule, including small molecule chemical compounds and polypeptides. For example, the effector moiety can be a known drug to cancer.

[0103] According to specific embodiments, the antibody is a monoclonal antibody.

[0104] According to another embodiment, the antibody is part of a polyclonal sample.

[0105] Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos.

[0106] 4,036,945 and 4,33,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 9-26 (959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[0107] Fv fragments comprise an association of VH and VE chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (9720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs aredescribed, for example, by [Whitlow and Filpula, Methods 2: 97-05 (99); Bird et al., Science 242:423-426 (988); Pack et al., Bio / Technology :27-77 (993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

[0108] Another form of an antibody fragment is a peptide coding for a single complementaritydetermining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 06-0 (99)].

[0109] It will be appreciated that for human therapy or diagnostics, humanized antibodies and human antibodies are preferably used.

[0110] According to preferred embodiments, the antibody is a human antibody, such as that derived from cancer patients and disclosed in Table 1.

[0111] When referring to humanized antibodies the meaning is to implant the CDRs of the human antibodies on a backbone of a human antibody e.g., human constant region.

[0112] According to specific embodiments, the antibody is a humanized antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigenbinding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 32:522-525 (986); Riechmann et al., Nature, 332:323-329 (988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (992)].

[0113] According to a specific embodiment, the human antibody carries human Vh,Dh, Jh, VI, J, gene segments such as in germ line antibodies or natural variants thereof. Although synthetic antibodies are also contemplated.As used herein “antigenic determinant of MET” refers to an epitope which is located on the MET receptor.

[0114] According to some embodiments, the epitope is located in the extracellular domain of MET. According to some embodiments, the extracellular domain of MET is up to residue 908 in the MET protein sequence.

[0115] According to some embodiments, the epitope is located within the 515 amino acid semaphorin (SEMA) domain of MET.

[0116] According to some embodiments, the epitope overlaps with or is proximal to the HGF binding site on MET, thereby the antibody competes with HGF binding to MET.

[0117] According to some embodiments, the epitope is preferentially presented on cancer cells relative to adjacent healthy tissue.

[0118] According to some embodiments, the epitope is a conformational or linear in nature, as determined by binding under native or denaturing conditions, respectively.

[0119] According to some embodiments, the epitope is shared by proteins other than MET, facilitating the polyreactive nature of the antibody.

[0120] Hence the antibody may bind a plurality of proteins which are presented by cancer cells or the tumor microenvironment but not healthy cells.

[0121] Despite their polyreactive nature, the antibodies described herein demonstrate preferential and significantly higher binding to MET-expressing cancer cells and tumor tissue relative to healthy adjacent tissue, as determined by flow cytometry, ELISA, and immunofluorescence.

[0122] According to a specific embodiment, the antibody is a homolog comprising an amino acid sequence at least 80 %, 81 %, 82%, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the VH chain and / or VL chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 92L204, 92L205 and 92L236, the antibody is capable of binding an antigenic determinant of MET (and / or is characterized by any or all other functional features described herein).

[0123] According to preferred embodiments, the antibody is a human antibody, such as that derived from cancer patients. According to a specific embodiment, the human antibody carries human VH, DH, JH, VL and JL gene segments, such as in germline antibodies or natural variants thereof. Synthetic antibodies are also contemplated, wherein, for example, the CDRs are implanted on human or humanized scaffolds of interest.

[0124] According to some embodiments, the antibody is a recombinant antibody.

[0125] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the samewhen aligned. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have "sequence similarity" or "similarity." Means for making this adjustment are well-known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a nonconservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-10919]. Identity (e.g., percent homology) can be determined using any homology comparison software, including, for example, the BlastN or BlastP software of the National Center of Biotechnology Information (NCBI), using default parameters.

[0126] When referring to "at least 80 % identity," the claimed invention also refers to at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100% identity, where each represents a separate and distinct embodiment.

[0127] According to a specific embodiment, the level of identity is at least 90% over the entire sequence of any of the VH and / or VL chains described herein, as determined by the methods described herein.

[0128] According to a specific embodiment, the level of identity is at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % over at least one (or at least 2, 3, 4, 5, or 6) of the CDR sequences, at least one framework or the entire sequence of an antibody selected from those listed in Table 1, as described herein.

[0129] According to some embodiments, the antigen binding domain of the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 87B156.

[0130] According to some embodiments, the antigen binding domain of the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 69B287.

[0131] According to an aspect of the invention there is provided a method of producing an antibody, the method comprising:(a) expressing in a host cell a heterologous polynucleotide encoding the antibody as described herein; and optionally

[0132] (b) recovering the antibody from the host cell.

[0133] Thus, a polynucleotide comprising a nucleic acid sequence encoding an antibody of some embodiments of the invention. According to some embodiments, the nucleic acid sequence is cloned into an expression construct selected according to the expression system used. Exemplary polynucleotide sequences are provided in SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86.

[0134] A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the antibody of some embodiments of the invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence. Mammalian expression systems can also be used to express the antibodies of some embodiments of the invention.

[0135] Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3. (+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3, pSinRep5, DH26S, DHBB, pNMT, pNMT4, pNMT8, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0136] Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTOO / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0137] Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (990) Methods in Enzymol. 85:60-89).In yeast, a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447. Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.

[0138] In cases where plant expression vectors are used, the expression of the coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S RNA and 9S RNA promoters of CaMV [Brisson et al. (984) Nature 30:5-54], or the coat protein promoter to TMV [Takamatsu et al. (987) EMBO J. 6:307-3] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (984) EMBO J. 3:-680 and Brogli et al., (984) Science 224:838-843] or heat shock promoters, e.g., soybean hsp7.5-E or hsp7.3-B [Gurley et al. (986) Mol. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach, 988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 42-463.

[0139] Other expression systems such as insects and mammalian host cell systems which are well known in the art and are further described hereinbelow can also be used by some embodiments of the invention.

[0140] According to a specific embodiment, antibodies are expressed in Expi293F™ cells such as by using cationic lipid-based composition as the transfection reagent.

[0141] It will be appreciated that antibodies can also be produced in in-vivo systems such as in mammals, e.g., goats, rabbits etc.

[0142] Recovery of the recombinant antibody is effected following an appropriate time (in culture). The phrase "recovering the antibody” refers to collecting the whole fermentation medium containing the antibody and need not imply additional steps of separation or purification. Notwithstanding the above, antibodies of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

[0143] Once antibodies are obtained, they may be tested for activity.

[0144] Thus, antibodies described herein may be tested and / or characterized using a variety of methods. Such methods may be used to determine a variety of characteristics that may include, but are not limited to, antibody affinity; specificity; and activity (e.g., MET signaling, cell migration, cell proliferation). Antibody testing may further include testing in vivo (e.g., in animal and / or human studies) for one or more of toxicity, therapeutic effect, pharmacodynamics, pharmacokinetics,absorption, deposition, metabolism, and excretion. Testing in animals may include, but is not limited to, testing in mice, rats, rabbits, guinea pigs, pigs, primates (e.g., cynomolgus monkeys), sheep, goats, horses, and cattle.

[0145] In some embodiments, antibodies of the present invention may be tested or characterized through the use of one or more cell-based assays. Such cell-based assays may be carried out in vitro with cells in culture. In some cases, cell-based assays may be carried out in vivo. Examples of cellbased in vivo assays include tumor models in which tumor cells are injected or otherwise introduced into a host.

[0146] In some cases, cell-based assays used herein may include the use of cancer cells. Many cancer cell lines are available for experiments to test antibodies of the invention. Such cells preferably express the target antigen e.g., MET (such as detected at the protein and / or at the mRNA level). Additionally, cancer cell lines may be used to test antibodies of the invention.

[0147] According to some embodiments, the cancer cell lines are cloned from breast cancer, gastric cancer, hepatocellular cancer or lung cancer.

[0148] Examples of cancer cell lines include but are not limited to CAL-51, MCF-7, MDA-MB-468 and HCC70.

[0149] According to some embodiments, the cancer cell is hormone receptor positive (HR+) breast cancer.

[0150] As used herein “hormone receptor-positive (HR+) breast cancer” refers to breast cancer in which tumor cells express one or both of the following receptors, as determined by standard clinical immunohistochemical (IHC) assay:

[0151] (i) estrogen receptor alpha (ERa; ESRI), wherein HR+ status is defined as >1% of tumor cell nuclei staining positive by IHC, in accordance with the 2010 ASCO / CAP guidelines (Hammond et al., J. Clin. Oncol. 2010, 28(16):2784-2795); and / or

[0152] (ii) progesterone receptor (PR; PGR), wherein HR+ status is defined as >1% of tumor cell nuclei staining positive by IHC.

[0153] According to some embodiments, the cancer cell is triple negative breast cancer (TNBC). It will be appreciated that TNBC express high levels of MET. In other embodiments, the cancer is HR+. As shown in the Examples section a clinical association between HR+ status and anti-MET serological response was evident, reflecting the immune context of HR+ patients rather than a restriction of MET expression to HR+ tumors. The inhibitory activity demonstrated in TNBC lines supports the broad therapeutic potential of antibodies of the present invention regardless of hormone receptor status.

