Anti-MET antibodies, antibody-drug conjugates, compositions, and their use

Abstract

The present invention relates to a novel recombinant antibody or antigen-binding site that targets MET, an ADC containing the antibody or antigen-binding site, a composition containing the antibody or antigen-binding site, a composition containing the anti-MET antibody or ADC containing the anti-MET antibody or antigen-binding site, and the use of the antibody or antigen-binding site, the ADC, or the composition containing the anti-MET antibody or anti-MET ADC.

Description

[Background technology]

[0001] Background of the Invention Mesenchymal-epithelial conversion factor (MET or cMET) is a receptor tyrosine kinase containing a 50 kDa α-subunit and a 145 kDa β-subunit. The only known ligand for MET is hepatocyte growth factor (HGF), also known as cell dispersal factor. Binding of MET by hepatocyte growth factor leads to receptor dimerization and autophosphorylation of residues Y1349 and Y1356 of the β-subunit, activating downstream signaling pathways, including the phosphoinositol 3-kinase (PI3K)-protein kinase B (Akt) pathway, the signaling and transcriptional activator (STAT) pathway, the mitogenic factor-activated protein kinase (MAPK) pathway, and the activated B cell nuclear factor kappa light chain enhancer (NFκB) pathway. Ultimately, this increases mitosis, cell proliferation, cell survival, and cell motility. Deregulation of MET or hepatocyte growth factor activity can occur, for example, through MET overexpression, gene amplification, mutation, or alternative splicing, or through hepatocyte growth factor ligand-induced autocrine / paracrine loop signaling. Such deregulation plays a role in many cancers by promoting cancer invasiveness, angiogenesis, metastasis, and tumor growth, leading to a more aggressive cancer phenotype and a worse prognosis.

[0002] MET is also known to interact with signaling pathways involving other receptors such as EGFR (epidermal growth factor receptor), TGF (tumor growth factor)-β, and HER3, and may play a role in resistance to treatments targeting these receptors. Therefore, MET inhibitors, such as anti-MET antibodies, may be effective in overcoming resistance phenotypes when combined with other receptor inhibitors.

[0003] Current MET inhibitors include both monoclonal antibodies that can target either MET or its ligand, hepatocyte growth factor, and small molecule kinase inhibitors. Known anti-MET small molecule receptor tyrosine kinase inhibitors include tivantinib, cabozantinib, foretinib, golvatinib, and crizotinib. However, there are no anti-cMET antibodies approved for therapeutic use. Known antibodies that target the cMET pathway include onartuzumab (Genentech, International Publication No. 2006 / 015371), ARGX-111 (Argenix, International Publication No. 2012 / 059561), emibetuzumab (LY2875358; Eli Lilly, International Publication No. 2010 / 059654), SAIT-301 (Samsung, U.S. Patent Application Publication No. 2014 / 0154251), terisotuzumab (ABT-700, Abbott / AbbVie, International Publication No. 2017 / 201204), and Sym015 (Symphogen, International Publication No. 2016 / 042412). MET-targeted bispecific antibodies, such as amivantamab (JNJ-61186372, Janssen Biotech, U.S. Patent No. 9593164), a bispecific antibody that targets both EGFR and MET, are also described.

[0004] Given its role in cancer biology and its overexpression in several types of cancer, the MET receptor is an effective target for cancer treatment and an attractive target for the development of anti-MET therapeutic antibodies and antibody-drug conjugates. [Overview of the project]

[0005] Summary of the Invention This invention relates to melanoma, uveal melanoma, kidney cancer, thyroid cancer, mesothelioma, liver hepatocellular carcinoma, lung cancer (including non-small cell lung cancer and small cell lung cancer), and gastric cancer. This includes cancers such as pancreatic cancer, colorectal cancer, esophageal cancer, cholangiocarcinoma, head and neck cancer (including oral cancer), cervical and intracervical cancer, bladder and urothelial cancer, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, adrenal cortical cancer, brain cancer, spleen cancer, kidney cancer, thymoma, multiple myeloma, plasmacytic myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancies originating from T cells or B cells, hematological cancers, myeloid leukemia, or myeloma. This invention relates to novel recombinant antibodies or their antigen-binding moieties that target MET, as well as antibody-drug conjugates (ADCs) containing the antibody or its antigen-binding moiety, compositions containing the antibody or its antigen-binding moiety, compositions containing the antibody-drug conjugate (ADC) containing the antibody or its antigen-binding moiety, the use of the antibody or its antigen-binding moiety or the antibody-drug conjugate (ADC), and the use of compositions containing the anti-MET antibody or its antigen-binding moiety or the anti-MET antibody-drug conjugate (ADC). Compared to currently available treatments for such cancers, including treatment with antibodies or antibody-drug conjugates, the antibodies of the present invention, antibody-drug conjugates (ADCs) containing the antibodies of the present invention, and compositions containing the antibodies or antibody-drug conjugates of the present invention are considered to be remarkably effective against cancer cells.

[0006] In one embodiment, the present invention provides an anti-MET antibody or its antigen-binding moiety. In some embodiments, the antibody or its antigen-binding moiety competes for binding to human MET with an antibody in which its H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 contain or comprise the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. In some embodiments, the anti-MET antibody or its antigen-binding moiety binds to the same human MET epitope as an antibody in which its H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 contain or comprise the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0007] In one embodiment, the present invention provides an anti-MET antibody or its antigen-binding moiety comprising H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the anti-MET antibody or its antigen-binding moiety comprises a heavy chain variable domain (VH) whose sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-MET antibody or its antigen-binding moiety comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 7. In some embodiments, the anti-MET antibody or its antigen-binding moiety comprises a heavy chain (HC) comprising or consisting of the amino acid sequence of SEQ ID NO: 11 or 13.

[0008] In some embodiments, the anti-MET antibody or its antigen-binding moiety includes L-CDR1, L-CDR2, and L-CDR3, which contain or consist of the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In some embodiments, the anti-MET antibody or its antigen-binding moiety includes a light chain variable domain (VL) whose sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-MET antibody or its antigen-binding moiety includes a VL containing or consisting of the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-MET antibody or its antigen-binding moiety includes a light chain (LC) containing or consisting of the amino acid sequence of SEQ ID NO: 12 or 14.

[0009] In some embodiments, the anti-MET antibody or its antigen-binding moiety includes H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. In some embodiments, the anti-MET antibody or its antigen-binding moiety includes VH, whose sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, and VL, whose sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-MET antibody or its antigen-binding moiety includes VH, which contains or comprises the amino acid sequence of SEQ ID NO: 7, and VL, which contains or comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-MET antibody or its antigen-binding moiety includes HC, which contains or comprises the amino acid sequence of SEQ ID NO: 11 or 13, and LC, which contains or comprises the amino acid sequence of SEQ ID NO: 12 or 14. In some embodiments of the antibody or its antigen-binding moiety, the anti-MET antibody or its antigen-binding moiety comprises HC and LC containing or comprising the amino acid sequences of SEQ ID NOs. 11 and 12, respectively, or HC and LC containing or comprising the amino acid sequences of SEQ ID NOs. 13 and 14, respectively.

[0010] In some embodiments of the antibody or its antigen-binding moiety described herein, the antibody may be of isotype IgG. In certain embodiments, the antibody is of isotype subclass IgG1. In certain embodiments, the anti-MET antibody is of isotype subclass IgG2.

[0011] The present invention also provides a multispecific (e.g., bispecific) binding molecule comprising the antigen-binding moiety of an anti-MET antibody described herein and the antigen-binding moiety of another distinctly different antibody, such as another anti-MET antibody or an antibody that targets a different protein. In certain embodiments, the bispecific binding molecule comprises the antigen-binding moiety of an antibody, wherein its H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 comprise the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0012] In some embodiments, this disclosure provides novel antibody-drug conjugate (ADC) compounds comprising the anti-MET antibody of the present invention. ADC compounds may exhibit biological activity against cancer cells, slowing, inhibiting, and / or reversing tumor growth in mammals, and / or may be useful in treating human cancer patients. More specifically, this disclosure relates, in some embodiments, to ADC compounds capable of binding to and killing cancer cells. In some embodiments, the ADC compounds may also be capable of internalizing into target cells after binding.

[0013] In some embodiments, the ADC may be represented by Ab-(LD)p, where Ab is an anti-Met antibody or its antigen-binding portion; D is a payload or drug or any compound to be linked to Ab using a linker L; L is a linker that covalently attaches Ab to D; and p is an integer from 1 to 16.

[0014] In certain embodiments, the ADC includes an anti-Met antibody or its antigen-binding moiety, wherein H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 each contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

[0015] As used herein, "LD" refers to a linker-drug, linker-payload, or linker-compound. In some embodiments, p is an integer from 1 to 16. The linker (L) may be a cleavable linker or an incleavable linker. D refers to the drug portion or payload and is selected from Eg5 inhibitors, type V ATPase inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatins (such as monomethyl auristatin E, i.e., MMAE), dorastatins, meitansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear export of protein CRM1, DPPIV inhibitors, inhibitors of mitochondrial phosphate transfer reactions, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, proteasome inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, RNA polymerase inhibitors, amanitin, spliceosome inhibitors, topoisomerase inhibitors, DHFR inhibitors, and apoptosis promoters.

[0016] The present invention also provides antibody compositions comprising an anti-MET antibody or its antigen-binding moiety as described herein. For example, the antibody composition comprises an antibody in which H-CDR1, H-CDR2, H-CDR3, and L-CDR1, L-CDR2, and L-CDR3 each contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, and an anti-MET antibody or its antigen-binding moiety that competes for binding to human MET. In some embodiments, the antibody composition comprises an anti-MET antibody or its antigen-binding moiety that binds to the same human MET epitope as the antibody in which H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3 each contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

[0017] In some embodiments, the antibody composition includes an anti-MET antibody or its antigen-binding moiety, comprising H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each comprising the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively. In some embodiments, the antibody composition includes an anti-MET antibody or its antigen-binding moiety, comprising VH and VL, each having at least 90% identical sequences to the amino acid sequences of SEQ ID NOs: 7 and 8, respectively. In some embodiments, the antibody composition includes an anti-MET antibody or its antigen-binding moiety, comprising VH and VL, each comprising the amino acid sequences of SEQ ID NOs: 7 and 8, respectively. In some embodiments, the antibody composition includes an anti-MET antibody or its antigen-binding moiety, comprising HC and LC, each comprising the amino acid sequences of SEQ ID NOs: 11 and 12, respectively, or HC and LC, each comprising the amino acid sequences of SEQ ID NOs: 13 and 14, respectively. In some embodiments, the antibody composition comprises an anti-MET antibody or its antigen-binding moiety, comprising HC and LC containing the amino acid sequences of SEQ ID NOs. 11 and 12, respectively, or HC and LC containing the amino acid sequences of SEQ ID NOs. 13 and 14, respectively.

[0018] The present invention also provides an ADC composition comprising an anti-MET antibody or an antigen-binding portion thereof as described herein. For example, the ADC composition comprises an ADC in which the anti-MET antibody or an antigen-binding portion thereof comprises H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each comprising the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6. In some embodiments, the ADC composition comprises an ADC comprising an anti-MET antibody or an antigen-binding portion thereof, comprising VH and VL that are at least 90% identical in sequence to the amino acid sequences of SEQ ID NOs: 7 and 8, respectively. In some embodiments, the ADC composition comprises an ADC comprising an anti-MET antibody or an antigen-binding portion thereof, comprising VH and VL that comprise the amino acid sequences of SEQ ID NOs: 7 and 8, respectively. In some embodiments, the ADC composition comprises an ADC comprising an anti-MET antibody or an antigen-binding portion thereof, comprising HC and LC that are at least 90% identical in sequence to the amino acid sequences of SEQ ID NOs: 11 and 12, respectively, or HC and LC that are at least 90% identical in sequence to the amino acid sequences of SEQ ID NOs: 13 and 14, respectively. In some embodiments, the ADC composition comprises an ADC comprising an anti-MET antibody or an antigen-binding portion thereof, comprising HC and LC that comprise the amino acid sequences of SEQ ID NOs: 11 and 12, respectively, or HC and LC that comprise the amino acid sequences of SEQ ID NOs: 13 and 14, respectively.

[0019] The present invention also provides a pharmaceutical composition comprising the anti-MET antibody composition as described herein or the ADC composition as described herein and a pharmaceutically acceptable excipient.

[0020] The present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain of the anti-MET antibody as described herein or an antigen-binding portion thereof, a nucleotide sequence encoding the light chain thereof or an antigen-binding portion thereof, or both. In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 or 10.

[0021] The present invention also provides a vector comprising the isolated nucleic acid molecule, wherein the vector further comprises an expression control sequence.

[0022] The present invention also provides a host cell comprising a nucleotide sequence encoding the heavy chain or its antigen-binding portion of the anti-MET antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both. In some embodiments, the host cell comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9 or 10.

[0023] The present invention also provides a non-human transgenic animal or plant comprising a nucleotide sequence encoding the heavy chain or its antigen-binding portion of an anti-MET antibody described herein, a nucleotide sequence encoding the light chain or its antigen-binding portion, or both, wherein the animal or plant expresses the nucleotide sequence(s). In some embodiments, the animal or plant comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 9 or 10.

[0024] The present invention also provides a method for producing the anti-MET antibody or its antigen-binding moiety described herein, comprising providing the host cells described above, culturing the host cells under conditions suitable for the expression of the antibody or moiety, and isolating the obtained antibody or moiety. A method for producing an ADC compound comprising the anti-MET antibody of the present invention is also disclosed.

[0025] The present invention also provides a method for treating a patient with MET-mediated disorder, comprising administering to the patient an anti-MET antibody or its antigen-binding moiety as described herein, an ADC containing an anti-MET antibody or its antigen-binding moiety as described herein, an antibody composition containing an anti-MET antibody or its antigen-binding moiety, or an ADC containing an anti-MET antibody as described herein, or a pharmaceutical composition containing an anti-MET antibody composition or an ADC containing an anti-MET antibody composition.

[0026] In this specification, in some embodiments, therapeutic uses of the described anti-MET antibodies or their antigen-binding moieties, of ADC compounds comprising the described anti-MET antibodies or their antigen-binding moieties, and of compositions are further provided, for example, for treating cancer. In some embodiments, the disclosure provides methods for treating cancer (e.g., cancer expressing a MET antigen targeted by the antibody or antigen-binding moiety of the ADC). In some embodiments, the disclosure provides methods for reducing or delaying the expansion of a cancer cell population in a subject.

[0027] The present invention also provides a method for treating a patient who has or is suspected of having cancer, comprising administering to the patient an anti-MET antibody or its antigen-binding moiety, an ADC containing an anti-Met antibody or its antigen-binding moiety as described herein, or a composition or pharmaceutical composition containing an anti-Met antibody or its antigen-binding moiety, or an ADC containing an anti-Met antibody or its antigen-binding moiety. Another exemplary embodiment is a method for reducing or inhibiting tumor growth in a patient, comprising administering to a target therapeutically effective amount of an anti-MET antibody or its antigen-binding moiety, an ADC containing an anti-Met antibody or its antigen-binding moiety, or a composition or pharmaceutical composition containing an anti-Met antibody or its antigen-binding moiety. Another exemplary embodiment is a method for reducing or delaying the expansion of a cancer cell population in a patient, comprising administering to a target therapeutically effective amount of an anti-MET antibody or its antigen-binding moiety, an ADC containing an anti-Met antibody or its antigen-binding moiety, or a composition or pharmaceutical composition containing an anti-Met antibody or its antigen-binding moiety. In some embodiments, the cancer depends on the activation and / or expression of MET. In some embodiments, the cancer includes melanoma, uveal melanoma, kidney cancer, thyroid cancer, mesothelioma, liver hepatocellular cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), and gastric cancer. The cancers include pancreatic cancer, colorectal cancer, esophageal cancer, cholangiocarcinoma, head and neck cancer (including oral cancer), cervical and intracervical cancer, bladder and urothelial cancer, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, adrenocortical cancer, brain cancer, spleen cancer, kidney cancer, thymoma, multiple myeloma, plasmacytotic myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancies originating from T cells or B cells, hematological cancers, myeloid leukemia, or myeloma. In some embodiments, the cancers are lung cancer, pancreatic cancer, or gastric cancer.In some embodiments, the cancer is a hematological cancer, such as chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, non-Hodgkin lymphoma, or myelodysplastic syndrome (MDS).

[0028] Another exemplary embodiment is a composition or pharmaceutical composition for use in the treatment of a patient having or suspected of having cancer, comprising an anti-Met antibody or its antigen-binding moiety, an ADC containing an anti-Met antibody or its antigen-binding moiety as described herein, or an ADC containing an anti-Met antibody or its antigen-binding moiety. Another embodiment is the use of a composition or pharmaceutical composition for use in the treatment of a patient having or suspected of having cancer, comprising an anti-MET antibody or its antigen-binding moiety, an ADC containing an anti-Met antibody or its antigen-binding moiety as described herein, or an ADC containing an anti-Met antibody or its antigen-binding moiety. In some embodiments, the cancer depends on the activation and / or expression of MET. In some embodiments, the cancer is melanoma, uveal melanoma, kidney cancer, thyroid cancer, mesothelioma, liver hepatocellular cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), gastric cancer (stomach cancer). The cancers include pancreatic cancer, colorectal cancer, esophageal cancer, cholangiocarcinoma, head and neck cancer (including oral cancer), cervical and intracervical cancer, bladder and urothelial cancer, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, adrenocortical cancer, brain cancer, spleen cancer, kidney cancer, thymoma, multiple myeloma, plasmacytotic myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancies originating from T cells or B cells, hematological cancers, myeloid leukemia, or myeloma. In some embodiments, the cancers are lung cancer, pancreatic cancer, or gastric cancer.In some embodiments, the cancer is a hematological cancer, such as chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, non-Hodgkin lymphoma, or myelodysplastic syndrome (MDS).