[0154] According to some embodiments, the antibody inhibits cancer cells growth.Inhibition of cancer cell growth may be determined by one or more assays known in the art, including but not limited to a cell viability assay (e.g., MTT, MTS, WST-1, or CellTiter-Glo luminescent cell viability assay), a cell proliferation assay (e.g., BrdU or EdU incorporation assay, Ki-67 staining, or real-time cell proliferation monitoring by impedance-based assay such as xCELLigence), a colony formation assay or a cell counting assay performed at one or more time points following antibody treatment. In certain embodiments, inhibition of cancer cell growth is expressed as a percentage reduction in cell viability or proliferation relative to an isotype control antibody-treated or untreated control population or as an IC50 value representing the antibody concentration required to inhibit cell growth by 50% relative to control. In certain embodiments, inhibition of cancer cell growth is assessed in a MET-expressing cancer cell line, including but not limited to those described in the Examples herein, following treatment with the antibody at one or more concentrations over a defined time period.

[0155] As used herein a biological effect described as a “reduction”, “inhibition”, “decrease” in a certain phenotype, such as inhibition of cell growth refers to a statistically significant reduction in the phenotype such as cell viability or proliferation relative to control as described herein.

[0156] Properties of antibodies of some embodiments of the invention (e.g., 69B287 and 87B156) can be improved such as by affinity maturation.

[0157] According to some embodiments, the antibody binds semaphorin (SEMA) domain of MET. According to some embodiments, the antibody competes with hepatocyte growth factor (HGF) binding to MET, such as determined by ELISA.

[0158] According to some embodiments, the antibody does not bind a healthy tissue as determined by immunofluorescence.

[0159] Assays for determining binding of an antibody to a target antigen include, but are not limited to, ELISA and surface plasmon resonance (SPR).

[0160] As used herein “binding” or “binds” refers to an antibody-antigen mode of binding, which is generally, in the range of KD below 500 nM. The affinity of the antibodies to MET was determined by ITC, revealing KD values of 2.87 x 10 M and 3.27 x 10 M for 69B287 and 87B 156, respectively. Accordingly, the contemplated KD range for binding MET is below 10 pM, such as between 1-10 pM, as determined by ITC, such as determined by ELISA.

[0161] According to another specific embodiment, the affinity of the antibody to its antigen is determined by Surface Plasmon Resonance (SPR).

[0162] As used herein the term “KD” refers to the equilibrium dissociation constant between the antigen binding domain and its respective antigen.According to a specific embodiment, the KD for binding the target (e.g., MET) is typically in the range of 0.1-500 nM.

[0163] For example, between 1-10 nM, 1-50 nM, 0.1-10 nM, 0.1-50 nM, 0.1-100 nM, 0.1-10 pM, 0.5-5 pM, 1-5 pM or 1-10 pM

[0164] According to some embodiments, the antibody is soluble.

[0165] According to some embodiments, the antibody is insoluble.

[0166] Non-soluble antibodies may be a part of a particle (synthetic e.g., liposome or non-synthetic, e.g., exosome) or a cell (e.g., CAR-T cells, in which the antibody is part of a chimeric antigen receptor (CAR) typically as an scFv fragment).

[0167] According to some embodiments, the antibody is labeled. The attachment of a label may be for research in vitro use or clinical (in vivo) use. According to some embodiments, labels suitable for in vitro research applications include, but are not limited to, fluorescent labels (e.g., FITC, PE, Alexa Fluor dyes), enzymatic labels (e.g., horseradish peroxidase, alkaline phosphatase), and radioactive isotopes (e.g.,3H,14C), which may be used in applications such as flow cytometry, immunofluorescence, immunohistochemistry, and EEISA. Labels suitable for clinical in vivo use include, but are not limited to, radioisotopes suitable for positron emission tomography (PET) imaging (e.g.,89Zr,64Cu), single-photon emission computed tomography (SPECT) imaging (e.g., "mTc,H1In), and therapeutic radionuclides (e.g.,90Y,177Lu), as well as near-infrared fluorescent dyes suitable for intraoperative imaging. In certain embodiments, the labeled antibody of the present invention may be used for diagnostic imaging of MET-expressing tumors, for monitoring treatment response.

[0168] Increasing the cytotoxic activity of an antibody where necessary can also be achieved such as by using an antibody-drug conjugate (ADC) concept. In such a configuration the antibody is attached to a heterologous effector moiety that can be used to increase its toxicity or to render it detectable.

[0169] In some embodiments, antibodies of the invention may be developed for antibody drug conjugate (ADC) therapeutic applications. ADCs are antibodies in which one or more cargo (e.g., therapeutic agents) are attached [e.g. directly or via linker (e.g. a cleavable linker or a non-cleavable linker)]. ADCs are useful for delivery of therapeutic agents (e.g., drugs or cytotoxic agents) to one or more target cells or tissues (Panowski, S. et al., 204. mAbs 6:, 34-45). In some cases, ADCs may be designed to bind to a surface antigen on a targeted cell. Upon binding, the entire antibody-antigen complex may be internalized and directed to a cellular lysosome. ADCs may then be degraded, releasing the bound cargo.

[0170] It will be appreciated that also polyclonal antibodies can be formulated as ADCs and as such are envisaged herein.The therapeutic agent may be a small molecule drug, a proteinaceous agent, a nucleic acid agent, radio-isotopes and carbohydrate and the like. These can serve as cytotoxic agents, e.g., chemotherapy.

[0171] According to a specific embodiment, the therapeutic agent is a nucleic acid sequence (e.g., DNA or RNA, e.g., mRNA) which codes for a viral antigen, in order to elicit an anti viral immune response against the tumor. Examples of viral antigens include, but are not limited to CMV antigens, EBV antigens, Coronavirus antigens and the like. Generally, any mRNA for stimulating an immune response can be used.

[0172] Where the cargo is a cytotoxic agent, the target cell will be killed or otherwise disabled. Cytotoxic agents may include, but are not limited to cytoskeletal inhibitors [e.g., tubulin polymerization inhibitors, and kinesin spindle protein (KSP) inhibitors], DNA damaging agents (e.g., calicheamicins, duocarmycins, and pyrrolobenzodiazepine dimers such as talirine and tesirine), topoisomerase inhibitors [e.g., camptothecin compounds or derivatives such as 7-ethyl-0-hydroxycamptothecin (SN-38) and exatecan derivative DXd], transcription inhibitors (e.g., RNA polymerase inhibitors such as amanitin), and kinase inhibitors [e.g., phosphoinositide 3-kinase (PI3K) inhibitors or mitogen-activated protein kinase kinase (MEK) inhibitors].

[0173] Tubulin polymerization inhibitors may include, but are not limited to, maytansines (e.g., emtansine [DM] and ravtansine [DM4]), auristatins, tubulysins, and vinca alkaloids or derivatives thereof. Exemplary auristatins include auristatin E (also known as a derivative of dolastatin-O), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F and dolastatin. Exemplary tubulysin compounds include naturally occurring tubulysins A, B, C, D, E, F, G, H, I, U, and V, and tubulysin analogs such as pretubulysin D (PTb-D43) and N.sup.4-desacetoxytubulysin H (Tbl). Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). In some embodiments, cytotoxic agents may include auristatin derivatives [e.g. -aminopropan-2-yl-auristatin F, auristatin F-hydroxypropylamide, auristatin F-propylamide, auristatin F phenylenediamine (AFP)]; tubulysin derivatives; vinca alkaloid derivatives [e.g. N-(3-hydroxypropyl)vindesine (HPV)], and any of those described in U.S. Pat. Nos. 8,524,24; 8,685,383; 8,808,9; and 9,254,339; US Patent Application Publications US205034008A, US2060220696A and US2060022829A; the contents of each of which are herein incorporated by reference in their entirety.

[0174] Examples of gold-standard chemotherapy and targeted therapy useful for the treatment of HR+ breast cancer include, but are not limited to, Tamoxifen (Nolvadex), Letrozole (Femara), Anastrozole (Arimidex), Exemestane (Aromasin), Fulvestrant (Faslodex), Palbociclib (Ibrance), Ribociclib (Kisqali), Abemaciclib (Verzenio), and Everolimus (Afinitor).Examples of gold-standard chemotherapy and targeted therapy useful for the treatment of HER2+ breast cancer include, but are not limited to, Trastuzumab (Herceptin), Pertuzumab (Perjeta), Trastuzumab emtansine (Kadcyla), Trastuzumab deruxtecan (Enhertu), Lapatinib (Tykerb), Neratinib (Nerlynx), Tucatinib (Tukysa), Docetaxel (Taxotere), and Paclitaxel (Taxol).

[0175] Examples of gold-standard chemotherapy useful for the treatment of triple-negative breast cancer (TNBC) include, but are not limited to, Paclitaxel (Taxol), Docetaxel (Taxotere), Doxorubicin (Adriamycin), Cyclophosphamide (Cytoxan), Carboplatin (Paraplatin), Sacituzumab govitecan (Trodelvy), Pembrolizumab (Keytruda), and Olaparib (Lynparza).

[0176] Examples of gold-standard chemotherapy and targeted therapy useful for the treatment of non-small cell lung cancer (NSCLC) include, but are not limited to, Cisplatin (Platinol), Carboplatin (Paraplatin), Paclitaxel (Taxol), Pemetrexed (Alimta), Erlotinib (Tarceva), Gefitinib (Iressa), Osimertinib (Tagrisso), Crizotinib (Xalkori), Pembrolizumab (Keytruda), and Bevacizumab (Avastin).

[0177] Examples of gold-standard chemotherapy and targeted therapy useful for the treatment of small cell lung cancer (SCLC) include, but are not limited to, Etoposide (Toposar), Cisplatin (Platinol), Carboplatin (Paraplatin), Irinotecan (Camptosar), Atezolizumab (Tecentriq), and Durvalumab (Imfinzi).

[0178] Examples of gold-standard chemotherapy and targeted therapy useful for the treatment of hepatocellular carcinoma (HCC) include, but are not limited to, Sorafenib (Nexavar), Lenvatinib (Lenvima), Regorafenib (Stivarga), Cabozantinib (Cabometyx), Atezolizumab (Tecentriq), Bevacizumab (Avastin), Nivolumab (Opdivo), Pembrolizumab (Keytruda), and Doxorubicin (Adriamycin).

[0179] In some embodiments, antibody-drug conjugates (ADCs) of the invention may further comprise one or more polymeric carrier connecting the antibody and the therapeutic agents (e.g., antibody-polymer-drug conjugates). As used herein, the term "polymeric carrier" refers to a polymer or a modified polymer, which may be covalently attached to one or more therapeutic agents and / or antibodies. Polymeric carriers may provide additional conjugation sites for therapeutic agents, increasing the drug-to-antibody ratio and enhancing therapeutic effects of ADCs. In some embodiments, polymeric carriers used in this invention may be water soluble and / or biodegradable. Such polymeric carriers may include, but are not limited to poly(ethylene glycol) (PEG), poly(N-(2-hydroxypropyl)methacrylamide) (polyHPMA), poly(.alpha.-amino acids) [e.g., poly(L-lysine), poly(L-glutamic acid), and poly ((N-hydroxy alky )glutamine)], carbohydrate polymers [e.g., dextrins, hydroxyethylstarch (HES), and polysialic acid], glycopolysaccharides (e.g., homopolysaccharide such as cellulose, amylose, dextran, levan, fucoidan, carraginan, inulin, pectin, amylopectin, glycogenand lixenan; or homopolysaccharide such as agarose, hyluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin), glycolipids, glycoconjugates, polyglycerols, polyvinyl alcohols, poly(acrylic acid), polyketal and polyacetal [e.g., poly(l -hydroxymethylethylene hydroxymethylformal), also known as PHF or FLEXIMER®., described in U.S. Pat. Nos. 5,811,501; 5,863,990; and 5,958,398; the contents of each of which are herein incorporated by reference in their entirety], and derivatives, dendrimers, copolymers and mixtures thereof. For example, the polymeric carrier may include a copolymer of a polyacetal / polyketal (e.g., PHF) and a hydrophilic polymer such as poly acrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, polypeptides, and derivatives thereof.

[0180] In some embodiments, therapeutic agents are attached (e.g., covalently bonded) to antibodies of the invention directly or via linkers. In some embodiments, therapeutic agents are attached to polymeric carriers directly or via linkers, and the polymeric carriers are attached to the antibodies directly or via linkers. In some embodiments, linkers may comprise an oxalic, malonic, succinic, glutaric, adipic, pimelic, suberic, azelaic, sebacic, phthalic, isophthalic, terephthalic, diglycolic acid, tartaric, glutamic, fumaric, or aspartic moiety, including amide, imide, or cyclic-imide derivatives of each thereof, and each optionally substituted. Exemplary linkers may include any of those disclosed in U.S. Pat. Nos. 8,524,241; 8,685,383; 8,808,911; 9,254,339; and / or 9,555,2 the contents of each of which are herein incorporated by reference in their entirety.

[0181] In some embodiments, linkers may be cleavable linkers. Cleavable linkers may break down under certain conditions (such as changes in pH, temperature, or reduction) or cleaved by enzymes (e.g., proteases and glucuronidases) to allow release of therapeutic agents from ADCs. Such linkers may include a labile bond such as an ester bond, amide bond, or disulfide bond. Non-limiting cleavable linkers may include pH-sensitive linkers (e.g., hydrazone, semicarbazone, thiosemicarbazone, cisaconitic amide, thioether, orthoester, acetal, or ketal); reduction-sensitive linkers [e.g., N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-S-acetylthioacetate (SATA) and N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene or 2,5-dioxopyrrolidin— yl 4-(-(pyridin-2-yldisulfanyl)ethyl)benzoate (SMPT)]; photosensitive linkers; and enzymatically cleavable linkers [e.g., peptide linkers such as valine-citrulline, valine-citrulline-p-aminobenzoyloxycarbonyl (vc-PAB ) , maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB), linkers cleavable by glucuronidases, such as glucuronide-MABC, or linkers cleavable by esterases].In other embodiments, linkers may be non-cleavable linkers. Non-cleavable linkers may increase plasma stability of the ADCs compared to cleavable linkers. Exemplary non-cleavable linkers include maleimide alkane and maleimide cyclohexane (MCC).

[0182] Antibody-drug conjugates (ADCs) of the invention may be prepared using any method known in the art. For example, therapeutic agents may be modified to contain a functional group that can react with a functional group on the antibody. Antibody-drug conjugates (ADCs) may be prepared by reacting the two functional groups to form a conjugate. In some cases, polymeric carriers may be modified to contain functional groups that can react with the functional group on the therapeutic agents and the functional group on the antibody under different chemical conditions. Antibodies, polymeric carriers, and therapeutic agents may be linked to form the antibody-polymer-drug conjugates through sequential chemical reactions. Conjugation to antibodies may employ a lysine or a cysteine residue as the conjugation site. In some embodiments, antibodies may be engineered to have additional lysine or cysteine residues. Such approaches may avoid disruption of antibody structure (e.g., interchain disulfide bonds) and maintain antibody stability and / or activity.

[0183] In some embodiments, antibodies of the invention may be tested for their ability to promote cell death per se or when developed as ADCs.

[0184] In some embodiments, antibody sequences of the invention may be used to develop a chimeric antigen receptor (CAR). CARs are transmembrane receptors expressed on immune cells that facilitate recognition and killing of target cells (e.g. tumor cells). CARs typically include three basic parts. These include an ectodomain (also known as the recognition domain), a transmembrane domain and an intracellular (signaling) domain. Ectodomains facilitate binding to cellular antigens on target cells, while intracellular domains typically include cell signaling functions to promote the killing of bound target cells. Further, they may have an extracellular domain with one or more of the antibody variable domains described herein or fragments thereof. CARs of the invention also include a transmembrane domain and cytoplasmic tail. CARs may be designed to include one or more segments of an antibody, antibody variable domain and / or antibody CDR, such that when such CARs are expressed on immune effector cells, the immune effector cells bind and clear any cells that are recognized by the antibody portions of the CARs.

[0185] Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.CARs engineered to target tumors may have specificity for MET according to some embodiments of the invention. In some embodiments, ectodomains of these CARs may include one or more antibody variable domains or a fragment thereof. In some embodiments, CARs are expressed in T cells, and may be referred to as "CAR-engineered T cells" or "CAR-Ts". CAR-Ts may be engineered with CAR ectodomains having one or more antibody variable domains.

[0186] In some embodiments, antibodies of the present invention may bind more than one epitope. As used herein, the terms "multibody" or "multispecific antibody" refer to an antibody wherein two or more variable regions bind to different epitopes. The epitopes may be on the same or different targets. In certain embodiments, a multi- specific antibody is a "bispecific antibody," which recognizes two different epitopes on the same or different antigens.

[0187] Bispecific antibodies are capable of binding two different antigens. Such antibodies typically comprise antigen-binding regions from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies, thus allowing the BsAb to bind to two different types of antigen. One common application for this technology is in cancer immunotherapy, where BsMAbs are engineered to simultaneously bind to a cytotoxic cell (using a receptor like CD3) and a target like a tumor cell to be destroyed.

[0188] Bispecific antibodies may include any of those described in Riethmuller, G., 202. Cancer Immunity. 2:2-8; Marvin, J. S. et al., 2005. Acta Pharmacologic a Sinica. 26(6):649-58; and Schaefer, W. et al., 20. PNAS. 08(27):87-92, the contents of each of which are herein incorporated by reference in their entirety.

[0189] New generations of BsMAb, called "trifunctional bispecific" antibodies, have been developed. These consist of two heavy and two light chains, one each from two different antibodies, where the two Fab regions (the arms) are directed against two antigens, and the Fc region (the foot) comprises the two heavy chains and forms the third binding site.