[0029] Furthermore, the present invention provides the use of an anti-MET antibody or its antigen-binding moiety, an ADC containing an anti-Met antibody or its antigen-binding moiety as described herein, or a composition or pharmaceutical composition containing an anti-Met antibody or its antigen-binding moiety or an ADC containing an anti-Met antibody or its antigen-binding moiety, in a method for producing a pharmacopoeia for treating a patient having or suspected of having cancer. In some embodiments, the cancer depends on the activation and / or expression of MET. In some embodiments, the cancer is melanoma, uveal melanoma, kidney cancer, thyroid cancer, mesothelioma, liver hepatocellular cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), gastric cancer (stomach cancer). The cancers include pancreatic cancer, colorectal cancer, esophageal cancer, cholangiocarcinoma, head and neck cancer (including oral cancer), cervical and intracervical cancer, bladder and urothelial cancer, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, adrenocortical cancer, brain cancer, spleen cancer, kidney cancer, thymoma, multiple myeloma, plasmacytotic myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancies originating from T cells or B cells, hematological cancers, myeloid leukemia, or myeloma. In some embodiments, the cancers are lung cancer, pancreatic cancer, or gastric cancer. In some embodiments, the cancer is a hematological cancer, such as chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, non-Hodgkin lymphoma, or myelodysplastic syndrome (MDS).

[0030] In some embodiments of the treatment methods described herein, the patient is a mammal. In certain embodiments, the patient is a primate. In certain embodiments, the patient is a human.

[0031] Furthermore, the present invention provides the use of the antibody for detecting and / or measuring the level of MET in a sample derived from a patient. The present invention further encompasses a kit (e.g., a diagnostic kit) comprising the antibody described herein. [Brief explanation of the drawing]

[0032] [Figure 1A] Figure 1 shows the dose-response binding affinity of either IgG1 (Figure 1A) or IgG2 (Figure 1B) anti-MET antibodies against either human cMET or cynomolgus monkey cMET in CHO-S cells: 9006 (black circle), 8902 (black square), 9338 (black diamond), terizotuzumab (half-white diamond), amibantamab (half-white triangle), and isotype control (white circle). As a negative control, CHO-S cells were transfected using an empty vector, which is referred to as a mock. Data are expressed as mean ± standard error. [Figure 1B] Same as above [Figure 2A]Figure 2 shows the dose-dependent effect on cell viability in MET-amplified or MET-non-amplified cell lines exposed for 120 hours to either an IgG1-type (Figure 2A) or IgG2-type (Figure 2B) anti-MET monoclonal antibody (Mab) or anti-MET ADC. Figure 2A: Terisotuzumab monoclonal antibody (black square), terisotuzumab ADC (white circle), 9006 monoclonal antibody (black star), 9006 ADC (white square), 9338 monoclonal antibody (X mark), 9338 ADC (white triangle), 8902 monoclonal antibody (plus mark), 8902 ADC (black circle). The activity of the monoclonal antibody or ADC was compared to that of the payload MMAE alone (circle including X marks). Figure 2B: Terisotuzumab monoclonal antibody (black circle), terisotuzumab ADC (black square), 9006 monoclonal antibody (white triangle), 9006 ADC (black star), 9338 monoclonal antibody (plus mark), 9338 ADC (white circle), 8902 monoclonal antibody (X mark), and 8902 ADC (white square). The activity of the monoclonal antibody or ADC was compared with that of the payload MMAE alone (circle including the X mark). Data are expressed as the mean ± standard error of IC50. [Figure 2B] Same as above [Figure 3A] Figure 3A shows the mean tumor volume of SCID mice inoculated with SNU-5 cells, either untreated (black circles) or after treatment with 8902 antibody (black diamonds). Inverted triangles indicate the time of antibody administration. Data are expressed as mean ± standard error. Figure 3B shows the mean body weight volume of SCID mice inoculated with SNU-5 cells, either untreated (black circles) or after treatment with 8902 antibody (black squares). Inverted triangles indicate the time of antibody administration. Data are expressed as mean ± standard error. [Figure 3B] Same as above [Modes for carrying out the invention]

[0033] Detailed explanation

[0034] General definition

[0035] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have meanings generally understood by those skilled in the art. Methods and materials similar to or equivalent to those described herein may also be used in carrying out or testing the present invention, but exemplary methods and materials are described below.

[0036] Furthermore, the singular forms “a,” “an,” and “the” also include the plural form unless otherwise clearly indicated in the context. The terms “is,” “including,” and “contain,” as found in “comprising,” “possess,” and “are a chemical formula,” should be considered open terms (i.e., “including but not limited to”) unless otherwise specified. Moreover, whenever “contains” or another open-ended term is used in an embodiment, it should be understood that the embodiment can be claimed more narrowly using the intermediate term “substantially consisting” or the closed term “consisting of.”

[0037] Unless otherwise specified, "MET" as used herein refers to human MET (also known as human c-MET, cMET, MET proto-oncoreceptor tyrosine kinase, or hepatocyte growth factor receptor, i.e., HGF receptor). The human MET polypeptide sequence is 1390 amino acids long and is available under Uniprot reference number P08581, shown herein as SEQ ID NO: 31. Unless otherwise specified, MET refers to the amino acid sequence of SEQ ID NO: 31. Human MET also exists in different isoforms (isoform 2, shown in SEQ ID NO: 32, and isoform 3, shown in SEQ ID NO: 33). "MET" also encompasses functional variants or fragments of human MET, including but not limited to splice variants, allele variants, and isoforms that retain one or more biological functions of human MET. MET may be isolated from humans, recombinantly produced, or synthesized. The human MET extracellular domain consists of amino acid residues 1-927 and is described herein as MET ECD (extracellular domain).

[0038] The term “antibody” (Ab) is used in its broadest sense to refer to an immunoglobulin (Ig) molecule that recognizes, specifically binds to, and has binding specificity to, a target, such as a protein, polypeptide, glycan, polynucleotide, lipid, or combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural sources or recombinant sources. As used herein, “intact” antibody or immunoglobulin refers to a tetramer containing two heavy chains (H) (approximately 50-70 kDa) and two light chains (L) (approximately 25 kDa), linked to each other by disulfide bonds. Each heavy chain contains a heavy chain variable domain (VH) and a heavy chain constant domain (CH). Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL domains can be further subdivided into highly variable regions called "complementarity-determining regions" (CDRs), which are dotted with more conserved regions called "framework regions" (FRs). Each VH and VL consists of three CDRs (H-CDRs refer to heavy-chain derived CDRs; L-CDRs refer to light-chain derived CDRs) and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to each region may follow the definition of IMGT® (Lefranc et al., Dev Comp Immunol 27(1):55-77 (2003)); or Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); or Chothia et al., Nature 342:878-883 (1989). The variable regions of the heavy and light chains contain binding domains that interact with the antigen.The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system (C1q). The antibody can be a monoclonal antibody, a human antibody, a humanized antibody, a camelized antibody or a chimeric antibody. The antibody can be of any isotype (such as IgG, IgE, IgM, IgD, IgA and IgY), class (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. The antibody can be an intact antibody or its antigen-binding portion.

[0039] The term "recombinant antibody" refers to an antibody expressed from a cell or cell line containing a nucleotide sequence (group) encoding the antibody, where the nucleotide sequence (group) is not naturally associated with the cell.

[0040] As used herein, the term "antigen-binding portion" (or simply "antibody portion") of an antibody refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (such as human MET or a portion thereof). It has been shown that specific portions or fragments of a full-length antibody can perform the antigen-binding function of the antibody. Examples of binding fragments included within the term "antigen-binding portion" include: (i) Fab fragment: a monovalent fragment consisting of V L , V H , C L and C H domains; (ii) F(ab’)2 fragment: a bivalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region; (iii) Fd fragment consisting of V H domain and C H 1 domain; (iv) Fv fragment consisting of V L domain of a single arm of an antibody and V H domain; (v) dAb fragment consisting of V H domain; and (vi) an isolated complementarity-determining region (CDR) that can specifically bind to an antigen. Further, V L and V H , the two domains of the Fv fragmentThese are encoded by separate genes, but they can be combined into a single protein chain (V) using recombination. L Region and V H The region can be linked by a synthetic linker that allows it to be fabricated as a monovalent molecule (known as single-stranded Fv(scFv)) by pairing the regions. H and / or V L There are also antigen-binding molecules that contain V H In this case, the molecule may also contain one or more of the CH1 region, hinge region, CH2 region, or CH3 region. Such single-chain antibodies are also intended to be included within the term "antigen-binding portion" of the antibody. Other forms of single-chain antibodies, such as diabodies, are also included. Diabodies are bivalent, bispecific antibodies, and here V H Domain and V L The domain is expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain. This forces the domain to pair with a complementary domain on another chain, creating two antigen-binding sites.

[0041] Antibody moieties, such as Fab and F(ab')2 fragments, can be prepared from a complete antibody using conventional techniques, such as digestion of the complete antibody with papain or pepsin. Furthermore, antibodies, antibody moieties, and immunoadhesin molecules can be obtained using standard recombinant DNA techniques, for example, as described herein.

[0042] In one embodiment, the antibody of the present invention is a monoclonal antibody. As used herein, the acronym "mAb" refers to a monoclonal antibody, i.e., an antibody synthesized and secreted by an individual clonal cell population. The clonal population may be a clonal population of immortalized cells. In some embodiments, the immortalized cells within the clonal population are typically hybrid cells (hybridoms) created by fusing individual B lymphocytes derived from an immunized animal with individual cells derived from a lymphocytic tumor.

[0043] The class (isotype) and subclass of an antibody can be determined by any method known in the art. Generally, the class and subclass of an antibody can be determined using an antibody that is specific to a particular class and subclass of the antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blotting, and other techniques. Alternatively, the class and subclass may be determined by sequencing all or part of the constant domains of the heavy and / or light chain of the antibody, comparing their amino acid sequences with known amino acid sequences of various classes and subclasses of immunoglobulins, and then determining the class and subclass of the antibody.

[0044] The term "antibody composition" refers to a combination of two or more antibodies or their antigen-binding moieties. An antibody composition may be monoclonal (i.e., consisting of identical antibody or antigen-binding moiety molecules) or polyclonal (i.e., consisting of two or more different antibodies or antigen-binding moieties that react with the same or different epitopes on the same antigen or even on distinct antigens).

[0045] The terms “isolated protein,” “isolated polypeptide,” or “isolated antibody” refer to a protein, polypeptide, or antibody that, for reasons of its origin or source, (1) is not associated with any naturally associated components that are present in its natural state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Therefore, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cells of its natural origin is “isolated” from its naturally associated components. Proteins may also be made substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0046] As used herein, the term “binding specificity” refers to the ability of an individual antibody or antigen-binding moiety to preferentially react with one antigenic determinant from among various antigenic determinants. Specificity indicates the degree to which an antibody or moiety preferentially binds to one antigenic determinant from among various antigenic determinants. Furthermore, as used herein, the terms “specific,” “specifically binds,” and “binds specifically” refer to the binding reaction between an antibody or antigen-binding moiety (e.g., anti-Met antibody) and a target antigen (e.g., MET) in heterogeneous protein populations and other biological products. A “specific antibody” or “target-specific antibody” is an antibody that binds only to a target antigen (e.g., MET) but does not bind to (or shows minimal binding to) other antigens.

[0047] The term "affinity" refers to a measure of attraction between an antigen and an antibody. The term "kon" or "ka" refers to the binding rate constant at which the antibody and antigen associate to form an antibody / antigen complex. The rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assays. The term "koff" or "kd" refers to the dissociation rate constant at which the antibody dissociates from the antibody / antigen complex. This rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assays. D The term "K" refers to the equilibrium dissociation constant of a specific antibody-antigen interaction. D This is calculated by ka / kd. The rate can be determined using standard assays such as surface plasmon resonance, biolayer interferometry, or ELISA assays. The intrinsic attractiveness of an antibody to an antigen is typically the K of a particular antibody-antigen interaction. D It is expressed as follows: Antibodies are K D It is said that when the concentration is 1 mM or less, preferably 100 nM or less, it specifically binds to the antigen.

[0048] As used herein, the term “epitope” refers to a portion of an antigen (determinant) that specifically binds to an antibody or related molecule, such as a bispecifically binding molecule. Epitope determinants generally consist of chemically active surface groups of a molecule, such as amino acids or sugar chains or sugar side chains, and generally possess specific three-dimensional structural features as well as specific charge characteristics. Epitopes may be “linear” or “stereostructural.” In linear epitopes, all interaction sites between a protein (e.g., antigen) and an interacting molecule (e.g., antibody) lie linearly along the primary amino acid sequence of the protein. In stereostructural epitopes, interaction sites are located between amino acid residues on the protein that are separated from each other within the primary amino acid sequence. Once a desired epitope on an antigen is determined, it is possible to produce an antibody against that epitope using techniques well known in the art. Furthermore, antibody production and characterization can elucidate information about the desired epitope. This information allows for competitive screening of antibodies for binding to the same or similar epitopes, for example, by conducting competitive testing to discover antibodies that compete for binding to the antigen. Whether an antibody binds to the same epitope or cross-competes with the anti-MET antibody can be determined using methods known in the art. In one embodiment, the anti-MET antibody of the present invention is enabled to bind to MET under saturated conditions, and then the ability of the test antibody to bind to MET is measured. If the test antibody can bind to MET simultaneously with the reference anti-MET antibody, the test antibody binds to a different epitope than the reference anti-MET antibody. However, if the test antibody cannot bind to MET simultaneously, the test antibody binds to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope that binds to the anti-MET antibody of the present invention. This experiment can be performed using ELISA, RIA (radioactive immunoassay), ViaCore®, BioLayer interferometry, or flow cytometry.To test whether an anti-MET antibody cross-competes with another anti-MET antibody, the above competitive method can be used in two directions: namely, to determine whether a known antibody blocks the test antibody and vice versa. In a preferred embodiment, the experiment is carried out using Octet®. Epitope binning can also be used to determine antibodies that share the same or overlapping epitopes. Competitive binding is then used to sort groups of binding proteins that share similar epitopes. Binding proteins that compete for binding can be “binned” as a group of binding proteins that have overlapping or nearby epitopes, while non-competitive binding proteins are placed in a separate group of binding proteins (a separate bin) that do not have overlapping or nearby epitopes.

[0049] As used herein, the terms “peptide,” “polypeptide,” and “protein” are synonymous and refer to polymers of amino acid residues. The term encompasses amino acid polymers containing two or more amino acids linked to each other by peptide bonds, amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, and natural and non-natural amino acid polymers. The term includes, among other things, biologically active fragments, substantially homogeneous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. The term also includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. Unless otherwise noted, a particular polypeptide sequence implicitly includes its conservatively modified variants.

[0050] As used herein, the term “variant” refers to a nucleic acid sequence or amino acid sequence that differs from the reference nucleic acid sequence or amino acid sequence, but retains one or more biological characteristics of the reference sequence. A variant may contain one or more amino acid substitutions, deletions, and / or insertions (or corresponding codon substitutions, deletions, and / or insertions) compared to the reference sequence. Changes in a nucleic acid variant may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid sequence, or they may result in amino acid substitutions, additions, deletions, fusions, and / or cleavage shortenings. In some embodiments, the nucleic acid variants disclosed herein encode the same amino acid sequence as that encoded by the unmodified nucleic acid, or they encode a modified amino acid sequence that retains one or more functional characteristics of the unmodified amino acid sequence. Since the changes in peptide variant sequences are typically limited or conserved, the sequences of the unmodified peptide and the variant are generally similar and identical in many regions. In some embodiments, the peptide variant retains one or more functional characteristics of the unmodified peptide sequence. Mutants and unmodified peptides may differ in their amino acid sequences by any combination of one or more substitutions, additions, or deletions. Nucleic acid or peptide variants may be native variants or variants not known to occur naturally. Nucleic acid and peptide variants may be prepared by mutagenesis, direct synthesis, or other techniques known in the art. In some embodiments, the variants exhibit high sequence identity (i.e., 80% or more nucleic acid or amino acid sequence identity) compared to the reference sequence.In some embodiments, peptide variants include polypeptides having amino acid substitutions, deletions, and / or insertions, insofar as the polypeptide has at least 80%, at least 85%, 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%, and at least 99% amino acid sequence identity with respect to the reference sequence or the corresponding segment (e.g., a functional fragment) of the reference sequence (e.g., a variant that also retains one or more functions of the reference sequence). In some embodiments, the nucleic acid variants include polynucleotides having amino acid substitutions, deletions, and / or insertions, insofar as the polynucleotide has at least 80%, at least 85%, 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%, and at least 99% nucleic acid sequence identity with respect to a reference sequence or to a corresponding segment (e.g., a functional fragment) of the reference sequence.