[0190] Other types of bispecific antibodies have been designed to overcome certain problems, such as short half-life, immunogenicity and side-effects caused by cytokine liberation and are contemplated herein. They include chemically linked Fabs, consisting only of the Fab regions, and various types of bivalent and trivalent single-chain variable fragments (scFvs), fusion proteins mimicking the variable domains of two antibodies. The furthest developed of these newer formats are the bi-specific T-cell engagers (BiTEs) and mAb2's, antibodies engineered to contain an Fcab antigen-binding fragment instead of the Fc constant region.

[0191] In some embodiments, antibodies of the present invention may be diabodies. Diabodies are functional bispecific single-chain antibodies (bscAb). These bivalent antigen-binding molecules arecomposed of non-covalent dimers of scFvs, and can be produced in mammalian cells using recombinant methods. (See, e.g., Mack et al, Proc. Natl. Acad. Sci., 92: 702-7025, 995). Few diabodies have entered clinical development. An iodine-23 -labeled diabody version of the anti-CEA chimeric antibody cT84.66 has been evaluated for pre-surgical immuno scintigraphic detection of colorectal cancer in a study sponsored by the Beckman Research Institute of the City of Hope (Clinicaltrials(dot)gov NCT0064753) (Nelson, A. L., MAbs. 200. January-February; 2():77-83).

[0192] Also included are maxibodies (bivalent scFV fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG.

[0193] Bispecific T-cell-engager (BiTE) antibodies are designed to transiently engage cytotoxic T-cells for lysis of selected target cells. These typically include two scFvs (one binding to CD3 on Tcells and one binding to a target antigen on the surface of a cell being targeted for destruction). In some embodiments, the two scFvs are joined by a linker. In other embodiments, the two scFvs are different regions on an antibody. The clinical activity of BiTE antibodies corroborates findings that ex vivo expanded, autologous T-cells derived from tumor tissue, or transfected with specific T-cell receptors, have shown therapeutic potential in the treatment of solid tumors. While these personalized approaches prove that T-cells alone can have considerable therapeutic activity, even in late-stage cancer, they are cumbersome to perform on a broad basis. This is different for cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibodies, which facilitate generation of tumor- specific T-cell clones, and also for bi- and tri-specific antibodies that directly engage a large proportion of patients' T-cells for cancer cell lysis. The potential of global T-cell engagement for human cancer therapy by T-cell-engaging antibodies is under active investigation (Baeuerle P A, et al., Current Opinion in Molecular Therapeutics. 2009, ():22-30 and Baeuerle P A and Reinhardt C, Cancer Res. 2009, 69(2): 494-4, the contents of each of which are herein incorporated by reference in their entirety).

[0194] In a whole antibody, a therapeutic activity is intrinsic to the molecule since the Fc domain activates antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC is a mechanism of cell-mediated immune defense whereby an effector cell of the immune system actively lyses a target cell, whose membrane- surface antigens have been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain infection. Classical ADCC is mediated by natural killer (NK) cells; macrophages, neutrophils and eosinophils can also mediate ADCC. For example, eosinophils can kill certain parasitic worms known as helminths through ADCC mediated by IgE. ADCC is part of the adaptive immune response due to its dependence on a prior antibody response.

[0195] The term "Fc domain" or "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includesnative sequence Fc regions and variant Fc regions. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 99. An "Fc polypeptide" of a dimeric Fc as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, an Fc polypeptide of a dimeric IgG Fc comprises an IgG CH2 and an IgG CH3 constant domain sequence. An Fc can be of the class IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG, IgG2, IgG3, IgG4, IgA, and IgA2.

[0196] The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. For example, an FcR can be a native sequence human FcR. Generally, an FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the Fc gamma RI, Fc gamma RII, and Fc gamma RIII subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc gamma RII receptors include Fc gamma RIIA (an "activating receptor") and Fc gamma RUB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Immunoglobulins of other isotypes can also be bound by certain FcRs (see, e.g., Janeway et al., Immuno Biology: the immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 999)). Activating receptor Fc gamma RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor Fc gamma RUB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (reviewed in Daeron, Annu. Rev. Immunol. 5:203-234 (997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (99); Capel et al., Immunomethods 4:25-34 (994); and de Haas et al., J. Lab. Clin. Med. 26:330-4 (995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 7:587 (976); and Kim et al., J. Immunol. 24:249 (994)).

[0197] Modifications in the CH2 domain can affect the binding of FcRs to the Fc. A number of amino acid modifications in the Fc region are known in the art for selectively altering the affinity of the Fc for different Fc gamma receptors. In some aspects, the Fc comprises one or more modifications to promote selective binding of Fc-gamma receptors.

[0198] Exemplary mutations that alter the binding of FcRs to the Fc are listed below:

[0199] S298A / E333A / K334A, S298A / E333A / K334A / K326A (Lu Y, Vernes J M, Chiang N, et al. J Immunol Methods. 20 Feb. 28; 365(-2): 32-4);F243L / R292P / Y300L / V305 / P396L, F243L / R292P / Y300L / L235V / P396L (Stavenhagen J B, Gorlatov S, Tuaillon N, et al. Cancer Res. 2007 Sep. 5; (8):8882-90; Nordstrom J L, Gorlatov S, Zhang W, et al. Breast Cancer Res. 20 Nov. 30; 3(6):R23);

[0200] F243L (Stewart R, Thom G, Levens M, et al. Protein Eng Des Sei. 20 September; 24(9):-8.), S298A / E333A / K334A (Shields R L, Namenuk A K, Hong K, et al. J Biol Chem. 200 Mar. 2; 276(9):659-604);

[0201] S239D / I332E / A330L, S239D / I332E (Lazar G A, Dang W, Karki S, et al. Proc Natl Acad Sci USA. 2006 Mar. 4; 03():4005-0); S239D / S2E, S2E / L328F (Chu S Y, Vostiar I, Karki S, et al. Mol Immunol. 2008 September; 45(5):3926-33);

[0202] S239D / D265S / S298A / I332E, S239E / S298A / K326A / A327H, G237F / S298A / A330L / I332E, S239D / I332E / S298A, S239D / K326E / A330L / I332E / S298A, G236A / S239D / D270L / I332E, S239E / S2E / H268D, L 234F / S2E / N325L, G237F / V266L / S2D and other mutations listed in W020 / 2034 and W020 / 2035, herein incorporated by reference. Therapeutic Antibody Engineering (by William R. Strohl and Lila M. Strohl, Woodhead Publishing series in Biomedicine No, ISBN 907568379, October 202) lists mutations on page 283.

[0203] In some embodiments an antibody described herein includes modifications to improve its ability to mediate effector function. Such modifications are known in the art and include afucosylation, or engineering of the affinity of the Fc towards an activating receptor, mainly FCGR3a for ADCC, and towards Cq for CDC.

[0204] Methods of producing antibodies with little or no fucose on the Fc glycosylation site (Asn 297 EU numbering) without altering the amino acid sequence are well known in the art.

[0205] In some embodiments, an antibody has antibody-dependent cellular phagocytosis (ADCP) activity. ADCP can occur when antibodies bind to antigens on the surface of pathogenic or tumorigenic target-cells. Phagocytic cells bearing Fc receptors on their cell surface, including monocytes and macrophages, recognize and bind the Fc region of antibodies bound to target-cells. Upon binding of the Fc receptor to the antibody-bound target cell, phagocytosis of the target cell can be initiated. ADCP can be considered a form of ADCC.

[0206] Antibodies of some embodiments of the invention can be used in treating cancer.

[0207] Thus, according to an aspect of the invention there is provided an antibody comprising an antigen binding domain which binds an antigenic determinant of MET for use in preventing or treating cancer in a subject in need thereof, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selectedfrom the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

[0208] According to an additional aspect there is provided a method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236, thereby preventing or treating cancer in the subject.

[0209] The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and / or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

[0210] As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

[0211] As used herein, the term “subject” includes human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology.

[0212] According to a specific embodiment, the pathology is cancer.

[0213] According to a specific embodiment, the cancer is a solid tumor.

[0214] According to an embodiment, the cancer is a primary cancer.

[0215] According to an embodiment, the cancer is a metastatic cancer.

[0216] According to a specific embodiment, cells of the cancer express MET, as can be determined at the DNA (amplification or mutations), protein (e.g., such as biopsy staining) or RNA level. Expression of MET can be of wild type MET or a mutant MET (e.g., MET exon 14 skipping mutations, MET amplification, MET point mutations in the kinase domain (e.g., M1250T, Y1235D) or MET gain-of-function mutations associated with hereditary papillary renal cell carcinoma and other MET-driven malignancies). The expression is typically higher than that of healthy cells of the same tissue.

[0217] Thus, MET expression may be determined by any method known in the art, including, but not limited to, immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR) or Western blot. According to a specific embodiment, MET expression is determined by IHC usingan anti-MET antibody. According to some embodiments, MET positivity is defined by a clinically accepted scoring threshold. MET overexpression, MET amplification, and MET exon 14 skipping mutations each represent distinct molecular mechanisms by which MET signaling is dysregulated in cancer, and each is encompassed within the scope of the present embodiment. According to specific embodiments, the MET-expressing cancer include, but are not limited to, breast cancer, lung cancer, hepatocellular carcinoma, gastric cancer, colorectal cancer, renal cell carcinoma, and glioblastoma.