[0051] The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences across a comparison window for optimal alignment of the two sequences (where the amino acid sequence fragments within the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the reference sequence (which does not contain additions or deletions)). The ratio can be calculated by determining the number of positions where identical amino acid residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The result is the % identity of the target sequence to the question sequence. The % identity between two sequences is a function of the number of gaps that need to be introduced for optimal alignment of the two sequences, and the number of identical positions shared by the sequences, taking into account the length of each gap. Generally, the amino acid identity or homology between the proteins disclosed herein and their variants (including variants of target antigens (such as METs) and variants of antibody variable domains (including individual variant CDRs)) is at least 80% with respect to the sequences shown herein, for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, nearly 100%, or 100% identity or homology.

[0052] The terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are used synonymously and refer to one or more therapeutic compounds linked to one or more antibody or antigen-binding moieties. In some embodiments, an ADC is defined by the general formula: Ab-(LD)p, where Ab is an antibody or antigen-binding moiety (e.g., an anti-Met antibody or its antigen-binding moiety); L is the linker moiety; D is the drug moiety; and p is the number of drug moieties per antibody or antigen-binding moiety.

[0053] Anti-MET antibody

[0054] This invention relates to a novel anti-MET antibody 8902 directed against human MET, or to the antigen-binding portion of the antibody. The amino acid sequences of the variable domains (VH and VL) of the heavy and light chains of this antibody are provided in SEQ ID NOs: 7 and 8, respectively, and the corresponding nucleotide sequences are provided in SEQ ID NOs: 9 and 10, respectively. The full-length amino acid sequences of the heavy and light chains (HC and LC) are available in SEQ ID NOs: 11 and 12 (IgG1 chain) and SEQ ID NOs: 13 and 14 (IgG2 chain), respectively. The amino acid sequences of the heavy chain CDRs (H-CDR1, H-CDR2, and H-CDR3) and light chain CDRs (L-CDR1, L-CDR2, and L-CDR3) of the 8902 antibody are shown in SEQ ID NOs: 1, 2, and 3, and SEQ ID NOs: 4, 5, and 6, respectively. The CDR sequences were assigned according to the definition of IMGT®.

[0055] In certain embodiments, the present invention provides the following:

[0056] Antibodies having H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, respectively, which contain or consist of the amino acid sequences of SEQ ID NOs. 1, 2, 3, 4, 5, and 6, and anti-MET antibodies or their antigen-binding moieties that compete for binding to human MET; • Anti-MET antibodies or their antigen-binding moieties that bind to the same human MET epitopes as antibodies having H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, respectively, which contain or consist of the amino acid sequences of SEQ ID NOs. 1, 2, 3, 4, 5, and 6; An antibody having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8, and an anti-MET antibody or its antigen-binding moiety that competes for binding to human MET; An anti-MET antibody or its antigen-binding moiety that binds to the same human MET epitope as an antibody having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8; An antibody having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 11 and a light chain containing or comprising the amino acid sequence of SEQ ID NO: 12, and an anti-MET antibody or its antigen-binding moiety that competes for binding to human MET; An anti-MET antibody or its antigen-binding moiety that binds to the same human MET epitope as an antibody having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 11 and a light chain containing or comprising the amino acid sequence of SEQ ID NO: 12; An antibody having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 13 and a light chain containing or comprising the amino acid sequence of SEQ ID NO: 14, and an anti-MET antibody or its antigen-binding moiety that competes for binding to human MET; and An anti-MET antibody or its antigen-binding moiety that binds to the same human MET epitope as an antibody having a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 13 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 14.

[0057] In one embodiment, the anti-MET antibody or its antigen-binding moiety comprises H-CDR1, H-CDR2, and H-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0058] In one embodiment, the anti-MET antibody or its antigen-binding moiety comprises L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0059] In one embodiment, the anti-MET antibody or its antigen-binding moiety includes at least two, three, four, or five CDR sequences selected from the group consisting of H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, H-CDR3 of SEQ ID NO: 3, L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6.

[0060] In one embodiment, the anti-MET antibody or its antigen-binding moiety comprises H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and L-CDR1, L-CDR2, and L-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0061] In one embodiment, the anti-MET antibody or its antigen-binding moiety has a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7. In one embodiment, the anti-MET antibody or its antigen-binding moiety has a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8. In one embodiment, the anti-MET antibody or its antigen-binding moiety has a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8.

[0062] In certain embodiments, the anti-MET antibody or its antigen-binding moiety comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 11 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 12.

[0063] In certain embodiments, the anti-MET antibody or its antigen-binding moiety comprises a heavy chain containing or consisting of the amino acid sequence of SEQ ID NO: 13 and a light chain containing or consisting of the amino acid sequence of SEQ ID NO: 14.

[0064] In another embodiment, the present invention provides a variant or portion thereof of the antibody described above, wherein the variant differs from the antibody or portion thereof by only one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions.

[0065] In one embodiment, the present invention provides an anti-MET antibody or an antigen-binding moiety of said antibody, comprising a heavy-chain variable domain whose amino acid sequence is at least 90% identical to SEQ ID NO: 7. In a particular embodiment, the heavy-chain variable domain is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7. In one embodiment, the present invention provides an anti-MET antibody or an antigen-binding moiety of said antibody, comprising a light-chain variable domain whose amino acid sequence is at least 90% identical to SEQ ID NO: 8. In a particular embodiment, the light-chain variable domain is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8. The anti-MET antibody may also include any combination of the heavy-chain and light-chain variable domains referenced above.

[0066] In one embodiment, the present invention provides an anti-MET antibody comprising a heavy chain having at least 90% amino acid sequence identity with SEQ ID NO: 11 or 13, or an antigen-binding moiety of said antibody. In a particular embodiment, the heavy chain has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with SEQ ID NO: 11 or 13. In one embodiment, the present invention provides an anti-MET antibody comprising a light chain having at least 90% amino acid sequence identity with SEQ ID NO: 12 or 14, or an antigen-binding moiety of said antibody. In a particular embodiment, the light chain has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with SEQ ID NO: 12 or 14. The anti-MET antibody may also include any combination of the variable domains of the heavy and light chains referenced above.

[0067] The anti-MET antibody of the present invention may be an IgG molecule, an IgM molecule, an IgE molecule, an IgA molecule, or an IgD molecule, but is typically an IgG isotype, for example, an IgG subclass IgG1, IgG2, IgG3, or IgG4. In one embodiment, the antibody is IgG1. In another embodiment, the antibody is IgG2.

[0068] Sequence homology of polypeptides, also known as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications (including conserved amino acid substitutions). For example, GCG includes programs such as "Gap" or "Bestfit," which, when used with default parameters, can determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between wild-type proteins and their mutaines. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA; a program within GCG version 6.1, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignment and % sequence identity of regions that best overlap between the query sequence and the search sequence (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000)). Another preferred algorithm for comparing the sequences of the present invention with a database containing numerous sequences from different organisms is the computer program BLAST, particularly blastp or tblastn, using default parameters. See, for example, Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997).

[0069] The length of polypeptide sequences compared for homology is generally at least about 16 amino acid residues, usually at least about 20 residues, more commonly at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.

[0070] According to the present invention, certain amino acid substitutions that can be performed are chemically active changes from one or more cysteines in an antibody to another residue (such as, but not limited to, alanine or serine). In one embodiment, this is a substitution of a non-standard cysteine. The substitution may occur within the CDR region or framework region of a variable domain, or within the constant domain of the antibody. In some embodiments, the cysteine ​​is standard.

[0071] Another type of amino acid substitution that can be performed is the removal of candidate protein lysis sites within the antibody. Such sites may be located within the CDR region or framework region of the variable domain, or within the constant domain of the antibody. Cysteine ​​residue substitution and removal of protein lysis sites can reduce the risk of heterogeneity in the antibody product and thus improve its uniformity.

[0072] Another type of amino acid substitution involves altering one or both residues to eliminate asparagine-glycine pairs that form candidate deamidation sites.

[0073] Another type of amino acid substitution that may be performed in one of the variants described in the present invention is a conservative amino acid substitution. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having an R side chain group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other due to a conservative substitution, the degree of sequence identity or homology may be up-adjusted to modify the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994).

[0074] Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0075] In one embodiment, one or both of the amino acid residues at positions 234 and 235 may be mutated, for example, from Leu (leucine) to Ala (alanine) (L234A / L235A). These mutations reduce the effector function of the Fc region of the IgG1 antibody.

[0076] In some embodiments, if the antibody is an IgG4 subclass, it may contain the S228P mutation, i.e., having a proline at position 228, where the amino acid position is numbered according to the EU numbering or IMGT® numbering system. This mutation is known to reduce undesirable Fab arm replacement.

[0077] In some embodiments, the amino acid residue at position 233 can be mutated to, for example, Pro (proline), the amino acid residue at position 234 can be mutated to, for example, Val (valine), and / or the amino acid residue at position 235 can be mutated to, for example, Ala (alanine). The amino acid positions are numbered according to the EU numbering system or the IMGT® numbering system.

[0078] Alternatively, a conservative permutation can be defined as any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256:1443-45 (1992). A "moderately conservative" permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0079] In certain embodiments, amino acid substitutions to the antibody or antigen-binding moiety of the present invention (1) reduce sensitivity to protein lysis, (2) reduce sensitivity to oxidation, (3) alter binding affinity for protein complex formation, and (4) confer or modify other physicochemical or functional properties of such analogues while retaining specific binding to human MET. Analogues may include a variety of substitutions to commonly existing peptide sequences. For example, one or more amino acid substitutions, preferably conservative amino acid substitutions, may be made within commonly existing sequences, for example, within polypeptide moieties outside domains(s) that form intermolecular contacts. Amino acid substitutions that can improve polypeptide activity may also be made within domains(s) that form intermolecular contacts. Conservative amino acid substitutions should not substantially alter the structural features of the parent sequence; for example, the substituted amino acid should not alter the antiparallel β-sheets that constitute the immunoglobulin-binding domains present in the parent sequence, or disrupt other types of secondary structures that characterize the parent sequence. Generally, glycine and proline are not used in antiparallel β-sheets. Examples of secondary and tertiary structures of polypeptides recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354:105 (1991).

[0080] In another aspect of the present invention, the antibody may be deimmunized to reduce its immunogenicity, for example, by using the techniques described in International Publication No. 98 / 52976 and International Publication No. 00 / 34317.

[0081] In some embodiments, the antibodies or antibody moieties disclosed herein include modified or engineered amino acid residues, such as one or more cysteine ​​residues, as sites for conjugation with the drug moiety (Junutula JR, et al., Nat Biotechnol 2008, 26:925-932). In one embodiment, the disclosure provides a modified antibody or antibody moiety comprising the substitution of one or more amino acids using cysteine. The sites for cysteine ​​substitution are within a constant region of the antibody or antibody moiety and are therefore applicable to a wide variety of antibodies or antibody moieties, and the sites are selected to give a stable and uniform conjugate. The modified antibody or antibody moiety may have one, two or more cysteine ​​substitutions, and these substitutions may be used in combination with other modification and conjugation methods as described herein. Methods for inserting cysteine ​​into specific sites of antibodies are known in the art. For example, see Lyons et al., (1990) Protein Eng., 3:703-708.

[0082] As used herein (for example, referring to cells), the term “inhibits proliferation” is intended to include any measurable decrease in cell proliferation (increase in cell number) or metabolism upon contact with the anti-MET antibody or antigen-binding moiety or anti-MET antibody composition, compared to the proliferation of the cells in the absence of the anti-MET antibody or composition, e.g., at least about 10%, preferably more than that, e.g., at least about 20% or 30%, more preferably at least about 40% or 50%, e.g., at least about 60%, 70%, 80%, 90%, 95%, or 99%, or even about 100% inhibition of cell culture proliferation. Proliferation inhibition may be determined in relevant cancer cell lines, for example, as described in the following examples.

[0083] The class of the anti-MET antibody obtained by the method described herein may be switched with another class. In one aspect of the present invention, a nucleic acid molecule encoding VL or VH is isolated using a method well known in the art such that it does not contain a nucleic acid sequence encoding CL or CH. The nucleic acid molecule encoding VL or VH is operably ligated to a nucleic acid sequence encoding CL or CH, respectively, derived from an immunoglobulin molecule of a different class. This may be achieved using a vector or nucleic acid molecule containing a CL chain or a CH chain, as described above. For example, an anti-MET antibody that was originally IgM may be class-switched to IgG. Furthermore, class switching may be used to convert one subclass of IgG to another, for example, from IgG1 to IgG2. A preferred method for producing the antibody of the present invention having a desired isotype includes isolating nucleic acid molecules encoding the heavy chain and light chain of the anti-MET antibody; obtaining a variable domain of the heavy chain; ligating the variable domain of the heavy chain with the constant domain of the heavy chain of the desired isotype; expressing the heavy chain ligated with the light chain in cells; and recovering the anti-MET antibody having the desired isotype.

[0084] The anti-MET antibody of the present invention may be an IgG molecule, an IgM molecule, an IgE molecule, an IgA molecule, or an IgD molecule. In one embodiment, the anti-MET antibody is an IgG molecule, and is an IgG1, IgG2, IgG3, or IgG4 subclass. In a particular embodiment, the antibody is an IgG1 subclass. In a particular embodiment, the antibody is an IgG2 subclass.

[0085] In certain embodiments, the antibody or antigen-binding moiety of the present invention may be part of a larger immunoadhesin molecule formed by the covalent or non-covalent association of the antibody or antibody moiety with one or more other proteins or peptides. Examples of such immunoadhesin molecules include the creation of tetrameric scFv molecules using a streptavidin core region (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101 (1995)), and the creation of divalent and biotinylated scFv molecules using cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags (Kipriyanov et al., Mol. Immunol. 31:1047-1058 (1994)). Other examples include the incorporation of one or more CDRs derived from an antibody into a molecule, either covalently or non-covalently, so that the molecule becomes an immunoadhesin that specifically binds to the antigen of interest. In such embodiments, the CDR(group) may be incorporated as part of a larger polypeptide chain, covalently linked to another polypeptide chain, or incorporated noncovalently.

[0086] In another embodiment, a fusion antibody or immunoadhesin may be prepared, comprising all or part of the anti-MET antibody of the present invention linked to another polypeptide. In a particular embodiment, only the variable domain of the anti-MET antibody is linked to the polypeptide. In a particular embodiment, the VH domain of the anti-MET antibody is linked to a first polypeptide, while the VL domain of the anti-MET antibody is linked to a second polypeptide associated with the first polypeptide, so that the VH and VL domains can interact with each other to form an antigen-binding site. In another preferred embodiment, the VH domain is separated from the VL domain by a linker (e.g., a single-chain antibody) so that the VH and VL domains can interact with each other. The VH-linker-VL antibody is then linked to the polypeptide of choice. Furthermore, a fusion antibody may be prepared in which two (or more) single-chain antibodies are linked to each other. This is useful when it is desired to produce a bivalent or polyvalent antibody on a single polypeptide chain, or to produce a bispecific antibody.

[0087] To produce single-chain antibodies (scFv), DNA fragments encoding VH and VL are operably linked to another fragment encoding a mobile linker, for example, the amino acid sequence (Gly4-Ser)3, thereby allowing the VH and VL sequences to be expressed as a continuous single-chain protein (with the VL and VH domains linked by a mobile linker). See, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature 348:552-554 (1990). Single-chain antibodies can be monovalent when using only one VH and VL; bivalent when using two VH and VL; or polyvalent when using more than two VH and VL. Bispecific or polyvalent antibodies that specifically bind to human MET and, for example, to another molecule can be produced.

[0088] In other embodiments, other modified antibodies may be prepared using nucleic acid molecules encoding anti-MET antibodies. For example, "Kappa Bodies" (Ill et al., Protein Eng. 10:949-57 (1997)), "Mini Bodies" (Martin et al., EMBO J. 13:5303-9 (1994)), "Dia Bodies" (Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or "Janusine" (Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992)) may be prepared using standard molecular biological techniques in accordance with the teachings of the specification.

[0089] The anti-MET antibody or antigen-binding moiety of the present invention may be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or moiety is derivatized so that binding to MET is not negatively affected by derivatization or labeling. Accordingly, the antibodies and antibody moieties of the present invention are intended to include both intact and modified human anti-MET antibodies as described herein. For example, the antibody or antibody moiety of the present invention may be functionally linked (by chemical bonding, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a pharmaceutical, and / or a protein or peptide that can mediate the association of the antibody or antibody moiety with another molecule (e.g., a streptavidin core region or polyhistidine tag).

[0090] Certain derivatized antibodies are produced by cross-linking two or more antibodies (of the same or different types, for example, to produce a bispecific antibody). Suitable crosslinkers include heterobifunctional or homobifunctional (e.g., disuccinimidyl suberate) antibodies having two distinctly different reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimidyl).

[0091] Anti-MET antibodies may also be derivatized using chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar chain groups. These groups may be useful to improve the biological characteristics of the antibody, for example, to extend its serum half-life.

[0092] The antibodies described in the present invention may also be labeled. As used herein, the terms “labeling” or “labeled” refer to the incorporation of another molecule into the antibody. In one embodiment, labeling is the incorporation of a detectable marker, such as a radiolabeled amino acid, or the attachment of a biotinyl moiety to a polypeptide that is detectable by a marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity, detectable by optical or colorimetric methods). In another embodiment, the labeling or marker may be a therapeutic agent, such as a drug conjugate or toxin. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, and pre-determined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, secondary antibody binding sites, metals). Binding domains (epitope tags), magnetic materials, e.g., gadolinium chelates, toxins, e.g., pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogs or homologs. In some embodiments, the labels are attached by spacer arms of varying lengths to reduce the possibility of steric hindrance.

[0093] In certain embodiments, the antibody of the present invention may exist in a neutral form (including a zwitterionic form) or as a positively or negatively charged species. In some embodiments, the antibody may form a pharmaceutically acceptable salt by complexing with a counterion.

[0094] The term "pharmaceutically acceptable salt" refers to a salt that does not impair the biological activity and properties of the compound of the present invention and does not cause significant irritation to the subject to which it is administered. Examples of such salts include, but are not limited to, (a) acid addition salts formed using inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and nitric acid; and salts formed using organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid; and (b) salts formed from anionic elements such as chlorine, bromine, and iodine. See, eg, Haynes et al., “Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database,” J. Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al., “Pharmaceutical Salts,” J. Pharmaceutical Sciences, vol. 66, no. 1 (1977).

[0095] In some embodiments, depending on their charge, the antibody-drug conjugates (ADCs) described herein may contain a monovalent anion counterion. Any suitable anion counterion may be used. In certain embodiments, the monovalent anion counterion is a pharmaceutically acceptable monovalent anion counterion. In certain embodiments, the monovalent anion counterion can be selected from bromide ions, chloride ions, iodide ions, acetate ions, trifluoroacetate ions, benzoate ions, mesylate ions, tosylate ions, trifluoromethanesulfonate ions, formate ions, and the like.

[0096] Bispecific binding molecules In further embodiments, the antibody binding specificities disclosed herein may be combined within a single bispecific binding molecule. For example, the bispecific binding molecule may have binding specificity for anti-MET antibody 8902. For example, the bispecific binding molecule may contain anti-MET antibody 8902.

[0097] In some embodiments, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having L-CDR1, L-CDR2, and L-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and L-CDR1, L-CDR2, and L-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0098] In some embodiments, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0099] In some embodiments, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 11 and / or a light chain containing or comprising the amino acid sequence of SEQ ID NO: 12.

[0100] In some embodiments, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 13 and / or a light chain containing or comprising the amino acid sequence of SEQ ID NO: 14.

[0101] In one embodiment, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0102] In one embodiment, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11, and / or a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0103] In one embodiment, the bispecific binding molecule may include an anti-MET antibody or its antigen-binding moiety having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, and / or a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0104] In some embodiments, the bispecific binding molecule may be a dual variable domain antibody, where the two arms of the antibody contain two different variable domains, or it may be in the form of an antibody fragment such as a bispecific Fab fragment or a bispecific scFv. This is also useful when it is desired to create a bivalent or polyvalent antibody on a single polypeptide chain.

[0105] A bispecific binding molecule or a polyvalent antibody may have the binding specificity of the anti-MET antibody or its antigen-binding portion described herein, and the binding specificity of the same protein or another protein, such as an immune checkpoint protein, a cancer antigen, or another antibody whose activity targets cell surface molecules that mediate disease conditions such as cancer.

[0106] In some embodiments, the bispecific binding molecule has binding specificity to a first anti-Met antibody 8902 and a second antibody or its antigen-binding moiety. In some embodiments, the bispecific binding molecule has binding specificity to a first anti-Met antibody 8902 and a second anti-Met antibody 9006 or its antigen-binding moiety. In some embodiments, the bispecific binding molecule includes an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 12, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the bispecific binding molecule includes an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 14, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 18. In some embodiments, the bispecific binding molecule has binding specificity for the first anti-Met antibody 8902 and the second anti-Met antibody 9338 or their antigen-binding moieties. In some embodiments, the bispecific binding molecule includes an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 12, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the bispecific binding molecule includes an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 14, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0107] Antibody-drug conjugates (ADCs or ADC groups)

[0108] The antibody-drug conjugate (ADC) compounds of this disclosure comprise an anti-MET antibody or its antigen-binding moiety conjugated (i.e., covalently attached by a linker) to a drug moiety, wherein the drug moiety, when not conjugated with the antibody or antigen-binding moiety, exhibits cytotoxic or cytostatic effects. Furthermore, although we do not wish to be bound by theory, by conjugating a drug moiety to an antibody that binds to an antigen associated with expression in cancer, the ADC may offer improved activity, better cytotoxic specificity, and / or reduced off-target cell death compared to the drug moiety administered alone.

[0109] In some embodiments, the components of the ADC are therefore selected to (i) retain one or more therapeutic properties demonstrated by the antibody and drug moieties alone; (ii) maintain the specific binding properties of the antibody or antigen binding moiety; (iii) optimize drug loading and drug-to-antibody ratios; (iv) enable delivery of the drug moiety, e.g., intracellular delivery, via stable attachment to the antibody or antigen binding moiety; (v) maintain the stability of the ADC as an intact conjugate until transport or delivery to the target site; (vi) minimize aggregation of the ADC before or after administration; (vii) enable the therapeutic effect of the drug moiety, e.g., cytotoxic effect, after cleavage or other release mechanism in the intracellular environment; (viii) demonstrate in vivo anticancer efficacy equivalent to or better than that of the antibody and drug moieties alone; (ix) minimize off-target cell death by the drug moiety; and / or (x) exhibit desirable pharmacodynamic and pharmacokinetic properties, pharmacoagulability, and toxicological / immunological profiles. Each of these characteristics could provide an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

[0110] The ADC compounds of this disclosure can selectively deliver effective doses of cytotoxic or cytostatic agents to cancer cells or tumor tissue. In some embodiments, the cytotoxic and / or cytostatic activity of the ADC depends on the expression of the target antigen within the cell. In some embodiments, the disclosed ADC is particularly effective in killing cancer cells expressing the target antigen while minimizing off-target death. In some embodiments, the disclosed ADC does not exhibit cytotoxic and / or cytostatic effects on cancer cells that do not express the target antigen.

[0111] In this specification, in certain embodiments, ADC compounds are provided, comprising an anti-Met antibody or its antigen-binding moiety (Ab), a drug moiety (D), and a linker moiety (L) that covalently attaches Ab to D. In some embodiments, the antibody or antigen-binding moiety can bind to a tumor-associated antigen (e.g., MET) with, for example, high specificity and high affinity. In some embodiments, the antibody or antigen-binding moiety, upon binding, internally moves into the target cell, for example, into an intracellular degradation compartment. In some embodiments, the ADC, upon binding to the target cell, internally moves, undergoes degradation, and releases the drug moiety to kill the cancer cell. The drug moiety may be released from the antibody moiety and / or linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

[0112] An exemplary ADC is denoted as Ab-(LD)p, where Ab is the anti-Met antibody or antigen-binding moiety, L is the linker moiety, D is the drug moiety or payload (e.g., MMAE), and p is the number of drug moieties per antibody or its antigen-binding moiety. The term "p" may be used as a synonym for the terms "drug load," "drug:antibody ratio," "drug-to-antibody ratio," or "DAR." For example, if two drug moieties D are linked to the antibody or its antigen-binding moiety, then p=2. In a composition comprising multiple copies of an ADC, "mean p" refers to the average number of -LD moieties per antibody or antigen-binding moiety, and is also referred to as the "mean drug load." In some embodiments, the anti-Met antibody or its antigen-binding moiety in the antibody-drug conjugate of this disclosure is the anti-Met antibody 8902 or its antigen-binding moiety.

[0113] In one embodiment, the present invention provides an ADC comprising an anti-MET antibody or an antigen-binding moiety thereof, selected from the group consisting of:

[0114] • Anti-MET antibodies or their antigen-binding moieties having H-CDR1, H-CDR2, and H-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; • An anti-MET antibody or its antigen-binding moiety having L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; An anti-MET antibody or its antigen-binding moiety comprising at least two, three, four, or five CDR sequences selected from the group consisting of H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, H-CDR3 of SEQ ID NO: 3, L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6; An anti-MET antibody or its antigen-binding moiety having H-CDR1, H-CDR2, and H-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; • An anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain containing or consisting of the amino acid sequence of SEQ ID NO: 7; • An anti-MET antibody or its antigen-binding moiety having a light chain variable domain containing or consisting of the amino acid sequence of SEQ ID NO: 8; An anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8; An anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8; An anti-MET antibody or its antigen-binding moiety having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 11 and a light chain containing or comprising the amino acid sequence of SEQ ID NO: 12; An anti-MET antibody or its antigen-binding moiety having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 13 and a light chain containing or comprising the amino acid sequence of SEQ ID NO: 14; and An anti-MET antibody or its antigen-binding moiety having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12; and An anti-MET antibody or its antigen-binding moiety having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0115] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety having H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0116] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety having L-CDR1, L-CDR2, and L-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0117] In one embodiment, the ADC includes an anti-MET antibody or its antigen-binding moiety, which comprises at least two, three, four, or five CDR sequences selected from the group consisting of H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, H-CDR3 of SEQ ID NO: 3, L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6.

[0118] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety, having H-CDR1, H-CDR2, and H-CDR3 comprising or comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and L-CDR1, L-CDR2, and L-CDR3 comprising or comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0119] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0120] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 12.

[0121] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 14.

[0122] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0123] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety, having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0124] In one embodiment, the ADC comprises an anti-MET antibody or its antigen-binding moiety, having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0125] In some embodiments, the anti-MET antibody or its antigen-binding moiety of the ADC disclosed herein is an anti-Met bispecific binding molecule. For example, the bispecific binding molecule may have binding specificity to anti-MET antibody 8902. For example, the bispecific binding molecule may contain anti-MET antibody 8902.

[0126] In some embodiments, the ADC comprises a bispecific binding molecule comprising an anti-MET antibody or its antigen-binding moiety, having H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the ADC comprises a bispecific binding molecule comprising an anti-MET antibody or its antigen-binding moiety, having L-CDR1, L-CDR2, and L-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the ADC comprises a bispecific binding molecule comprising an anti-MET antibody or its antigen-binding moiety, having H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and L-CDR1, L-CDR2, and L-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0127] In some embodiments, the ADC comprises a bispecific binding molecule comprising an anti-MET antibody or its antigen-binding moiety, having a heavy chain variable domain comprising or comprising the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable domain comprising or comprising the amino acid sequence of SEQ ID NO: 8.

[0128] In some embodiments, the ADC comprises a bispecific binding molecule containing an anti-MET antibody or its antigen-binding moiety, having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 11 and / or a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 12.

[0129] In some embodiments, the ADC comprises a bispecific binding molecule containing an anti-MET antibody or its antigen-binding moiety, having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 13 and / or a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 14.

[0130] In one embodiment, the ADC comprises a bispecific binding molecule containing an anti-MET antibody or its antigen-binding moiety, having a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0131] In one embodiment, the ADC comprises a bispecific binding molecule containing an anti-MET antibody or its antigen-binding moiety, having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11, and / or a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0132] In one embodiment, the ADC comprises a bispecific binding molecule containing an anti-MET antibody or its antigen-binding moiety, having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, and / or a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0133] In some embodiments, the ADC includes a bispecific binding molecule that may have the binding specificity of the anti-MET antibody or its antigen-binding moiety described herein, and the binding specificity of the same or different protein, such as an immune checkpoint protein, a cancer antigen, or another antibody whose activity targets a cell surface molecule that mediates a disease condition such as cancer.

[0134] In some embodiments, the ADC comprises an anti-Met antibody 8902 and a bispecific binding molecule having binding specificity to a secondary antibody or its antigen-binding moiety.

[0135] In some embodiments, the ADC comprises a bispecific binding molecule having binding specificity to a first anti-Met antibody 8902 and a second anti-Met antibody 9006 or their antigen-binding moieties. In some embodiments, the ADC comprises a bispecific binding molecule comprising an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 12, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 15 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 16. In some embodiments, the ADC comprises a bispecific binding molecule comprising an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 14, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 17 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 18.

[0136] In some embodiments, the ADC comprises a bispecific binding molecule having binding specificity to a first anti-Met antibody 8902 and a second anti-Met antibody 9338 or their antigen-binding moieties. In some embodiments, the ADC comprises a bispecific binding molecule comprising an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 12, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 19 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 20. In some embodiments, the ADC comprises a bispecific binding molecule comprising an antigen-binding moiety of a first antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 13 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 14, and an antigen-binding moiety of a second antibody having a heavy chain variable domain (VH) containing the amino acid sequence of SEQ ID NO: 21 and a light chain variable domain (VL) containing the amino acid sequence of SEQ ID NO: 22.

[0137] As used herein, the terms “drug portion” or “payload” refer to a chemical portion that is conjugated to or suitable for conjugation of an antibody or antigen-binding fragment, which may include any therapeutic or diagnostic agent, and metabolites of antibody-drug conjugates disclosed herein (which have desired therapeutic or diagnostic properties, such as, for example, anticancer agents, anti-inflammatory agents, anti-infective agents (e.g., antifungal agents, antibacterial agents, antiparasitic agents, antiviral agents), or anesthetic agents). In certain embodiments, the drug portion is selected from Eg5 inhibitors, V-type ATPase inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, drastatin, meitansinoids, MetAP (methionine aminopeptidase), inhibitors of nuclear export of protein CRM1, DPPIV inhibitors, inhibitors of mitochondrial phosphate transfer reactions, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, proteasome inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, RNA polymerase inhibitors, amanitin, spliceosome inhibitors, topoisomerase inhibitors, DHFR inhibitors, and apoptosis promoters.

[0138] In some embodiments, the linker L within the ADC is sufficiently stable extracellularly to be therapeutically effective. In some embodiments, because the linker is stable extracellularly, the ADC remains intact when present under extracellular conditions (e.g., before intracellular transport or delivery). As used in the context of ADCs, the term “intact” means that the antibody or antigen-binding portion remains attached to the drug portion or payload D. In some embodiments, the ADCs disclosed herein may remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours. Whether the linker is stable extracellularly can be determined, for example, by exposing the ADC to plasma for a predetermined time (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying the amount of free drug portion present in the plasma. Stability can allow time for the ADC to localize to target cancer cells and prevent premature release of the drug moiety (which can reduce the therapeutic index of the ADC by damaging both normal and cancerous tissues indiscriminately). In some embodiments, the linker is stable outside the target cell and, once inside the cell, releases the drug moiety from the ADC so that the drug can bind to its target. Thus, an effective linker (i) maintains the specific binding properties of the antibody or antigen binding moiety; (ii) enables delivery of the drug moiety, e.g., intracellular delivery, via stable attachment to the antibody or antigen binding moiety; (iii) remains stable and intact until the ADC is transported or delivered to its target site; and (iv) enables the therapeutic effect of the drug moiety, e.g., cytotoxic effect, after cleavage or an alternative release mechanism.

[0139] Linkers can affect the physical and chemical properties of ADCs. Since many cytotoxic drugs are hydrophobic, aggregation can occur when they are linked to antibodies with additional hydrophobic moieties. ADC aggregates are insoluble and often limit the achievable drug load on the antibody, which can negatively impact the potency of the ADC. Generally, protein aggregates of biological agents are also associated with increased immunogenicity. As shown below, the ADCs disclosed herein have low aggregation levels and desired levels of drug loading. Linkers may be "cleavable" or "incleavable" (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release the drug moiety when exposed to certain environmental factors, for example, upon internal migration into target cells, while incleavable linkers generally rely on the degradation of the antibody or its antigen-binding moiety. For example, the linker MC-VC-PAB is a protease-cleavable linker.

[0140] In some embodiments, an intermediate, which is a precursor of the linker moiety, is reacted with the drug moiety or payload under appropriate conditions. In some embodiments, a reactive group is used on the drug or payload (e.g., MMAE means monomethyl auristatin E) and / or the intermediate or linker. The reaction product between the drug or payload and the intermediate, i.e., the derivatized drug or payload (drug / payload plus linker), is subsequently reacted with the antibody or antigen-binding moiety under conditions that promote conjugation between the drug and intermediate i.e., the derivatized drug / payload and the antibody or antigen-binding moiety. Alternatively, the intermediate or linker is first reacted with the antibody or antigen-binding moiety, or a derivatized antibody or antigen-binding moiety, and then reacted with the drug or derivatized drug. Many different reactions are available for the covalent attachment of the drug moiety and / or linker moiety to the antibody or antigen-binding moiety. This is often accomplished by the reaction of one or more amino acid residues of the antibody or antigen-binding moiety using techniques known to those skilled in the art.