[0218] According to a specific embodiment, the cancer is MET positive, optionally HR+.

[0219] According to some embodiments, the cancer is triple negative breast cancer.

[0220] According to a specific embodiment, the level of MET expression in the cancer is elevated relative to the corresponding non-cancerous tissue of the same subject, consistent with the finding described herein that the antibodies of the present invention demonstrated significantly higher binding to cancer tissue relative to adjacent healthy tissue as determined by tissue array analysis.

[0221] According to a specific embodiment, the level of MET expression in a cancer of a subject is determined ex vivo or in vitro. According to a specific embodiment, the level of MET expression in a cancer of a subject is determined by analysis of a biopsy sample obtained from the subject, including but not limited to a core needle biopsy, fine needle aspirate, excisional biopsy, or liquid biopsy comprising circulating tumor cells (CTCs) or cell-free tumor DNA (ctDNA). In certain embodiments, MET expression is determined in vivo by diagnostic imaging using a labeled anti-MET antibody of the present invention, such as a radiolabeled antibody suitable for PET or SPECT imaging, wherein the level and distribution of MET expression in the tumor may be assessed non-invasively and in real time. Determination of MET levels prior to and following treatment may further serve as a pharmacodynamic biomarker of treatment response, consistent with the monitoring method as described herein. It will be appreciated that any of the antibodies described herein or any other anti-MET antibody can be used for analyzing the level of MET either for diagnosing, prognosing or treatment of the pathology. The antibodies of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

[0222] Thus, according to an aspect of the invention there is provided a pharmaceutical composition comprising the antibody, cell, polynucleotide, construct as described herein. Also contemplated herein are combinations or a plurality of antibodies (e.g., 2, 3) for augmenting treatment efficacy.

[0223] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.Herein the term "active ingredient" refers to the antibody, cell, polynucleotide, construct accountable for the biological effect.

[0224] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0225] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0226] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0227] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.

[0228] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

[0229] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0230] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0231] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0232] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0233] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0234] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0235] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0236] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0237] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0238] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0239] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0240] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (antibody, cell, polynucleotide, construct) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0241] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0242] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0243] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 975, in "The Pharmacological Basis of Therapeutics", Ch. p.).

[0244] Dosage amount and interval may be adjusted individually to provide effective tissue levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0245] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0246] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0247] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0248] Treatment may be augmented by the use of other treatment modules such as chemotherapy, radiotherapy, biological therapy (other than the claimed antibodies or their conjugated) or surgery.

[0249] Antibodies of some embodiments of the invention may also find use in diagnostic and treatment selection.

[0250] Thus, according to an aspect of the invention there is provided a method of diagnosing cancer in a subject in need thereof, the method comprising:

[0251] (a) contacting cells of the subject with an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236;

[0252] (b) detecting an immunocomplex formation between said antibody and said cells, wherein presence of said immunocomplex is indicative of cancer.

[0253] As used herein the term “diagnosing” refers to determining presence or absence of a pathology (e.g., a disease, disorder, condition or syndrome), classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and / or prospects of recovery and screening of a subject for a specific disease.

[0254] In the context of the present teachings, the pathology is cancer.

[0255] According to a specific embodiment, the subject is symptomatic for cancer. Symptoms indicative of cancer, which may prompt diagnostic evaluation using the antibodies and methods of the present invention, include, but are not limited to, a palpable mass or lump such as in the breast, axilla or lung field, unexplained weight loss, persistent cough or hemoptysis, fatigue, night sweats, jaundice, hepatomegaly, and abnormal findings on routine imaging studies such as mammography, ultrasound, computed tomography (CT) or magnetic resonance imaging (MRI). In certainembodiments, the subject has been referred for biopsy or further diagnostic workup based on one or more of the foregoing clinical presentations.

[0256] In other embodiments, the subject is asymptomatic for cancer. In some embodiments, the subject is identified as being at elevated risk for cancer. In certain embodiments, the subject carries a germline or somatic mutation in the MET gene. In certain embodiments, the subject is participating or is referred to a routine cancer screening program.

[0257] According to some embodiments, contacting of the antibodies with the suspected cancer tissue is performed in vitro (such as on tissue biopsy) or in vivo, as described above.

[0258] According to some embodiments, immunocomplex formation is scored as positive or negative arbitrarily (by indication of presence) or by reference to one or more controls, including a positive control comprising a tissue or sample known to express MET at a clinically relevant level, a negative control comprising a tissue or sample known to lack MET expression, and / or a predetermined threshold value established from a reference population. In certain embodiments, a sample is scored as MET -positive when the level of immunocomplex formation exceeds that of the negative control by a statistically significant margin or exceeds a pre-defined cutoff value such as expressed as an IHC score, optical density value, mean fluorescence intensity or equivalent quantitative measure. In certain embodiments, the level of immunocomplex formation is further correlated with MET expression level in a semi-quantitative manner, for example using a standardized IHC scoring system such as an H-score or a 0-3+ scoring scale, wherein a score of 2+ or 3+ is considered MET-positive for the purposes of patient selection or diagnostic determination.

[0259] Positive diagnosis may be followed by treatment.

[0260] According to another aspect of the invention there is provided a method of treating cancer in a subject in need thereof, the method comprising:

[0261] (a) diagnosing cancer as described herein; and

[0262] (b) treating the cancer with an anti-cancer treatment, optionally wherein the anti-cancer treatment comprises the antibody which is used for diagnosis.

[0263] According to other embodiments the diagnosis may be followed by selecting the appropriate treatment which is tailored for subjects clinical or molecular presentation.

[0264] Thus, according to an aspect there is provided a method of selecting treatment to cancer in a subject diagnosed with cancer, the method comprising:

[0265] (a) contacting cells of the subject with an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selectedfrom the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236;

[0266] (b) detecting an immunocomplex formation between said antibody and said cells, wherein presence of said immunocomplex is indicative of cancer cells which express MET; and

[0267] (c) selecting a treatment which is efficacious for MET expressing cancer, wherein said treatment is optionally with said antibody such as described herein (e.g., 69B287 or 87B156).

[0268] In other embodiments, the treatment may be with other medicaments which are specific for MET or its signaling pathway.

[0269] Examples of treatments which target MET or MET / HGF signaling are well known in the art. Examples of MET kinase inhibitors include, but are not limited to, K252a, SU11274, PHA-665752, and Tivantinib. These small molecules block ATP binding to the MET receptor, preventing its activation and downstream signaling. Some inhibitors, like K252a, target multiple receptor tyrosine kinases, while others, such as SU 11274 and PHA-665752, are more specific to MET. These inhibitors have shown varying levels of efficacy in treating cancers associated with MET activation, such as lung, gastric, and renal cancers.

[0270] Examples of HGF inhibitors include, but are not limited to, NK4, neutralizing anti-HGF antibodies, and uncleavable HGF. NK4 competes with HGF by binding MET without activating it, while anti-HGF antibodies block HGF binding to MET and prevent receptor activation. Uncleavable HGF is an engineered form that prevents HGF maturation, thereby inhibiting MET-induced biological responses. These inhibitors aim to block HGF-dependent MET activation and are being explored for their potential in treating MET-driven cancers.

[0271] Examples of decoy MET receptors include, but are not limited to, CGEN241, which binds to MET and prevents both ligand binding and receptor dimerization, inhibiting MET activation. In the immunotherapy, examples of monoclonal antibodies targeting MET include, but are not limited to, DN30 and OA-5D5.

[0272] The antibodies can also be used for determining treatment efficacy of MET positive cancer patients.

[0273] Thus, according to an aspect of the invention there is provided a method of monitoring treatment of cancer in a subject in need thereof, the method comprising:

[0274] (a) treating the subject with an anti-cancer treatment, wherein said cancer is characterized by MET expression;

[0275] (b) determining a level of said MET prior to and following said treating using an antibody, wherein a reduction in MET is indicative of an efficacious treatment, wherein at least one of said anti-cancer treatment and said antibody, comprises the antibody as described herein e.g., 69B287, 87B 156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236 or a homolog thereof.

[0276] As used herein the term “diagnosing” refers to determining presence or absence of a pathology (e.g., a disease, disorder, condition or syndrome), classifying a pathology or a symptom, determining a severity of the pathology, monitoring pathology progression, forecasting an outcome of a pathology and / or prospects of recovery and screening of a subject for a specific disease.

[0277] In the context of the present teachings, the pathology is cancer.

[0278] As used herein the term “about” refers to ± 10 %.

[0279] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0280] The term “consisting of’ means “including and limited to”.

[0281] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0282] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0283] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0284] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0285] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques andprocedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0286] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0287] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0288] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0289] EXAMPLES

[0290] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0291] Methods

[0292] Study cohort and study approval

[0293] Seventy-five breast cancer patients and twenty-five lung cancer patients were recruited from the Oncology Institute in Sheba Medical Center, Ramat Gan, Israel. The breast cancer patients had metastatic disease or were undergoing neoadjuvant therapy, while the lung cancer patients had metastatic disease or were receiving chemoradiation therapy. Serum samples were initially collected from each patient to screen for anti-MET antibodies. Additional blood was drawn two to four times from patients with high anti-MET serum activity to provide a total of 150 mL of whole blood. Serumsamples from healthy donors, used as controls, were obtained from the Israeli blood bank. All studies involving patient enrollment, sample collection, and clinical follow-up were approved by the Tel Aviv University Institutional Review Board (IRB) under protocol number 002606-3. Patients were recruited and followed at the Sheba Cancer Center, which received Helsinki Committee approval under number 5841-19-SMC. Written informed was obtained from all patients before first blood collection.