[0141] The drug load is denoted by p and referred to herein as the drug-to-antibody ratio (DAR). The drug load may range from 1 to 16 drug moieties per antibody or antigen-binding moiety. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, p is 2. In some embodiments, p is 4. The drug load may be limited by the number of attachment sites on the antibody or antigen-binding moiety. In some embodiments, the linker moiety (L) of the ADC is attached to the antibody or antigen-binding moiety through a chemically active group on one or more amino acid residues on the antibody or antigen-binding moiety. For example, the linker may attach to the antibody or antigen-binding moiety via a free amino group, imino group, hydroxyl group, thiol group, or carboxyl group (e.g., from the N-terminus to the C-terminus, to the epsilon-amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine ​​residues). The site to which the linker attaches may be a native residue in the amino acid sequence of the antibody or antigen-binding moiety, or it may be introduced to the antibody or antigen-binding moiety by, for example, DNA recombination technology (e.g., by introducing cysteine ​​residues into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis). In some embodiments, conjugation is carried out probabilistically on the native antibody. In some embodiments, the number of drug moieties that can be conjugated to the antibody or antigen-binding moiety is limited by the number of free cysteine ​​residues. For example, if the attachment is a cysteinethiol group, the antibody may have only one or more cysteinethiol groups, or only one or more sufficiently reactive thiol groups through which the linker may be attached. Generally, antibodies do not contain many free and reactive cysteinethiol groups that can be linked to the drug moiety. In fact, most cysteinethiol residues in antibodies are involved in interchain or intrachain disulfide bonds.Therefore, in some embodiments, conjugation to cysteine ​​may require at least partial reduction of the antibody. Excessive attachment of linker-toxin to the antibody may destabilize the antibody by reducing cysteine ​​residues available for disulfide bond formation. Therefore, the optimal drug:antibody ratio should increase the potency of the ADC without destabilizing the antibody or antigen-binding moiety (by increasing the number of drug moieties attached per antibody). In some embodiments, the optimal ratio may be 2, 4, 6, or 8. In some embodiments, the optimal ratio may be 2 or 4. In some embodiments, the antibody or antigen-binding moiety is exposed to reducing conditions before conjugation to generate one or more free cysteine ​​residues. In some embodiments, the antibody may be reduced under partial or complete reducing conditions using a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) to generate reactive cysteinethiol groups. Unpaired cysteine ​​can be generated through partial reduction using a limited molar equivalent of TCEP, which can reduce the interchain disulfide bonds linking the light and heavy chains (one pair per heavy-chain-light pair formation) and the interchain disulfide bonds linking two heavy chains within the hinge region (two pairs per heavy-chain-heavy pair formation in the case of human IgG1), while the intrachain disulfide bonds remain intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In the embodiments, disulfide bonds within the antibody are electrochemically reduced, for example, by using a working electrode to which reduction and oxidation potentials are alternately applied. This approach may enable the reduction of disulfide bonds to be linked online to analytical instruments (e.g., electrochemical detectors, NMR (nuclear magnetic resonance) spectroscopy, or mass spectrometers) or chemical separation instruments (e.g., liquid chromatographs (e.g., high-performance liquid chromatography) or electrophoresis (see, for example, U.S. Patent Application Publication No. 2014 / 0069822)). In some embodiments, antibodies are subjected to denaturing conditions to expose reactive nucleophiles on amino acid residues, such as cysteine.The drug loading of an ADC can be controlled in various ways, for example, by (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody; (ii) limiting the reaction time or temperature of the conjugation; (iii) by partial or limited reducing conditions for cysteine ​​thiol modification; and / or by engineering the amino acid sequence of the antibody by recombinant technology, thereby modifying the number and position of cysteine ​​residues to control the number and / or position of linker-drug attachments. In some embodiments, free cysteine ​​residues are introduced into the amino acid sequence of the antibody or antigen-binding moiety. For example, an engineered cysteine ​​antibody can be prepared in which one or more amino acids of the parent antibody are substituted with cysteine ​​amino acids. Any form of antibody can be engineered, i.e., mutated, in this way. For example, an engineered cysteine ​​Fab, referred to as "ThioFab," can be formed by engineering a parent Fab antibody fragment. Similarly, a "ThioMab" (monoclonal antibody) can be formed by engineering a parent monoclonal antibody. A single-site mutation generates one engineered cysteine ​​residue within the thioFab, while a single-site mutation generates two engineered cysteine ​​residues within the thioMab due to the dimeric nature of the IgG antibody. In some embodiments, the parent antibody is engineered by introducing cysteine ​​mutations into the heavy chain by substituting serine at position 400 (EU numbering) of the peptide backbone and into the light chain by substituting valine at position 205 (EU numbering) (HC S400C and LC V205C, respectively). In some embodiments, if one or more free cysteine ​​residues are already present in the antibody or antigen-binding moiety without the use of engineering, the existing free cysteine ​​residues may be used to conjugate the antibody or antigen-binding moiety to the drug moiety.In a reaction mixture containing multiple copies of antibody or antigen-binding moieties and linker moieties, when one or more nucleophiles react with a drug-linker intermediate, or with a linker moiety followed by a drug moiety reagent, the resulting product may be a mixture of ADC compounds, in which one or more drug moieties attached to each copy of the antibody or antigen-binding moiety are dispersed. In some embodiments, the drug load in the mixture of ADCs resulting from the conjugation reaction ranges from 1 to 16 drug moieties attached to each antibody or antigen-binding moiety. The average number of drug moieties per antibody or antigen-binding moiety (i.e., average drug load, i.e., mean p) can be calculated by any conventional method known in the art, such as mass spectrometry (e.g., liquid chromatography-mass spectrometry (LC-MS)) and / or high-performance liquid chromatography (e.g., HIC (hydrophobic interaction chromatography)-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding moiety is determined by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody or antigen-binding moiety is 1.5–3.5, 2.5–4.5, 3.5–5.5, 4.5–6.5, 5.5–7.5, 6.5–8.5, or 7.5–9.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding moiety is 2–4, 3–5, 4–6, 5–7, 6–8, 7–9, 2–8, or 4–8. In some embodiments, the average number of drug moieties per antibody or antigen-binding moiety is 2, 3, 4, 5, or 6. Individual ADC compounds or "species" in the mixture can be identified by mass spectrometry, separated by, for example, ultra-high-performance liquid chromatography (UPLC) or HPLC, or by, for example, hydrophobic interaction chromatography (HIC-HPLC). In some embodiments, a homogeneous or nearly homogeneous ADC product with a single loading value can be isolated from the conjugation mixture by, for example, electrophoresis or chromatography. This disclosure includes methods for producing ADCs as described in Example 5 and Table 6. The prepared ADCs may be subjected to a purification step.The purification process may include any biochemical method known in the art for the purification of proteins, or any combination thereof. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, chromatography based on any charge or isoelectric point, mixed-mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

[0142] Anti-MET antibodies and anti-MET ADC compositions

[0143] In one embodiment, the present invention provides an antibody composition comprising the anti-MET antibody of the present invention or its antigen-binding moiety.

[0144] In another embodiment, the present invention provides an ADC composition comprising an anti-MET ADC, wherein the ADC comprises the anti-MET antibody of the present invention or its antigen-binding moiety.

[0145] In one embodiment, the composition is an antibody composition comprising an anti-MET antibody or its antigen-binding moiety selected from the group consisting of: Antibodies having H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, respectively, which contain or consist of the amino acid sequences of SEQ ID NOs. 1, 2, 3, 4, 5, and 6, and anti-MET antibodies or their antigen-binding moieties that compete for binding to human MET; Antibodies having H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, respectively, containing or consisting of the amino acid sequences of SEQ ID NOs. 1, 2, 3, 4, 5, and 6, and anti-MET antibodies or their antigen-binding moieties that bind to the same human MET epitopes; An antibody having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8, and an anti-MET antibody or its antigen-binding moiety that competes for binding to human MET; and An antibody having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8, and an anti-MET antibody or its antigen-binding moiety that binds to the same human MET epitope.

[0146] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety selected from the group consisting of:

[0147] • Anti-MET antibodies or their antigen-binding moieties having H-CDR1, H-CDR2, and H-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; • An anti-MET antibody or its antigen-binding moiety having L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; An anti-MET antibody or its antigen-binding moiety comprising at least two, three, four, or five CDR sequences selected from the group consisting of H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, H-CDR3 of SEQ ID NO: 3, L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6; An anti-MET antibody or its antigen-binding moiety having H-CDR1, H-CDR2, and H-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs. 1, 2, and 3, respectively, and L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs. 4, 5, and 6, respectively; • An anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain containing or consisting of the amino acid sequence of SEQ ID NO: 7; • An anti-MET antibody or its antigen-binding moiety having a light chain variable domain containing or consisting of the amino acid sequence of SEQ ID NO: 8; An anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8; An anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8; An anti-MET antibody or its antigen-binding moiety having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 11 and a light chain containing or comprising the amino acid sequence of SEQ ID NO: 12; An anti-MET antibody or its antigen-binding moiety having a heavy chain containing or comprising the amino acid sequence of SEQ ID NO: 13 and a light chain containing or comprising the amino acid sequence of SEQ ID NO: 14; An anti-MET antibody or its antigen-binding moiety having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12; and An anti-MET antibody or its antigen-binding moiety having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0148] In one embodiment, the antibody composition comprises antibodies having H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, and an anti-MET antibody or its antigen-binding moiety that competes for binding to human MET.

[0149] In one embodiment, the antibody composition comprises antibodies having H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, and L-CDR3, each containing or comprising the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively, and an anti-MET antibody or its antigen-binding moiety that binds to the same human MET epitope.

[0150] In one embodiment, the antibody composition comprises an antibody having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8, and an anti-MET antibody or its antigen-binding moiety that competes for binding to human MET.

[0151] In one embodiment, the antibody composition comprises an antibody having a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 8, and an anti-MET antibody or its antigen-binding moiety that binds to the same human MET epitope.

[0152] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having H-CDR1, H-CDR2, and H-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively.

[0153] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having L-CDR1, L-CDR2, and L-CDR3, each comprising or consisting of the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0154] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, which comprises at least two, three, four, or five CDR sequences selected from the group consisting of H-CDR1 of SEQ ID NO: 1, H-CDR2 of SEQ ID NO: 2, H-CDR3 of SEQ ID NO: 3, L-CDR1 of SEQ ID NO: 4, L-CDR2 of SEQ ID NO: 5, and L-CDR3 of SEQ ID NO: 6.

[0155] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having H-CDR1, H-CDR2, and H-CDR3 each comprising or comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and L-CDR1, L-CDR2, and L-CDR3 each comprising or comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0156] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having a heavy chain variable domain comprising or comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable domain comprising or comprising the amino acid sequence of SEQ ID NO: 8.

[0157] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 11 and a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 12.

[0158] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 13 and a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 14.

[0159] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having a heavy chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and a light chain variable domain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0160] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0161] In one embodiment, the composition is an antibody composition or ADC composition comprising an anti-MET antibody or its antigen-binding moiety, having a heavy chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, and a light chain that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[0162] In one embodiment, the present invention provides a bispecific antibody composition comprising a bispecific antibody containing the anti-MET antibody of the present invention or its antigen-binding moiety.

[0163] For example, the bispecific antibody composition may include a bispecific binding molecule containing the anti-MET antibody 8902. In some embodiments, the bispecific antibody composition includes an anti-MET antibody or its antigen-binding moiety having H-CDR1, H-CDR2, and H-CDR3 comprising or comprising the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively, and / or L-CDR1, L-CDR2, and L-CDR3 comprising or comprising the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively.

[0164] In some embodiments, the bispecific antibody composition comprises an anti-MET antibody or its antigen-binding moiety having a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0165] In some embodiments, the bispecific antibody composition comprises an anti-MET antibody or its antigen-binding moiety having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 11 and / or a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 12.

[0166] In some embodiments, the bispecific antibody composition comprises an anti-MET antibody or its antigen-binding moiety having a heavy chain comprising or comprising the amino acid sequence of SEQ ID NO: 13 and / or a light chain comprising or comprising the amino acid sequence of SEQ ID NO: 14.

[0167] In some embodiments, the bispecific antibody composition may include a bispecific binding molecule having a dual variable domain antibody, i.e., the two arms of the antibody may contain two different variable domains, or it may be in the form of an antibody fragment such as a bispecific Fab fragment or a bispecific scFv. Such a bispecific antibody composition may include a bispecific binding molecule or a polyvalent antibody having the binding specificity of the anti-MET antibody or its antigen-binding moiety described herein, and the binding specificity of the same or different protein, such as an immune checkpoint protein, a cancer antigen, or another antibody whose activity targets a cell surface molecule that mediates a disease condition such as cancer.

[0168] In some embodiments, the bispecific antibody composition comprises a first anti-Met antibody 8902 and a bispecific binding molecule having binding specificity to a second antibody or its antigen-binding moiety. In some embodiments, the bispecific antibody composition comprises a first anti-Met antibody 8902 and a second anti-Met antibody 9006 or its antigen-binding moiety having binding specificity to a bispecific binding molecule. In some embodiments, the bispecific antibody composition comprises a first anti-Met antibody 8902 and a second anti-Met antibody 9338 or its antigen-binding moiety having binding specificity to a bispecific binding molecule.

[0169] nucleic acid molecules and vectors

[0170] The present invention also provides nucleic acid molecules and nucleic acid sequences encoding the anti-MET antibody or its antigen-binding moiety as described herein. In some embodiments, various nucleic acid molecules encode the amino acid sequences of the heavy and light chains of the anti-MET antibody or its antigen-binding moiety. In other embodiments, the same nucleic acid molecules encode the amino acid sequences of the heavy and light chains of the anti-MET antibody or its antigen-binding moiety.

[0171] References to nucleotide sequences, unless otherwise specified, include their complements. Therefore, references to nucleic acids having a particular sequence should be understood to include both their complementary sequence and their complementary strand. The term “polynucleotide” as used herein means a nucleotide of either a polymer form of ribonucleotide or deoxyribonucleotide, or any modified form of nucleotide, with a length of at least 10 nucleotides.

[0172] The aforementioned terms include single-stranded and double-stranded forms.

[0173] The present invention also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to one or more nucleotide sequences listed above, or to nucleotide sequences encoding amino acid sequences selected from the group consisting of SEQ ID NOs. 7 and 8. In the context of nucleic acid sequences, the term "% sequence identity" refers to residues in two sequences that are identical when aligned to the maximum extent possible. The length of a sequence identity comparison may be a sequence of at least 9 nucleotides, typically at least about 18 nucleotides, more commonly at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, preferably at least about 36, 48, or more nucleotides. Many different algorithms that can be used to measure nucleotide sequence identity are known in the art. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs in the Wisconsin Package version 10.0 (Gene Computer Group (GCG), Madison, Wisconsin). FASTA (including, for example, the FASTA2 and FASTA3 programs) provides the best overlap region alignment and sequence identity between the query sequence and the search sequence (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998)). Unless otherwise noted, default parameters for a particular program or algorithm are used. For example, % sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word size 6 and NOPAM coefficients for the scoring matrix), or using Gap, as provided in GCG version 6.1, with its default parameters.

[0174] In one embodiment, the present invention provides a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 9 or 10. In some embodiments, the nucleic acid molecule may comprise the nucleotide sequences of SEQ ID NO: 9 and 10.

[0175] In any of the embodiments described above, the nucleic acid molecule may be isolated.

[0176] In further embodiments, the present invention provides a vector suitable for expressing one of the antibody or antigen-binding segments thereof as described herein. As used herein, the term “vector” means a nucleic acid molecule capable of transporting another nucleic acid to which the vector is ligated. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded DNA fragment, which may have additional DNA segments ligated therein. In some embodiments, the vector is a viral vector, in which additional DNA segments may be ligated within a viral genome. In some embodiments, the vector can autonomously replicate within a host cell into which the vector (e.g., a bacterial vector having a bacterial origin of replication and an episomal mammalian vector) is introduced. In other embodiments, the vector (e.g., a non-episomal mammalian vector) may be incorporated into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome. Furthermore, certain vectors can instruct the expression of a gene to which the vector is operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”).

[0177] The present invention provides a vector comprising a nucleic acid molecule encoding the anti-MET antibody of the present invention or its antigen-binding moiety's heavy chain, the anti-MET antibody of the present invention or its antigen-binding moiety's light chain, or both the heavy and light chains of the anti-MET antibody of the present invention or its antigen-binding moiety. The present invention further provides a vector comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, an antibody fragment, and its probe.

[0178] Nucleic acid molecules encoding the heavy and / or light chains of an anti-MET antibody or a portion thereof can be isolated from any source producing such an antibody or portion. In various embodiments, the nucleic acid molecules are isolated from B cells expressing an anti-MET antibody isolated from an animal immunized with a human MET antigen, or from immortalized cells produced from such B cells. Methods for isolating nucleic acids encoding antibodies are well known in the art. mRNA can be isolated and used to generate cDNA for use in polymerase chain reaction (PCR) or cDNA cloning of the antibody gene. In certain embodiments, the nucleic acid molecules of the present invention can be synthesized rather than isolated.

[0179] In some embodiments, the nucleic acid molecule of the present invention may include a nucleotide sequence encoding a VH domain derived from the anti-MET antibody of the present invention or its antigen-binding moiety, in-frame to a nucleotide sequence encoding a heavy chain constant domain of any origin. Similarly, the nucleic acid molecule of the present invention may include a nucleotide sequence encoding a VL domain derived from the anti-MET antibody of the present invention or its antigen-binding moiety, in-frame to a nucleotide sequence encoding a light chain constant domain of any origin.

[0180] In a further embodiment of the present invention, nucleic acid molecules encoding variable domains of the heavy chain (VH) and / or light chain (VL) may be "converted" into a full-length antibody gene. In one embodiment, a nucleic acid molecule encoding a VH domain or a VL domain is converted into a full-length antibody gene by insertion into an expression vector already encoding a heavy chain constant (CH) domain or a light chain constant (CL) domain, respectively, so that the VH segment is operably ligated to the CH segment(s) in the vector, and / or the VL segment is operably ligated to the CL segment in the vector. In another embodiment, a nucleic acid molecule encoding a VH domain and / or a VL domain is converted into a full-length antibody gene, for example, by ligating the nucleic acid molecule encoding the VH domain and / or VL domain to a nucleic acid molecule encoding the CH domain and / or CL domain using standard molecular biological techniques. The full-length heavy chain and / or light chain encoding nucleic acid molecules can then be expressed from the cells into which they are introduced, and an anti-MET antibody can be isolated.

[0181] Nucleic acid molecules may be used to recombinantly express large quantities of anti-MET antibodies. Nucleic acid molecules may also be used to generate chimeric antibodies, bispecific antibodies, single-chain antibodies, immunoadhesins, diabodynes, mutant antibodies, and antibody derivatives as described herein.

[0182] In another embodiment, the nucleic acid molecule of the present invention is used as a probe or PCR primer for a specific antibody sequence. For example, the nucleic acid can be used as a probe in a diagnostic method, or as a primer for amplifying a DNA region that can be used, in particular, to isolate additional nucleic acid molecules encoding the variable domain of an anti-MET antibody. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the oligonucleotide is derived from the highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotide encodes all or part of one or more CDRs of the anti-MET antibody of the present invention or its antigen-binding moiety, as described herein. In another embodiment, a mutated anti-MET antibody can be produced using the nucleic acid molecule and vector. The antibody may be mutated in the variable domains of the heavy and / or light chain, for example, to change the binding properties of the antibody. For example, the K of the anti-MET antibody D In order to increase or decrease k off To increase or decrease or to alter the binding specificity of the antibody, one or more mutations may be introduced in the CDR region. In another embodiment, one or more mutations are introduced in amino acid residues known to be altered compared to germline in the monoclonal antibody of the present invention. The mutations may occur in the CDR region or framework region of the variable domain, or in the constant domain. In a preferred embodiment, the mutations occur in the variable domain. In some embodiments, one or more mutations are introduced in amino acid residues known to be altered compared to germline in the CDR region or framework region of the variable domain of the antibody of the present invention or its antigen-binding portion.

[0183] In another embodiment, the framework region(s) are mutated such that the resulting framework region(s) have the amino acid sequence of the corresponding germline gene. Mutations may be introduced within the framework region or constant domain to extend the half-life of the anti-MET antibody. See, for example, International Publication No. 00 / 09560. Mutations may also be introduced within the framework region or constant domain to alter the immunogenicity of the antibody and / or to provide a site for covalent or non-covalent bonding to another molecule. According to the present invention, a single antibody may have mutations in one or more of the CDR region or framework regions of the variable domain, or within the constant domain.

[0184] In some embodiments, the anti-MET antibody of the present invention or its antigen-binding moiety is expressed by inserting DNA encoding part or the full length of the light and heavy chains obtained as described above into an expression vector, thereby operably ligating the gene to necessary expression regulatory sequences, such as transcriptional and translational regulatory sequences. Examples of expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs (yeast artificial chromosomes), and EBV (Epstein-Barr virus)-derived episomes. The antibody gene may be ligated into the vector so that the transcriptional and translational regulatory sequences within the vector perform their intended functions of regulating the transcription and translation of the antibody gene. The expression vector and expression regulatory sequences may be selected to be compatible with the host cell used for expression. The antibody light chain gene and the antibody heavy chain gene may be inserted into separate vectors. In one embodiment, both genes are inserted into the same expression vector. The antibody gene may be inserted into the expression vector by a standard method (for example, ligation of the antibody gene fragment and complementary restriction enzyme sites on the vector, or ligation of the blunt ends if no restriction enzyme sites are present).

[0185] Conventional vectors are vectors encoding a functionally complete human CH or CL immunoglobulin sequence, along with appropriate restriction enzyme sites engineered to allow for the easy insertion and expression of any VH or VL sequence as described above. In such vectors, splicing typically occurs between a splice donor site in the inserted J region and a splice acceptor site preceding the human C domain, and also in splice regions present within human CH exons. Polyadenylation and transcription termination may occur at native chromosomal sites downstream of the coding region. Recombinant expression vectors may also encode a signal peptide that promotes the secretion of antibody chains from host cells. The antibody chain gene may be cloned into the vector such that the signal peptide is in-frame ligated to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0186] In addition to the antibody chain gene, the recombinant expression vector of the present invention may have regulatory sequences that control the expression of the antibody chain gene in host cells. It will be understood by those skilled in the art that the design of the expression vector (including the selection of regulatory sequences) may depend on factors such as the selection of host cells to be transformed and the desired level of protein expression. Preferred regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as retroviral LTRs (long repeat sequences), cytomegalovirus (CMV) (CMV promoter / enhancer), simian virus 40 (SV40) (SV40 promoter / enhancer, etc.), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma-derived promoters and / or enhancers, and potent mammalian promoters, such as innate immunoglobulin and actin promoters. For further descriptions of viral regulatory elements and their sequences, see, for example, U.S. Patents 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants (including descriptions of promoters and vectors) and for transforming plants are well known in the art. See, for example, U.S. Patent No. 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0187] In addition to the antibody chain gene and regulatory sequence, the recombinant expression vector of the present invention may have additional sequences, such as sequences that regulate vector replication in host cells (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, typically, selection marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Examples of selection marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-negative host cells with selection / amplification by methotrexate), the neo gene (for selection by G418), and the glutamate synthase gene.

[0188] As used herein, the term “regulatory sequence” means a polynucleotide sequence that is necessary for the expression and processing of the ligated coding sequence. Examples of regulatory sequences include appropriate start, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kossack common sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such regulatory sequences varies depending on the host organism; in prokaryotes, such regulatory sequences generally include promoters, ribosome binding sites, and termination sequences; in eukaryotes, such regulatory sequences generally include promoter and termination sequences. The term “regulatory sequence” is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion pair sequences.

[0189] Hybridoma method for producing antibodies according to the present invention

[0190] In certain embodiments, the present invention provides a method for producing a cell line that produces human monoclonal antibodies or antigen-binding moieties that are directed against MET, comprising: (a) immunizing a non-human transgenic animal with MET, a part of MET, or cells or tissues expressing MET; (b) enabling the transgenic animal to initiate an immune response against MET; (c) isolating antibody-producing cells from the transgenic animal; (d) immortalizing the antibody-producing cells; (e) creating individual monoclonal populations of immortalized antibody-producing cells; and (f) identifying antibodies directed against MET by screening the immortalized antibody-producing cells.

[0191] In another embodiment, the present invention provides a cell line that produces human anti-MET antibodies. In some embodiments, the cell line is a hybridoma cell line. In some embodiments, the hybridoma is a mouse hybridoma as described above. In other embodiments, the hybridoma is produced in a non-human, non-mouse species, such as a rat, sheep, pig, goat, cattle, or horse. In yet another embodiment, the hybridoma is a human hybridoma.

[0192] In another embodiment, transgenic animals are immunized with a MET antigen, and primary cells, such as spleen cells or peripheral blood B cells, are isolated from the immunized transgenic animals to identify individual cells that produce antibodies specific to the desired antigen. Polyadenylated mRNA from each individual cell is isolated, and reverse transcription polymerase chain reaction (RT-PCR) is performed using forward primers that anneal to the variable domain sequence, such as degenerate primers that recognize most or all of the FR1 region of human heavy and light chain variable domain genes, and reverse primers that anneal to the constant or connecting region sequence. Subsequently, the cDNA of the heavy and light chain variable domains is cloned and expressed in any suitable host cells, such as myeloma cells, as chimeric antibodies having the heavy chain and the respective immunoglobulin constant regions, such as the κ or λ constant domain. See Babcook et al., Proc Natl Acad Sci USA 93:7843-48 (1996). Subsequently, anti-MET antibodies can be identified and isolated as described herein.

[0193] Phage display library

[0194] The present invention provides a method for producing an anti-MET antibody or its antigen-binding moiety, comprising the steps of synthesizing a human antibody library on a phage, screening the library using MET or its antigen-binding moiety, isolating MET-binding phages, and obtaining antibodies from the phages. For example, one method for preparing an antibody library for use in phage display technology includes the steps of: inducing an immune response by immunizing a non-human animal with MET or its antigenic moiety; extracting antibody-producing cells from the immunized animal; isolating RNA encoding the heavy and light chains of the present invention from the extracted cells; generating cDNA by reverse transcription of the RNA; amplifying the cDNA using primers; and inserting the cDNA into a phage display vector to express the antibody on a phage. The recombinant anti-MET antibody of the present invention may be obtained in this way.

[0195] The recombinant human anti-MET antibody of the present invention may be isolated by screening a recombinant combinatorial antibody library. Preferably, the library is an scFv phage display library prepared using human VL and VH cDNA prepared from mRNA isolated from B cells. Methods for preparing and screening such libraries are known in the art. Kits for preparing phage display libraries are commercially available (e.g., Pharmacia Recombinant Phage Antibody Systems, catalog no. 27-9400-01; and Stratagene's SurfZAP® Phage Display Kit, catalog no. 240612).Other methods and reagents are also available for the preparation and screening of antibody display libraries (e.g., U.S. Patent No. 5,223,409; International Publication Nos. 92 / 18619, 91 / 17271, 92 / 20791, 92 / 15679, 93 / 01288, 92 / 01047, and 92 / 09690; Fuchs et al., Bio / Technology 9:1370-1372 (1991); Hay et al., Hum Antibod Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989); McCafferty et al., Nature 348:552-554 (1990); Griffiths et al., EMBO J 12:725-734 (1993); Hawkins et al., J Mol Biol 226:889-896 (1992); Clackson et al., Nature 352:624-628 (1991); Gram et al., Proc Natl Acad Sci USA 89:3576-3580 (1992); Garrad et al., Bio / Technology 9:1373-1377 (1991); Hoogenboom et al., Nuc Acid Res 19:4133-4137 (1991); and Barbas et al., Proc Natl Acad Sci USA 88:7978-7982 (1991)).

[0196] In one embodiment, to isolate and produce a human anti-MET antibody having the desired characteristics, a human anti-MET antibody as described herein is first used to select human heavy and light chain sequences having similar binding activity to MET using the epitope imprinting method described in International Publication No. 93 / 06213, incorporated herein by reference. The antibody library used in this method is preferably an scFv library prepared and screened as described in International Publication No. 92 / 01047, McCafferty et al., Nature 348:552-554 (1990); and Griffiths et al., EMBO J 12:725-734 (1993). The scFv antibody library is preferably screened using human MET as the antigen.

[0197] Once the initial human VL and VH domains are selected, a “mix and match” experiment can be performed, in which different pairs of the initially selected VL and VH segments are screened for binding to MET, thereby selecting a preferred VL / VH pair combination. Furthermore, to further improve antibody quality, the VL and VH segments of the preferred VL / VH pair(s) may be randomly mutated, preferably within the CDR3 regions of VH and / or VL, in a process similar to the in vivo somatic mutation process involved in antibody affinity maturation during the innate immune response. This in vitro affinity maturation can be achieved by amplifying the VH and VL domains using PCR primers complementary to the CDR3 of VH or VL, respectively, in which a random mixture of four nucleotide bases is “added” to specific positions, so that the resulting PCR product encodes the VH and VL segments with random mutations introduced into the CDR3 regions of VH and / or VL. These randomly mutated VH and VL segments may be screened again for binding to MET.

[0198] Following screening and isolation of the anti-MET antibodies of the present invention from a recombinant immunoglobulin display library, the nucleic acids encoding the selected antibodies can be recovered from the display package (e.g., from a phage genome) and subcloned into other expression vectors using standard recombinant DNA techniques. If desired, the nucleic acids may be further manipulated to produce other antibody forms of the present invention, as described herein. To express recombinant human antibodies isolated by combinatorial library screening, the DNA encoding the antibodies is cloned into a recombinant expression vector and introduced into mammalian host cells as described herein.

[0199] Methods for producing non-hybridoma host cells, antibodies, and antibody compositions.

[0200] Additional aspects of the present invention relate to antibody compositions and methods for producing antibodies and their antigen-binding moieties. One embodiment of this aspect of the present invention relates to a method for producing antibodies as defined herein, comprising providing recombinant host cells capable of expressing an antibody, culturing the host cells under conditions suitable for the expression of the antibody, and isolating the resulting antibody. The antibody produced by such expression in such recombinant host cells is referred to herein as a “recombinant antibody.” The present invention also provides progeny cells of such host cells and antibodies produced therefrom.

[0201] As used herein, the term “recombinant host cell” (or simply “host cell”) means a cell into which a recombinant expression vector has been introduced. The present invention provides a host cell which may, for example, contain the vector described in the present invention above. The present invention also provides a host cell which may, for example, contain the anti-MET antibody of the present invention or the heavy chain or nucleotide sequence encoding the antigen-binding portion thereof, the light chain or nucleotide sequence encoding the antigen-binding portion thereof, or both. It should be understood that “recombinant host cell” and “host cell” mean not only a specific target cell but also the offspring of such a cell. Such offspring may, in fact, not be identical to the parent cell because certain modifications may occur in subsequent generations due to mutation or environmental influences, but are still included within the scope of the term “host cell” as used herein.

[0202] Nucleic acid molecules encoding anti-MET antibodies, and vectors containing these nucleic acid molecules, can be used for transfection of suitable mammalian, plant, bacterial, or yeast host cells. Transformation is achieved by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Furthermore, nucleic acid molecules may be introduced into mammalian cells via viral vectors. Methods for transforming cells are well known in the art. See, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, bioristic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial cells and yeast cells are also well known in the art.

[0203] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, e.g., Sf9 cells or Sf21 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a sufficient amount of time to allow for antibody expression within the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is growing. The antibody can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, and potato. Bacterial host cells include Escherichia coli and Streptomyces species. Yeast host cells include fission yeast (Schizosaccharomyces pombe), Saccharomyces cerevisiae, and Pichia pastoris.

[0204] Furthermore, the expression of the antibodies of the present invention or their antigen-binding moieties from production cell lines can be enhanced using many known techniques. For example, the glutamine synthase gene expression system (GS system) is a common approach for enhancing expression under specific conditions. The GS system has been discussed, in whole or in part, in connection with European Patent Nos. 0216846, 0256055, 0323997, and 0338841.

[0205] Antibodies expressed by different cell lines or in transgenic animals are highly likely to have different glycosylation patterns. However, all antibodies encoded by the nucleic acid molecules provided herein, or containing the amino acid sequences provided herein, are part of the present invention regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications.

[0206] The antibodies or their antigen-binding portions or antibody compositions of the present invention can be produced by methods generally known in the art for the production of recombinant monoclonal antibodies or polyclonal antibodies. Therefore, in the case of producing a single antibody of the present invention, any method known in the art for the production of recombinant monoclonal antibodies can be used. For the production of antibody compositions of the present invention comprising an antibody mixture, the individual antibodies may be produced separately, i.e., each antibody may be produced in a separate bioreactor, or the individual antibodies may be produced together in a single bioreactor. If the antibody composition is produced in more than one bioreactor, a purified antibody composition can be obtained by pooling antibodies obtained from individually purified supernatants from each bioreactor. Various approaches for the production of polyclonal antibody compositions in multiple bioreactors (where cell lines or antibody preparations are combined before or during upstream or downstream processing at a later point in time) are described in International Publication No. 2009 / 129814.

[0207] When producing individual antibodies within a single bioreactor, this can be carried out as described, for example, in International Publication No. 2004 / 061104 or International Publication No. 2008 / 145133. The method described in International Publication No. 2004 / 061104 is based on site-specific incorporation of antibody coding sequences into the genomes of individual host cells, while the method described in International Publication No. 2008 / 145133 includes an alternative approach for producing antibodies within a single bioreactor using random incorporation.

[0208] Further information regarding suitable methods for preparing the antibodies and compositions of the present invention can be found in International Publication No. 2012 / 059857.

[0209] Transgenic animals and plants

[0210] The anti-MET antibody and its antigen-binding moiety of the present invention can also be transgenically produced by producing a mammal or plant that is transgenic with respect to the heavy and light chain sequences of the immunoglobulin of interest, and by producing an antibody therefrom in a recoverable form. In connection with transgenic production in mammals, the anti-MET antibody and moiety can be produced in the milk of a goat, a cow, or another mammal and recovered therefrom. See, for example, U.S. Patents 5,827,690, 5,756,687, 5,750,172 and 5,741,957. In some embodiments, a non-human transgenic animal containing a human immunoglobulin locus is immunized with human MET or its immunogenic moiety as described above. Methods for producing antibodies in plants are described, for example, U.S. Patents 6,046,037 and 5,959,177.