[0294] Sex as a biological variable

[0295] For breast cancer patients, recruitment was limited to females due to the rarity of male breast cancer patients, who constitute less than 1% of all cases. For lung cancer patients, sex was not considered a biological variable. Serum samples from healthy donors were obtained without regard to gender.

[0296] Isolation of plasma and PBMCs from whole blood samples

[0297] Plasma was isolated from a 5 mL blood sample that was centrifuged at 2000 g for 10 minutes. The upper layer, containing the plasma, was carefully collected, aliquoted, and subsequently stored at -20°C. For PBMC isolation, whole blood was collected into K2EDTA tubes (BD Vacutainer, Becton, Dickinson and Company) and subsequently diluted threefold with RPMI 1640 medium (Gibco). This mixture was then carefully layered onto Ficoll-Paque PLUS (Cytiva) in a 1:1 ratio for phase separation, following the manufacturer’s guidelines. The lymphocyte-rich buffy coat layer was collected into a new tube, washed, and resuspended in fetal bovine serum (FBS) supplemented with 10% DMSO, then cryopreserved in liquid nitrogen.

[0298] Expression of MET and SEMA

[0299] A plasmid encoding the human MET was obtained. Sequences corresponding to the extracellular portion of MET and the ligand-binding domain, SEMA, were amplified using specific primers. These sequences were cloned into pcDNA3.1 expression vectors. Each vector was designed with an N-terminal IgK signal peptide sequence (METDTLLLWVLLLWVPGSTGD SEQ ID NO: 92) and incorporated two C-terminal tags for processing: a hexa-histidine sequence (HHHHHH, "His-tag", SEQ ID NO: 93) for protein purification, and a site-specific biotinylation sequence (GLNDIFEAQKIEWHE SEQ ID NO: 94, "Avi-Tag"). These constructs were used for transient transfection of Expi293F™ cells by using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific Inc). Seven days after transfection, the supernatant from the cultured cells was collected, passed through a 0.22 pm fdter, and incubated with Ni2+-NTA agarose beads (Cytvia) for 2 hours at ambient temperature. Proteins were eluted with 250 mM imidazole, buffer-exchanged to PBS xl, aliquoted, and preserved at -80°C. Biotinylation of the EC-MET protein was performed using theBirA biotin-protein ligase kit (Avidity LLC, Colorado, USA), in accordance with the manufacturer’s protocol.

[0300] ELISA

[0301] For serological screening by ELISA, high-binding 96- well ELISA plates (Coming #9018) were prepared by coating with 5 pg / mL of MET, SEMA, or gpl20 in PBS xl and overnight incubation at 4°C. The next day, residual coating solution was discarded, and the wells were rinsed with a washing buffer composed of PBS xl and 0.05% Tween 20 (Sigma). Subsequent blocking was performed at room temperature for 2 h using 200 pL of blocking buffer containing PBS xl, 3% BSA (MP Biomedicals), 20 mM EDTA, and 0.05% Tween 20. Plasma samples, diluted 1:50 in blocking buffer, or isolated IgG samples, diluted to 30 pg / mL in blocking buffer, were added to the plates and incubated for 1 h at room temperature. All dilutions were chosen after tittering experiments. After incubation, the plates underwent three wash cycles with the washing buffer. Secondary anti-IgG antibodies conjugated to horseradish peroxidase (HRP), sourced from Jackson ImmunoResearch (109-035-088) and diluted 1:5000 in blocking buffer, were then applied and allowed to incubate for 45 minutes at room temperature. Following an additional four washes, each well received 100 pL of TMB / E substrate (Millipore) and the absorbance was measured at 650 nm after 20 minutes using a BioTek 800 TS absorbance reader.

[0302] For mAb ELISA, high-binding 96-well ELISA plates were prepared by coating with 1 pg / mL of MET, SEMA, or gpl20, following the same washing and blocking procedures as described above. MAbs were initially added at a concentration of 64 pg / mL, followed by four successive four-fold dilutions in PBS xl, and incubation for 1 h at room temperature. The plates were then washed three times with washing buffer before addition of the secondary anti-IgG HRP-conjugated antibodies, as already described. After four additional washes, 100 pL of TMBZE substrate was applied to each well. The absorbance was measured at 650 nm after 20 minutes.

[0303] For competition ELISA, high-binding 96-well ELISA plates were prepared by coating with 1 pg / mL of MET, following the same washing and blocking procedures as described above. HGF was initially added at the concentration of 100, 1.56, 0.02 and 0 pg / mL, and incubation for 1 h at room temperature. The plates were then washed three times with washing buffer before addition of 15 pg of primary mAb for 10 minutes at room temperature, as described in the Figures Legend. The plates were washed three times with washing buffer before adding the secondary anti-IgG HRP-conjugated antibodies, as detailed earlier. After four additional washes, 100 pL of TMBZE substrate was applied to each well. The absorbance was measured at 650 nm after 20 minutes.

[0304] All ELIS As were replicated at least 3 times.c-MET (SP44) immunostaining

[0305] FFPE blocks were sectioned at 4 m and a positive control was added on the right edge of the slides. c-MET (SP44) (790-4430, Roche Tissue Diagnostics, USA) immunostaining was calibrated on a Benchmark Ultra staining module (Roche Tissue Diagnostics, USA). Slides were warmed up to 60 0 C for Ihour and were processed by a fully automated protocol. Briefly, after dewaxed and rehydrated, sections were exposed to CC1 HIER pretreatment (Roche Tissue Diagnostics, USA) for 64 minutes. c-MET (SP44) antibody (prediluted) was incubated at 370 C for 52 minutes. Detection was performed with UltraView detection kit (760-500, Roche Tissue Diagnostics, USA) and amplification with Amplification Kit (760-080, Roche Tissue Diagnostics, USA). Sections were counterstained with hematoxylin (790-2208, Roche Tissue Diagnostics, USA). At the end of the automated run, slides were dehydrated in graded ethanols (70%, 96%, and 100%), cleared in Xylene and film coverslipped. Stained sections were scanned and analyzed by a pathologist.

[0306] Flow-cytometry and direct single B cell sorting

[0307] PBMCs were quickly thawed at 37°C and subsequently washed in 50 mL of RPMI 1640 medium. These cells were resuspended in FACS buffer that included 1% BSA, and 2 mM EDTA in PBS xl, and were stained with anti-CD19-VioBlue (Miltenyi Biotec, 130-120-031), anti-IgG-FITC (Miltenyi Biotec, 130-118-340). All samples were then analyzed using a CytoFLEX-S4 flow cytometer (Beckman Coulter).

[0308] For single B cell sorting, B cells were enriched with anti-CD19 magnetic beads (Miltenyi Biotec, 130-050-301) according to the manufacturer's guidelines. These cells were resuspended in FACS buffer that included 1% BSA, and 2 mM EDTA in PBS xl, and were stained with anti-CD19-VioBlue (Miltenyi Biotec, 130-120-031), anti-IgG-FITC (Miltenyi Biotec, 130-118-340), and labeled MET via streptavidin-PE (Miltenyi Biotec, 130-106-790) and streptavidin- APC (Miltenyi Biotec, 130-106-792). Single CD19+IgG+MET+ cells were sorted to 96 well plates using a FACSAria III sorter (Becton Dickinson) as previously described(35, 49, 50).

[0309] Stimulation of B cells and indirect single B cell sorting

[0310] PBMCs were quickly thawed at 37°C and subsequently washed in 50 mL of RPMI 1640 medium. B cells were then isolated using anti-CD19 magnetic beads (Miltenyi Biotec, 130-050-301) according to the manufacturer's guidelines. B cells were cultured in complete RPMI medium supplemented with penicillin-streptomycin (Biowest) and 10 ng / mL human IL- 15 (PeproTech) in a 96-well plate (Coming). Post-isolation, streptavidin microspheres (Bangs Laboratories) coated with biotinylated CpG ODN 2006 (InvivoGen) and biotinylated MET were introduced to the B cells. Three days after stimulation, the culture was supplemented with 50 ng / mL human IL-6 (PeproTech). After threeadditional days, B cells were harvested and stained with anti-CD27-FITC (Miltenyi Biotec, 30-113-629) and anti-CD38-APC (Miltenyi Biotec, 130-113-429). Single CD27hlCD38hlcells were sorted to 96 well plates using a FACSAria III sorter (Becton Dickinson) as previously described(32).