[0211] In some embodiments, non-human transgenic animals or plants are produced by introducing one or more nucleic acid molecules encoding the anti-MET antibody or its antigen-binding moiety (e.g., any of the above nucleic acid molecules encoding the anti-MET antibody or its antigen-binding moiety) into an animal or plant using standard transgenic techniques. See, for example, U.S. Patent No. 6,417,429. The transgenic cells used to produce the transgenic animals may be embryonic stem cells, somatic cells, or fertilized eggs. Transgenic non-human organisms may be chimeras, non-chimeric heterozygotes, and non-chimeric homozygotes. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual 2. ndSee ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999). In some embodiments, the transgenic non-human animal has targeted disruption and substitution by a targeted construct encoding the heavy chain and / or light chain of interest. The non-human transgenic animal or plant may include, for example, a nucleotide sequence encoding the heavy chain or its antigen-binding moiety of the anti-MET antibody of the present invention, a nucleotide sequence encoding the light chain or its antigen-binding moiety, or both. In a preferred embodiment, the transgenic animal includes and expresses nucleic acid molecules encoding the heavy and light chains, or their antigen-binding moiety, that specifically bind to human MET. The anti-MET antibody or moiety may be produced in any transgenic animal. In a preferred embodiment, the non-human animal is a mouse, rat, sheep, pig, goat, cattle, or horse. Non-human transgenic animals may express the encoded polypeptide in, for example, blood, milk, urine, saliva, tears, mucus, and other bodily fluids.

[0212] Pharmaceutical composition

[0213] Another aspect of the present invention is a pharmaceutical composition comprising, as an active ingredient (or sole active ingredient), the anti-MET antibody or its antigen-binding moiety, an ADC containing the anti-MET antibody or its antigen-binding moiety, or an anti-MET antibody composition, or an anti-MET antibody-containing ADC composition. In some embodiments, the composition is intended for the remission, prevention, and / or treatment of MET-mediated disorders (e.g., disorders characterized by overexpression of MET) and / or cancer. In certain embodiments, the composition is intended for the remission, prevention, and / or treatment of non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, esophageal cancer, colorectal cancer, papillary renal cell carcinoma, glioblastoma, renal cell carcinoma, prostate cancer, and / or adrenocortical carcinoma.

[0214] Generally, the antibodies or antigen-binding moieties of the present invention are suitable for administration as formulations together with one or more pharmaceutically acceptable excipients(s). The term “excipients” is used herein to describe any component other than the compounds(s) of the present invention. The selection of excipients(s) depends to a considerable extent on factors such as the specific dosage form, the effect of the excipients on solubility and stability, and the properties of the dosage form. As used herein, “pharmaceutically acceptable excipients” include all physiologically compatible solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarders, etc. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. Examples of pharmaceutically acceptable additions include wetting agents or small amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives or buffers, which enhance the shelf life or efficacy of the antibody.

[0215] The pharmaceutical compositions of the present invention and methods for preparing them will be readily apparent to those skilled in the art. Such compositions and methods for preparing them can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Company, 1995). The pharmaceutical compositions are preferably manufactured under GMP (Good Manufacturing Practice) conditions.

[0216] The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk as single-dose units or as multiple single-dose units. The term "unit dose" as used herein refers to... This is a specific amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equivalent to the dose of the active ingredient administered to the subject, or a convenient proportion of such a dose, such as half or one-third of such a dose.

[0217] Any method for administering peptides, proteins, or antibodies approved in the art can be appropriately used for the antibody and antigen-binding portions of the present invention.

[0218] The pharmaceutical compositions of the present invention are typically suitable for parenteral administration. As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by the creation of a physical fissure in the target tissue and the administration of the pharmaceutical composition through the fissure in the tissue, and thus generally, administration into the bloodstream, intramuscularly, or directly into the internal organs. Therefore, parenteral administration includes, but is not limited to, the administration of the pharmaceutical composition by injection, application through surgical incision, or application through a non-surgical wound penetrating the tissue. In particular, parenteral administration is considered to include, but is not limited to, injection or infusion into the subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, subarachnoid space, intraventricular, intraurethral, ​​intracranial, and synovial membranes; and renal dialysis fluid replacement techniques. Local perfusion is also possible. Specific embodiments include intravenous and subcutaneous routes.

[0219] Formulations of pharmaceutical compositions suitable for parenteral administration typically comprise an active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in dosage forms suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit-dose dosage forms, such as ampoules, or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, and pastes. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizers, or dispersants. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a drying form (i.e., powder or granules) for reconstitution using a suitable vehicle (e.g., sterile, pyrogen-free water) before parenteral administration of the reconstituted composition. Parenteral formulations also include aqueous solutions that may contain excipients such as salts, carbohydrates, and buffers (preferably pH 3-9), but in some applications they may be more appropriately formulated as sterile non-aqueous solutions or as desiccants for use with a suitable vehicle such as sterile pyrogen-free water. Exemplary parenteral formulations include solutions or suspensions in sterile aqueous solutions, e.g., aqueous propylene glycol or dextrose solutions. Such formulations may be appropriately buffered if desired. Other useful parenterally administered formulations include those containing the active ingredient in microcrystalline or liposomal forms. Parenteral formulations may be formulated to be immediate-release and / or modified-release. Modified-release formulations include delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release, and programmed-release.

[0220] For example, in one embodiment, a sterile injection solution may be prepared by incorporating an anti-MET antibody, or its antigen-binding moiety, or an anti-MET antibody composition, in the required amount, into a suitable solvent, along with one or a combination of the components listed above, if necessary, and then sterile filtration. Generally, dispersants are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other components from those listed above as needed. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, which result in powders of the active ingredient and any additional desired components from the solution that has been sterile filtered beforehand. Appropriate fluidity of the solution can be maintained, for example, by the use of a coating agent such as lecithin, by maintaining the required particle size in the case of a dispersant, and by the use of a surfactant. Extended absorption of an injection composition may be achieved by including absorption-delaying substances, such as monostearate and gelatin, in the composition, and / or by using a modified release coating (e.g., a slow-release coating).

[0221] The antibodies of the present invention may also be administered intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (either alone, as a mixture, or as particles of the component mixed with a suitable pharmaceutically acceptable excipient), as an aerosol spray with or without the use of a suitable propellant from a pressurized vessel, pump, spray, atomizer (preferably an atomizer using electrohydrodynamics to produce a fine mist), or nebulizer, or as intranasal droplets.

[0222] The pressurized vessel, pump, spray, atomizer, or nebulizer generally contains a liquid or suspension of the antibody of the present invention, which includes, for example, a substance suitable for dispersing, solubilizing, or extending the release of an active propellant(s) as a solvent.

[0223] Before use in dry powder or suspension formulations, the chemicals are generally pulverized to a size suitable for inhalation delivery (typically less than 5 μm). This can be achieved by any suitable pulverization method, such as spiral jet milling, fluidized bed jet milling, supercritical fluid treatment to form nanoparticles, high-pressure homogenization, or spray drying.

[0224] Capsules, blisters, and cartridges for use in inhalers or insufflers may be formulated to contain a powder mixture of the compound of the present invention, a suitable powder base, and a performance modifier.

[0225] A solution formulation suitable for generating a fine mist using electrohydrodynamics in an atomizer may contain an appropriate dose of the antibody of the present invention per operation, and the operating volume may vary, for example, from 1 μL to 100 μL.

[0226] A suitable fragrance agent such as menthol or levomenthol, or a sweetener such as saccharin or sodium saccharin, may be added to such formulations of the present invention intended for inhalation / intranasal administration.

[0227] Formulations for inhalation / intranasal administration may be formulated to be immediate-release and / or modified-release. Modified-release formulations include delayed-release, sustained-release, pulsed-release, controlled-release, targeted-release, and programmed-release.

[0228] In the case of dry powder inhalers and aerosols, the dose unit is determined using a valve that delivers a measured amount. The units according to the present invention are typically adjusted to administer a measured dose or "puff" of the antibody of the present invention. The total daily dose is typically administered as a single dose, or more commonly, in divided doses throughout the day.

[0229] The antibody and antibody portion of the present invention may also be formulated for oral administration. Oral administration may include swallowing, which allows the compound to enter the gastrointestinal tract, and / or administration buccally, lingually, or sublingually (so that the compound enters the bloodstream directly from the mouth).

[0230] Formulations suitable for oral administration include solid, semi-solid, and liquid forms, such as tablets; soft or hard capsules containing multiparticles or nanoparticles; liquids or powders; lozenges (including those filled with liquid); chewable preparations; gels; rapidly disintegrating formulations; films; vaginal suppositories; sprays; and buccal preparations / mucosal adhesive patches.

[0231] Liquid formulations include suspensions, liquids, syrups, and elixirs. Such formulations may be used as fillers for soft or hard capsules (e.g., made from gelatin or hydroxypropyl methylcellulose) and typically comprise a carrier, such as water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifiers and / or suspending agents. Liquid formulations may also be prepared, for example, by restoring solids from sachets.

[0232] Immunoconjugate

[0233] Another option for the therapeutic use of the antibody composition and antibody and its antigen-binding moiety of the present invention is an immunoconjugate, i.e., an antibody or antigen-binding moiety conjugated with one or more drugs such as an anticancer agent.

[0234] Various types of anticancer agents, including cytotoxic agents (e.g., conventional chemotherapeutic agents and other small molecule anticancer agents), cytokines (in this case, the conjugate may be called an "immunocytokine"), toxins (in this case, the conjugate may be called an "immunotoxin"), and radionuclides, may be conjugated to the antibodies of the present invention. Several immunoconjugates have already been approved for clinical use. These include Zevalin® ( 90Mouse anti-CD20 antibody conjugated with Y (yttrium), Baxxar (registered trademark) 131 This includes mouse anti-CD20 antibody conjugated to iodine (I) and Mylotarg® (humanized anti-CD33 antibody conjugated to calicheamicin). Other immunoconjugates being tested in clinical trials include antibodies conjugated to doxorubicin or meitansinoid compounds. Immunotoxins being tested in clinical trials include several antibodies conjugated to cleaved and shortened Pseudomonas exotoxin A. Immune cytokines, including humanized EpCAM (epithelial cell adhesion molecule) antibody conjugated to IL (interleukin)-2, are also being tested.

[0235] In the case of antibodies of the present invention conjugated with cytotoxic agents, these may belong to any of the major classes of chemotherapeutic agents, including alkylating agents (e.g., carboplatin, cisplatin, oxaliplatin), antimetabolites (e.g., methotrexate, capecitabine, gemcitabine), anthracyclines (e.g., bleomycin, doxorubicin, mitomycin C), and plant alkaloids (e.g., taxanes, e.g., docetaxel and paclitaxel, and vinca alkaloids, e.g., vinblastine, vincristine, and vinorelbine). Since the use of immunoconjugates directs anticancer agents specifically to tumors, immunoconjugates based on antibodies of the present invention may advantageously be based on highly cytotoxic agents such as calichemycin or meitansine derivatives, or toxins such as bacterial toxins (e.g., Pseudomonas exotoxin A, diphtheria toxin) or plant toxins (e.g., lysine).

[0236] The conjugated anticancer drug within an immunoconjugate is generally relatively stable in serum, but is linked to the antibody using an unstable linker that enables drug release once the immunoconjugate moves into the target cell. Suitable linkers include, for example, chemical linkers that are stable at the neutral pH of serum but undergo acid hydrolysis under the slightly acidic conditions within lysosomes after internal migration, disulfide linkers that are cleaved by intracellular thiols, and peptide linkers that are stable in serum but undergo enzymatic cleavage in the intracellular compartment.

[0237] For further information on anti-cancer immunoconjugates, see Wu et al., Nature Biotechnology 23(9):1137-1146 (2005); Schrama et al., Nature Reviews / Drug Discovery 5:147-159 (2006); and Rohrer, Chimica Oggi / Chemistry Today 27(5):56-60 (2009).

[0238] Therapeutic use of antibodies and compositions of the present invention

[0239] In one embodiment, the anti-MET antibody and its antigen-binding moiety and anti-MET composition of the present invention are used to treat MET-mediated disorders. In some embodiments, MET-mediated disorders are conditions characterized by overexpression of MET. In certain embodiments, the pharmaceutical composition is for use in treating cancers such as non-small cell lung cancer, gastric cancer, hepatocellular carcinoma, esophageal cancer, colorectal cancer, papillary renal cell carcinoma, glioblastoma, adrenocortical carcinoma, renal cell carcinoma, prostate cancer, and other cancers that express or overexpress MET or rely on MET pathway activation.

[0240] In some embodiments, the antibody or antibody composition is used to treat disorders such as cancer characterized by abnormal MET overactivity. In some embodiments, the abnormal overactivity derives from gene amplification, protein overexpression, gene mutations that activate MET (e.g., point mutations or abnormal gene splicing events), or hepatocyte growth factor overexpression.

[0241] In certain embodiments, the anti-MET antibodies and their antigen-binding moieties and anti-MET compositions of the present invention may be used to treat patients who are resistant to treatment with agents targeting different tyrosine kinase receptors. In some embodiments, the patients are resistant to treatment with ErbB kinase inhibitors. In certain embodiments, the ErbB kinase inhibitor targets EGFR (epidermal growth factor receptor), ErbB2, ErbB3, or ErbB4. In certain embodiments, the ErbB kinase inhibitor targets EGFR. In other embodiments, the ErbB kinase inhibitor targets HER3. The ErbB kinase inhibitor may be selected from, for example, gefitinib, erlotinib, cetuximab, pantinumumab, trastuzumab, or any combination thereof.

[0242] The term “agent” is used herein to mean a compound, a mixture of compounds, a biological molecule, an extract prepared from a biological molecule, or a combination of two or more thereof. The term “therapeutic agent” or “drug” means an agent that can modulate a biological process and / or has biological activity.

[0243] The terms “chemotherapeutic agent” or “anticancer agent” are used herein to refer to all agents that are effective in treating cancer (regardless of their mechanism of action). Inhibition of metastasis or angiogenesis is frequently a characteristic of chemotherapeutic agents. Chemotherapeutic agents include antibodies, biological molecules, and small molecules. Chemotherapeutic agents may be cytotoxic agents or cytostatic agents. The term “cytostatic agent” refers to an agent that inhibits or suppresses cell proliferation and / or cell replication. The term “cytotoxic agent” refers to a substance that primarily causes cell death by interfering with the expression activity and / or function of cells.

[0244] As used herein, the term “cancer” refers to the presence of cells that possess characteristics typical of cancerous cells, such as excessive cell growth or proliferation, uncontrolled proliferation, immortalization, metastatic ability, rapid growth and proliferation rates, and / or specific morphological features. Often, cancer cells may be in the form of a tumor or mass, but such cells may exist alone within a subject or circulate in the bloodstream as independent cells, such as leukemia cells or lymphoma cells. The term “cancer” encompasses all types of cancer and cancer metastases, including hematological cancers, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumor cancers. Hematological cancers include B-cell malignancies, hematological cancers (leukemia), plasma cell cancers (myeloma, e.g., multiple myeloma), or lymph node cancers (lymphoma). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemias include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), and acute monocytic leukemia (AMoL). The terms "acute lymphoblastic leukemia" and "acute lymphocytic leukemia" can be used synonymously, and this refers to acute lymphoblastic leukemia (ALL). Lymphomas may include Hodgkin lymphoma, non-Hodgkin lymphoma, etc. Other hematological cancers may include myelodysplastic syndrome (MDS). Solid tumors may include cancers such as adenocarcinomas, such as breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, stomach cancer, cervical cancer, endometrial cancer, ovarian cancer, bile duct cancer, glioma, and melanoma.In some embodiments, cancer includes melanoma, uveal melanoma, renal cancer, kidney cancer, thyroid cancer, mesothelioma, liver hepatocellular cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), and gastric cancer. Cancers include, but are not limited to, pancreatic cancer, colorectal cancer, esophageal cancer, cholangiocarcinoma, head and neck cancer (including oral cancer), cervical and intracervical cancer, bladder and urothelial cancer, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, adrenocortical cancer, brain cancer, splenic cancer, thymoma, multiple myeloma, plasmacytotic myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia (including acute lymphoblastic leukemia), follicular lymphoma, lymphoid malignancies originating from T cells or B cells, hematological cancers, myeloid leukemia, or myeloma. In some embodiments, cancer is lung cancer, pancreatic cancer, or gastric cancer. The terms “tumor” and “cancer” may be used synonymously herein and refer to a mass of cells exhibiting excessive cell growth or proliferation. The terms “tumor cell” and “cancer cell” may be used as synonyms herein.

[0245] The terms “patient” and “subject” are used herein as synonyms and refer to any human or non-human animal requiring treatment. Non-human animals include all vertebrates (e.g., mammals and non-mammals), including any mammal. Non-limiting examples of mammals include humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs. Non-limiting examples of non-mammals include birds and fish. In some embodiments, the patient is human.

[0246] As used herein, the term “subjects in need of treatment” refers to subjects who would benefit from treatment (e.g., treatment with one or more exemplary antibodies or ADC compounds) biologically, medically, or in terms of quality of life.