[0311] Single B cell BCR sequencing

[0312] Single B cells were sorted into a 96-well plate containing 4 pL of lysis buffer, which comprised 12 units of RNasin Ribonuclease Inhibitor (Promega, N2511), 10 mM DTT, and PBS x0.5. Plates were immediately stored on dry ice prior to RNA reverse transcription and PCR amplification. For amplification, the lysed cells were thawed on ice. Ig genes were amplified as previously described (31, 35). cDNA synthesis was conducted with random hexamer primers (Invitrogen, 48190011) and SuperScript III Reverse Transcriptase (Invitrogen, 18080085). Antibody sequences for the gamma, kappa, and lambda chains were subsequently amplified through nested PCR, as previously described(35). The second round PCR products were then purified, sequenced, and analyzed using IgBLAST(51).

[0313] MAbs cloning and production

[0314] Antibodies were cloned and produced as previously described(35). Briefly, the V(D)J regions of the antibodies were amplified from the first round PCR products of sorted B cells and subsequently cloned into Ig expression vectors obtained from the Nussenzweig Lab(10, 52). For unmutated germline versions cloning compatible gBlocks were designed and ordered the synthesis from Azenta. Cloned mAb vectors for both heavy and light chains were co-transfected into Expi293F™ cells (Thermo Fisher Scientific Inc Seven days post-transfection, the cell supernatant was incubated with protein A-coated agarose beads (GE Life Sciences, 17519901). After several washing steps, antibodies were eluted from chromatography columns using 50 mM sodium phosphate (pH 3.0). Finally, antibodies were buffer exchanged into lx PBS, aliquoted, and stored at -80°C.

[0315] Immunofluorescence assay

[0316] Breast carcinoma tissue sections, along with matched breast tissue slides (BR251f, TissueArray) or slides with Breast carcinoma tissue sections and healthy normal tissues (BRI 191, TissueArray) were initially baked for 60 minutes at 60°C. Following baking, slides underwent deparaffinization and antigen retrieval in Tris-EDTA buffer (pH 9) for 15 minutes. Subsequently, slides were blocked using 1% bovine serum albumin for 120 minutes and then stained overnight at 4°C with biotinylated antibodies (69B287, 87B156, mGO.53). The slides were then incubated with phalloidin conjugated to FITC (1:300, Sigma) and Streptavidin conjugated to AF647 (1: 100, Miltenyi Biotec) for 2 hours at 4°C. Finally, slides were stained with DAPI (1:1000) and sealed using nonfluor mounting media (Prolong Gold Antifade, Invitrogen). Imaging was conducted using a Leica TCS SP8 Confocal Microscope (Leica Microsystems).Cell lines

[0317] The CAL-51 cell line was cultured in Coming Dulbecco’s Modified Eagle Media (DMEM), supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin (Biowest), 1% glutamine (Biowest) and 1% sodium pyruvate (Sartorius). The MCF-7 cell line was cultured in DMEM, supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin (Biowest), 1% glutamine (Biowest) and 1% sodium pyruvate (Sartorius). The MDA-MB-468 and HCC70 cell lines were cultured in RPMI, supplemented with 10% FBS (Gibco), 1% penicillin / streptomycin (Biowest), 1% glutamine (Biowest) and 1% sodium pyruvate (Sartorius). Cell lines were continuously maintained in an incubator set at 37°C with 5% CO2 atmosphere and were regularly tested for mycoplasma contamination.

[0318] Cancer cell lines flow-cytometry for mAb binding

[0319] Cultured cells were harvested using Cell Dissociation Solution (Sartorius, 03-071-1B) and centrifuged at 400 g for 5 minutes at 4°C. The resulting cell pellets were resuspended in FACS buffer and incubated for 30 minutes on ice with mAbs. Following a washing step, the cells were stained with anti-human IgG conjugated to APC (Miltenyi Biotec, 130-119-772). All samples were then analyzed using a CytoFEEX-S4 flow cytometer (Beckman Coulter).

[0320] Live cell imaging

[0321] 2xl04of CAL-51, MDA-MB-468 or HCC70 cells were seeded in a 96-well plate (Coming) together with 500nM of each mAb (87B156, 69B287, and mGO.53). The plates were then immediately placed in an Incucyte SX5 (Sartorius), and images were captured at 30 minutes intervals. Data were analyzed using Incucyte analysis software and subsequently exported to GraphPad Prism software for further analysis.

[0322] Live cell imaging was replicated 3 times.

[0323] For chemotactic cell migration and invasion experiment, 5000 of CAL-51 cells were seeded in the upper chamber of each well in a 96-well chemotaxis microplate and treated with 100 pM of monoclonal antibodies (as described in the Figures Legend). Epidermal growth factor (EGF, 1 pg / mL) was added to the lower chamber of all wells as a chemoattractant. Cell migration was monitored using live imaging, and the number of migrated CAL-51 cells was quantified.

[0324] Western blot

[0325] 2xl04CAL-51 cells were seeded in a 96-well plate (Corning) and incubated with 500 nM of each mAb (87B156 and 69B287) for varying durations. The cells were then lysed in radioimmunoprecipitation assay buffer (150 mM sodium chloride, 1% triton X-100, 0.5% sodium deoxycholate, 0.1 % sodium dodecyl sulfate, 50 mM Tris, pH 8.0, protease inhibitors, and phosphatase inhibitors) and incubated on ice for 20 minutes with vortex every 5 minutes. Lysateswere then centrifuged for 15 minutes at 20,000 g at 4 °C. Determination of protein concentration were made using Pierce™ BCA Protein Assay (Thermo Fisher). lOpg from each lysate were boiled with SDS reducing sample buffer for 5 minutes, separated by SDS-PAGE (Bio-Rad), and transferred to trans-blot turbo nitrocellulose membrane (Bio-Rad). Following transfer, blots were blocked with 3 % BSA in PBS XI for 1 hour at room temperature and incubated with specific primary antibodies overnight at 4 °C with gentle agitation. The following antibodies were used for biochemical studies: anti-ERKl + ERK2 antibody (abeam), anti-PERK antibody (abeam). HRP-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch) and ECL Reagent (Bio-Rad) were used for detection.

[0326] Statistics

[0327] Statistical significance was determined using a p-value threshold of less than 0.05. Analysis was conducted using GraphPad Prism 10 software and the rstatix package in R. All statistical tests applied were nonparametric, Mann- Whitney test for comparisons between two groups, the Kruskal-Wallis test for comparisons involving more than two groups, and the Spearman test for correlation analyses. Unless indicated otherwise, corrections for multiple comparisons were performed with the False Discovery Rate (FDR) method.

[0328] Example 1

[0329] Antibodies binding MET are detected in a subset of breast and lung cancer patients The underlying question in the study was whether cancer patients develop antibodies binding the oncoreceptor MET, and if so, to characterize this response at the serological and B cell levels. To this end, sera were collected from 100 diagnosed cancer patients (75 with breast cancer and 25 with lung cancer, Table 2). Most of the patients had metastatic disease (49 breast cancer and 17 lung cancer), 26 breast cancer patients had early-stage disease and were receiving neoadjuvant therapy, and 8 non-metastatic lung cancer patients were undergoing chemo-radiation therapy (summarized in Table 1, below).

[0330] Table 2

[0331]

[0332]

[0333] MET is recognized as a tumor antigen in both breast and lung cancers (28, 29), and its expression correlates with poor prognosis (28, 30). The present inventors first tested whether the cohort exhibits serum antibodies that are capable of binding MET. Patient sera were screened, by ELISA, against the recombinantly expressed 908-amino acid extracellular portion of MET, and the 536-amino acid ligand-binding domain of MET, known as SEMA (Figure 1). HIV-1 gpl20 antigen, produced in the same system and carrying the same tags as MET and SEMA, was used as a control antigen (all donors were HIV-1 negative). Patients bound MET and SEMA, with a significant correlation between serum binding to the two molecules (Figure 2A, r = 0.5005, p = 0.0344, Figure 3A), while there was no correlation between binding to MET and binding to gpl20 (r = 0.09411, p = 0.7103, Figure 2A). Compared to healthy donors (n = 51), patients showed significantly higher binding to MET and SEMA (p = 0.0094 and p < 0.0001, respectively), while binding to gpl20 was similar (Figure 3B). To assess overall MET response, the mean O.D. values were calculated for MET and SEMA binding. This analysis also showed significantly higher binding in patients compared to healthy donors (p = 0.0033, Figure 2B). MET response was defined as any score above the 95thpercentile of the healthy donor cohort scores. According to this threshold, 13 cancer patients exhibited serum binding to MET, 8 breast cancer patients (10.6 %) and 5 lung cancer patients (20 %) (Figure 2A, indicated by red asterisks). For 4 of the 13 patients with serological MET responses (Pl, P45, P76, and P72), tumor tissue sections were available, along with 3 additional sections from patients without serum MET binding. Surprisingly, in this small number of samples, no correlation was observed between MET expression and MET serum responses (Figure 3C).

[0334] The study cohort included both metastatic and non-metastatic breast and lung cancer donors, with a limited number for each subtype (Figure 2C). This heterogeneity limited the ability to identify specific patient features associated with anti-MET response. Therefore next steps focused on breast cancer patients (n=75), where 8 patients with anti-MET response were identified. Interestingly, all 8 breast cancer patients responding to MET were hormone receptor-positive (HR+), and HR+ status was significantly associated with anti-MET response (p = 0.0448, Figures 2C, D). Moreover, when focusing on the 32 metastatic breast cancer patients who are HR+, and for whom survival data was available, a trend of improved survival within the MET binding patients was identified, although itwas not statistically significant (p=0.28, Supplementary Figure 2D). For lung cancer patients, the presence of EGFR mutations was assessed. No clear association with anti-MET responses was found, as only two patients in the cohort had EGFR mutations, one of whom exhibited a response to MET.

[0335] Example 2

[0336] Peripheral MET B cell repertoire is polyclonal with less IgGl

[0337] To gain more insights about MET-targeting B cells, the MET reactive B cell repertoire was analyzed by sequencing B cells enriched for MET binding. To confirm serum MET activity, IgG was purified from the serum, and the ELISA was repeated with quantified IgG. Six cancer donors with the highest IgG binding to MET: Pl, P23, P45, P69, P87, and P92 were taken for further analysis (Figure 3E, shaded area, P72 was omitted due to limited sample availability). For these donors, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and MET-binding B cells were sorted using fluorescently tagged MET baits (31). Approximately 0.2 % of the IgG+CD19+ B cells were positive to MET, and these were single cell sorted (Figure 4A). In accordance with previous results (8), cancer patients in the study exhibited low frequencies of IgG+ B cells, with an average of 5.56 % (SD = 2.26) from total CD 19+ cells (Figure 5), resulting in a low number of MET-binding B cells. To increase the number of MET-binding B cells, a parallel approach was taken, where prior to sorting, B cells were cultured with recombinant MET, resulting in activation and expansion of MET reactive B cells through their antibodies (identified by CD38hland CD27hl). This technique was previously demonstrated to activate and increase the frequency of rare, antigenspecific, B cells, without inducing SHM or class switching (32, 33). These cells were then single cell sorted (Figure 4B). Using both strategies, 995 B cells were collected, resulting in 325 heavy chain and 430 light chain sequences, as detailed in Table 3, below.

[0338] Table 3: Detailed Patient characteristics

[0339]

[0340] Analysis of the immunoglobulin sequences of MET-enriched B cells revealed a diverse repertoire of VH and VL genes (Figure 4C). VH3 was the most frequent VH gene across all patients. No VHJH combinations were shared across all patients (not shown). Kappa light chain was more frequent than Lambda in 5 out of 6 studied patients (Figure 4C).MET-enriched B cells exhibited 67.5 % IgGl compared to 91.8 % in RBD-enriched B cells from donors without cancer (p = 0.0345, Figure 4E). To rule out any bias that could have been introduced by the culturing technique, the frequency of IgGl in MET B cells recovered from both methods was compared (direct sorting versus culturing prior to sorting) and no differences were found, indicating that the dominance of non-IgGl is not protocol related (not shown).

[0341] Example 3

[0342] Patient-derived monoclonal antibodies bind tumor cells

[0343] The properties of MET-binding antibodies were investigated by expressing representative monoclonal antibodies (mAbs). Nine mAbs 69B287, 87B156, 69B253, 1B277, 1B233, 23B307, 92L236, 92L205, and 92L204 demonstrated a detectable binding to MET and SEMA by ELISA (Figure 6A). mAbs 69B287 and 87B156, which exhibited the highest binding among the nine tested mAbs, competed with HGF for MET binding (Figure 6B). These mAbs also bound to human breast cancer tissue array. The antibody staining co-localized to the cell membrane and showed significantly lower binding to the adjacent healthy tissue and significantly higher binding compared to the isotype control antibody (Figure 6C-E).

[0344] Example 4

[0345] MET-binding antibodies exhibit an inhibitory effect on tumor cells in culture

[0346] The present inventors next aimed to assess the impact of the mAbs on three human MET-expressing breast cancer cell lines: CAL-51, MDA-MB-468, and HCC70 (38, 39) (Figure 7A). The cells were incubated with either 69B287 or 87B 156 and cell confluence was monitored using live cell imaging for 60 hours. All three cell lines grew slower in the presence of 69B287 or 87B 156, an effect that was not observed when the cells were cultured with the isotype control (Figure 7B and 7E). The present inventors next assessed the impact of 69B287 and 87B 156 on cell invasion using a chemotaxis assay (Figure 7C). Notably, treatment with 69B287 or 87B156 significantly inhibited the ability of CAL-51 cells to migrate across membrane, as opposed to isotype control mAb that had no effect. Moreover, mAb 87B156 disrupted downstream MET signaling, as assessed by reduced pERK phosphorylation (Figure 7D). These results suggest that human polyreactive antibodies developed in cancer patients, and that react with MET exhibit anti-cancer effects.

[0347] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.References

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Claims

WHAT IS CLAIMED IS:

1. An antibody comprising an antigen binding domain which binds an antigenic determinant of MET for use in preventing or treating cancer in a subject in need thereof, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

2. A method of preventing or treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236, thereby preventing or treating cancer in the subject.

3. A method of monitoring treatment of cancer in a subject in need thereof, the method comprising:(a) treating the subject with an anti-cancer treatment, wherein said cancer is characterized by MET expression;(b) determining a level of said MET prior to and following said treating using an antibody, wherein a reduction in MET is indicative of an efficacious treatment, wherein at least one of said anticancer treatment and said antibody, comprises an antibody which comprises an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

4. A method of diagnosing cancer in a subject in need thereof, the method comprising: (a) contacting cells of the subject with an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 andCDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236;(b) detecting an immunocomplex formation between said antibody and said cells, wherein presence of said immunocomplex is indicative of cancer.

5. A method of treating cancer in a subject in need thereof, the method comprising: (a) diagnosing cancer according to the method of claim 4;(b) treating said cancer with an anti-cancer treatment, optionally wherein said anti-cancer treatment comprises said antibody.

6. A method of selecting treatment to cancer in a subject diagnosed with cancer, the method comprising:(a) contacting cells of the subject with an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236;(b) detecting an immunocomplex formation between said antibody and said cells, wherein presence of said immunocomplex is indicative of cancer cells which express MET; and(c) selecting a treatment which is efficacious for MET expressing cancer, wherein said treatment is optionally with said antibody.

7. A method of producing an antibody capable of binding an antigenic determinant of MET, the method comprising:(a) expressing in a host cell a heterologous polynucleotide encoding an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236; and optionally(b) recovering the antibody from the host cell.

8. A vaccine comprising an effective amount of an antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

9. A monoclonal antibody comprising an antigen binding domain which binds an antigenic determinant of MET, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

10. An antibody comprising an antigen binding domain which binds an antigenic determinant of MET attached to a heterologous effector moiety or carrier, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain at least 80 % identical to those of an antibody selected from the group consisting of 69B287, 87B156, 1B217, 1B233, 23B307, 69B253, 2L204, 92L205 and 92L236.

11. The antibody, method or vaccine of any one of claims 1-10, wherein said antibody is a recombinant antibody.

12. The antibody, method or vaccine of any one of claims 1-11, wherein said at least 80 is at least 85 %.

13. The antibody, method or vaccine of any one of claims 1-11, wherein said at least 80 is at least 90 %.

14. The antibody, method or vaccine of any one of claims 1-11, wherein said at least 80 is at least 95 %.

15. The antibody, method or vaccine of any one of claims 1-11, wherein said at least 80 is 100 %.

16. The antibody, method or vaccine of any one of claims 1-15, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 69B287 or 87B156.

17. The antibody, method or vaccine of any one of claims 1-15, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 69B287.

18. The antibody, method or vaccine of any one of claims 1-15, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 87B156.

19. The antibody, method or vaccine of any one of claims 1-18, wherein said antibody inhibits cancer cells growth.

20. The antibody, method or vaccine of any one of claims 1-19, wherein said antibody binds semaphorin (SEMA) domain of MET.

21. The antibody, method or vaccine of any one of claims 1-20, wherein said antibody competes with hepatocyte growth factor (HGF) binding to MET, as determined by ELISA.

22. The antibody, method or vaccine of any one of claims 1-20, wherein said antibody preferentially binds a cancer tissue over a healthy tissue as determined by immunofluorescence.

23. The antibody, method or vaccine of any one of claims 1-22, wherein said cancer is selected from the group consisting of breast cancer, lung cancer, gastric cancer and hepatocellular cancer.

24. The antibody, method or vaccine of any one of claims 1-20, wherein said cancer is hormone receptor positive (HR+) breast cancer.

25. The antibody, method or vaccine of any one of claims 1-23, wherein said antibody comprises an antibody fragment.

26. The antibody, method or vaccine of any one of claims 1-23, wherein said antibody fragment comprises Fab and / or scFv.

27. The antibody, method or vaccine of any one of claims 1-23, wherein said antibody is a chimeric antibody.

28. The antibody, method or vaccine of any one of claims 1-23 and 25, wherein said antibody is a human antibody.

29. The antibody, method or vaccine of any one of claims 3-7 and 9-28, wherein said antibody is labeled.

30. The method of any one of claims 4, 6 and 11-29, being effected in-vivo.

31. The method of any one of claims 4, 6 and 11-29, being effected ex- vivo.

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