[0247] As used herein, the terms “to treat,” “to treat,” or “treatment” refer to any improvement of any outcome of a disease, disorder, or condition, such as an extended survival, lower morbidity, and / or reduction of side effects resulting from an alternative treatment. In some embodiments, treatment includes delay or remission of a disease, disorder, or condition (i.e., slowing, cessation, or reduction of the onset of at least one of the disease or its clinical symptoms). In some embodiments, treatment includes delay, mitigation, or remission of at least one physical parameter of a disease, disorder, or condition (including one that may not be identifiable by the patient). In some embodiments, treatment includes modulating a disease, disorder, or condition either physically (e.g., stabilization of identifiable symptoms), physically (e.g., stabilization of physical parameters), or both. In some embodiments, treatment includes administration to a subject, for example, a patient, of the described antibody or ADC compound or composition, thereby obtaining the benefits of the treatment enumerated herein. Treatment may cure, alleviate, delay, prevent, mitigate, modify, treat, relieve, reduce, improve, or affect a disease, disorder or condition (e.g., cancer), symptoms of a disease, disorder or condition (e.g., cancer), or predisposition to a disease, disorder or condition (e.g., cancer). In some embodiments, in addition to treating a subject having a disease, disorder or condition, the compositions disclosed herein may also be administered preventively to prevent or reduce the probability of developing such a disease, disorder or condition.

[0248] In this specification, the terms “prevent,” “prevent,” or “prevention” refer to preventive measures for a disease, disorder, or condition; or to delaying the onset or progression of a disease, disorder, or condition.

[0249] As used herein, the terms “therapeutic dose” or “therapeutic effective dose” refer to the amount of a compound described herein, such as an anti-MET antibody or its antigen-binding moiety, an ADC compound containing an anti-MET antibody or its antigen-binding moiety, or a composition of an anti-MET antibody or ADC compound, that produces a desired therapeutic outcome (i.e., reduction or inhibition of enzyme or protein activity, remission of symptoms, alleviation of symptoms or condition, delay of disease progression, regression of tumor size, inhibition of tumor growth, or prevention of metastasis). In some embodiments, the therapeutic dose is effective in terms of detectable killing, reduction, and / or inhibition of cancer cell proliferation or spread, tumor size or number, and / or other measures relating to the level, stage, progression, and / or severity of cancer. The term also applies to a dose that induces a specific response within target cells, such as reduction, slowing, or inhibition of cell proliferation. The therapeutic dose can be determined by administering a low dose first, and then gradually increasing the dose until the desired effect is achieved. In the case of cancer, a therapeutically effective dose of an anti-MET antibody or an ADC containing an anti-MET antibody may reduce the number of cancer cells, regress tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and / or alleviate one or more symptoms.

[0250] As used herein, the terms “preventive effective dose” or “preventively effective dose” refer to the amount of any of the compounds disclosed herein, such as the anti-MET antibody or its antigen-binding moiety, an ADC compound containing the anti-MET antibody or its antigen-binding moiety, or a composition of the anti-MET antibody or ADC compound, that is effective in the dose and over the period necessary to achieve the desired preventive outcome. Typically, since the preventive dose is used for a target before the onset of the disease or at an earlier stage of the disease, the preventive effective dose is less than the therapeutic effective dose. In some embodiments, the preventive effective dose can prevent the onset of symptoms of a disease, including symptoms associated with cancer.

[0251] The antibody composition or antibody or its antigen-binding moiety of the present invention may be administered alone or in combination with one or more other drugs or antibodies (or in any combination thereof). Thus, the pharmaceutical composition, method and use of the present invention also encompass embodiments of combination (co-administration) with other active substances, as detailed below.

[0252] In this specification, the terms “co-administered,” “co-administered,” and “in combination with” used to refer to an antibody composition and an antibody and its antigen-binding moiety together with one or more other therapeutic agents are intended to mean, and refer to and include: • Simultaneous administration of such combinations of the antibody composition / antibody / antigen binding moiety of the present invention and therapeutic agents(group) to a patient requiring treatment (when such components are formulated together into a single dosage form, this releases the components to the patient substantially at the same time), • Substantially simultaneous administration of such combinations of the antibody composition / antibody / antigen binding moiety of the present invention and therapeutic agents(group) to a patient requiring treatment (when such components are isolated from each other and formulated into separate dosage forms and taken by the patient substantially simultaneously, the components are released to the patient substantially at the same time). • Sequential administration of such combinations of antibody compositions / antibody / antigen binding moieties and therapeutic agents(groups) of the present invention to patients in need of treatment (such components are isolated from each other and formulated into separate dosage forms, which are taken by the patient consecutively with a considerable time interval between administrations, so that the components are released to the patient at substantially different times); and • Sequential administration of such combinations of antibody compositions / antibody / antigen binding moieties and therapeutic agents(groups) of the present invention to patients in need of treatment (when such components are formulated together into a single dosage form, this releases the components in a controlled manner, at which time the components are released simultaneously, sequentially, and / or overlapping to the patient at the same time and / or at different times, where each part may be administered by the same route or by different routes).

[0253] The antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC of the present invention may be administered without additional therapeutic treatment, i.e., as monotherapy. Alternatively, the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC of the present invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC of the present invention may be co-administered or formulated with another drug therapy / drug for the treatment of cancer. Additional therapeutic treatments may include, for example, chemotherapeutic agents, antineoplastic agents, anti-angiogenic agents, different anti-cancer antibodies, tyrosine kinase inhibitors, MET pathway inhibitors, and / or radiotherapy.

[0254] The efficacy of the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC, can be further improved by combining them with agents known to induce terminal differentiation of cancer cells. Such compounds may be selected from the group consisting of, for example, retinoic acid, trans-retinoic acid, cis-retinoic acid, phenyl butyrate, nerve growth factor, dimethyl sulfoxide, active vitamin D3, peroxisome proliferator-activated receptor γ, 12-O-tetradecanoylphorbol 13-acetate, hexamethylene-bis-acetamide, transforming growth factor β, butyrate, cyclic AMP, and vesnarinone. In some embodiments, the compound is selected from the group consisting of retinoic acid, phenyl butyrate, total trans-retinoic acid, and active vitamin D.

[0255] A pharmaceutical composition comprising the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC, and at least one other agent (e.g., a chemotherapeutic agent, an antineoplastic agent, or an anti-angiogenic agent) may be used as a combination treatment for simultaneous, separate, or sequential administration in cancer treatment. The other agent may be any agent suitable for the treatment of the specific cancer in question, such as alkylating agents, e.g., platinum derivatives, e.g., cisplatin, carboplatin, and / or oxaliplatin; plant alkaloids, e.g., paclitaxel, docetaxel, and / or irinotecan; antitumor antibiotics, e.g., doxorubicin (adriamycin), daunorubicin, epirubicin, idarubicin, mitoxantrone, dactinomycin, bleomycin, actinomycin, luteomycin, and / or mitomycin; topoisomerase inhibitors, e.g., topotecan; and / or antimetabolites, e.g., fluorouracil and / or other fluoropyrimidines.

[0256] The antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC, may also be used in adjuvant therapy in conjunction with tyrosine kinase inhibitors. These are synthetic, low molecular weight molecules mainly derived from quinazolines, which interact with the intracellular tyrosine kinase domain of the receptor and can inhibit ligand-induced receptor phosphorylation by competing with the intracellular magnesium (Mg)-ATP binding site. A pharmaceutical comprising the antibody composition of the present invention and at least one tyrosine kinase inhibitor targeting MET may also be used as a combination treatment for simultaneous, separate, or sequential administration in cancer therapy.

[0257] In certain embodiments, the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC may be administered in combination with another inhibitor of the MET pathway that can target MET or HGF (hepatocyte growth factor). In some embodiments, the inhibitor may be AMG102, AMG208, AMG458, ARQ197, AV299, BAY-853474, CGEN241, DN30, E7050, EMD1204831, EMD1214063, INCB28060, JNJ38877605, K252a, LY-2875358, MGCD265, MK-2461, MP-470, NK4, OA-5D5, PF- The following are selected, but are not limited to, the group consisting of 02341066, PF-04217903, PF-02341066, PHA-665752, SGX-523, SU5416, SU11274, TAK701, XL184, XL880, cabozantinib, crizotinib, ficratuzumab, foretinib, golbatinib, onartuzumab, amibantamab, rilotumumab, and tivantinib.

[0258] In some embodiments, the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC may be administered in combination with an ErbB inhibitor (such as gefitinib or erlotinib) or a heat shock protein 90 (hsp90) inhibitor (such as 17-AAG).

[0259] In other embodiments, the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC may be used in combination with other antibody therapeutics, such as antibodies against VEGF (vascular endothelial growth factor) (e.g., Avastin®). In yet another embodiment, the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding moiety or ADC may be used in combination with agents known to stimulate cells of the immune system, and such combination treatment enhances the immunomediated enhancement of the efficacy of the antibody composition of the present invention. Examples of such immunostimulants include recombinant interleukins (e.g., IL-21 and IL-2).

[0260] It is understood that the antibody or ADC composition of the present invention, the anti-MET antibody and its antigen-binding portion or ADC of the present invention may be used in, for use in, and / or for use in the manufacture of pharmaceuticals for, such treatments.

[0261] Dosage and route of administration

[0262] The antibody composition or ADC composition of the present invention is administered in an effective amount for treating the condition in question, i.e., in the dose and duration necessary to achieve the desired result. The therapeutically effective dose may be modified depending on factors such as the specific condition being treated, the patient's age, sex and weight, and whether the antibody or ADC is administered as a monotherapy or in combination with one or more additional anticancer treatments.

[0263] The dose regimen may be adjusted to produce the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and dose uniformity, it is particularly advantageous to formulate parenteral compositions in dose-unit dosage forms. As used herein, dose-unit dosage forms refer to physically distinct units suitable as unit doses for the patient / subject to be treated; each unit contains a predetermined amount of the active compound, along with the required pharmaceutically acceptable carrier, calculated to produce the desired therapeutic effect. The details of the dose-unit dosage forms of the present invention are generally indicated and directly dependent on (a) the inherent characteristics of the chemotherapeutic agent and the specific therapeutic or prophylactic effect to be achieved, and (b) the inherent limitations of the technique for formulating such active compounds for the treatment of hypersensitivity in the individual.

[0264] Accordingly, those skilled in the art will understand that, based on the disclosures provided herein, doses and regimens are adjusted according to methods well known in the therapeutic field. That is, the maximum tolerable dose can be easily established, the effective dose that provides a detectable therapeutic benefit to the patient can be determined, and the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the patient can be determined in the same way. Thus, although specific doses and administration regimens are illustrated herein, these examples do not limit in any way the doses and administration regimens that may be provided to a patient when carrying out the present invention.

[0265] The numerical dose may be modified along with the type and severity of the condition to be alleviated, and it should be noted that this may include single doses or multiple doses. For any particular subject, the specific dose regimen should be adjusted over time according to the individual needs and the professional judgment of the administerer or supervisor of the administration of the composition, and it should be further understood that the dose ranges shown herein are merely illustrative and are not intended to limit the range or implementation of the embodied composition. Furthermore, dose regimens using the compositions of the present invention may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, condition, severity of the condition, route of administration, and the specific antibody used. Thus, dose regimens can vary widely but can be conventionally determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or clinical laboratory values. Thus, the present invention includes intra-patient dose escalation as determined by those skilled in the art. The determination of appropriate dosages and regimens is well known in the relevant art and, once the teachings disclosed herein are provided, will be understood to be encompassed by those skilled in the art.

[0266] Appropriate doses of the antibody composition or ADC composition of the present invention may be 0.1 to 100 mg / kg, for example, about 0.5 to 50 mg / kg, for example, about 1 to 20 mg / kg. The antibody composition may be administered in doses of, for example, at least 0.25 mg / kg, for example at least 0.5 mg / kg, for example at least 1 mg / kg, for example at least 1.5 mg / kg, for example at least 2 mg / kg, for example at least 3 mg / kg, for example at least 4 mg / kg, for example at least 5 mg / kg; and for example up to 50 mg / kg, for example up to 30 mg / kg, for example up to 20 mg / kg, for example up to 15 mg / kg. Administration is usually repeated at appropriate intervals, for example once every week, once every two weeks, once every three weeks, or once every four weeks, as long as the physician deems it appropriate, and the physician may increase or decrease the dose as needed.

[0267] The effective dose for tumor therapy can be measured by its ability to reverse disease progression, for example by stabilizing disease progression and / or relieving symptoms in the patient, such as by regressing tumor size. The ability of the antibodies or compositions of the present invention to suppress cancer can be evaluated by in vitro assays, for example as described in the examples, and in appropriate animal models to predict efficacy in human tumors. An appropriate dose regimen is selected to provide the optimal therapeutic response in each specific situation and is administered, for example, as a single bolus or as a series of infusions, with the possibility of adjusting the dose as indicated by the urgency of each case.

[0268] Diagnostic Use and Composition

[0269] The antibodies of the present invention and the ADCs of the present invention are also useful in diagnostic processes (e.g., in vitro, ex vivo). For example, the antibodies and ADCs can be used to detect and / or measure the level of MET in patient-derived samples (e.g., tissue samples, or body fluid samples such as inflammatory exudates, blood, serum, intestinal fluid, saliva or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescent assay, radioimmunoassay and immunohistochemical examination. The present invention further encompasses kits (e.g., diagnostic kits) containing the antibodies described herein.

[0270] For a better understanding of the present invention, the following examples are provided. These examples are for illustrative purposes only and should in no way be construed as limiting the scope of the present invention.

[0271] For the purpose of clarity of understanding, the present invention has been described in some detail using examples and illustrations. However, it will be readily apparent to those skilled in the art that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended embodiments in view of the teachings of the present invention.

Examples

[0272] Example 1 - Cloning of Anti-MET Antibody Anti-MET antibodies were obtained using the Symplex® procedure, essentially as described in International Publication No. 2005 / 042774. Briefly, BALB / c, C57, and C3H mice were immunized every two weeks with either a human cancer cell line overexpressing MET (HCT-116), recombinant human MET protein (Sinobiological), recombinant human MET protein pre-incubated with a ligand (hepatocyte growth factor), or MET digested with trypsin. Mouse plasma cells obtained from the spleen and inguinal lymph nodes were sorted using FACS (fluorescence-activated cell sorting), and VH-coding sequences and VL-coding sequences were linked on the sorted plasma cells using a two-step PCR procedure based on nested PCR following one-step multiple overlap-extension reverse transcription PCR, after promoting congenital sequence pairing. The principle of linking congeneral VH and VL sequences is described in detail in International Publication No. 2005 / 042774 and Meijer et al., J Mol Biol 358(3):764-72(2006).

[0273] To identify antibodies with binding specificity to MET, the VH and VL coding sequences obtained above were expressed as full-length antibodies. This involved inserting the repertoire of VH and VL coding pairs into an expression vector and transfection into host cells using the method described in International Publication No. 2012 / 059858.

[0274] The specificity of the generated antibodies was determined by ELISA using either the extracellular domain of the MET protein or the extracellular domain of the MET protein translated into a human immunoglobulin Fc domain as the antigen. Nunc MaxiSorp plates (lot number 464718) were coated overnight at 4°C with 1 μg / ml recombinant MET protein diluted in PBS. The plates were washed once with PBS + 0.05% Tween20 (PBS-T) and then blocked in 50 μl of 2% milk-PBS-T. The plates were washed again once with PBS-T and then with 20 μl of 2% milk-PBS-T. 10 μl of supernatant derived from the FreeStyle293 transfect was added and incubated at room temperature for 1 hour, after which the plates were washed once with PBS-T. Secondary antibody (horseradiol peroxidase-goat-anti-human kappa light chain, Cerotec, lot number STAR100P), diluted 1:25000 in 2% milk-PBS-T, was added, and antibodies bound to the wells were detected. The mixture was incubated at room temperature for 1 hour. The plate was washed once with PBS-T, then 25 μl of substrate (Kem-En-Tec Diagnostics, lot number 4518) was added and incubated for 5 minutes. The reaction was stopped by adding 25 μl of 1 M sulfuric acid after incubation. A specific signal at 450 nm was detected on an ELISA reader. From the ELISA data, positive antibody clones (i.e., antibodies 9338, 8902, and 9006) were identified and selected for sequence analysis and validation of binding to MET.

[0275] The sequence numbers for the CDR, VH, and VL domains of a novel anti-MET antibody 8902 targeted against human MET, as well as the corresponding DNA sequences for the VH and VL domains, are provided herein. The amino acid sequences of the variable domains (VH and VL) of the heavy and light chains are provided in Sequence IDs 7 and 8, respectively, and the corresponding nucleotide sequences are provided in Sequence IDs 9 and 10, respectively. The full-length amino acid sequences of the heavy and light chains (HC and LC) are available in Sequence IDs 11 and 12 (IgG1 chain) and Sequence IDs 13 and 14 (IgG2 chain), respectively. The amino acid sequences of the heavy chain CDRs (H-CDR1, H-CDR2, and H-CDR3) and the light chain CDRs (L-CDR1, L-CDR2, and L-CDR3) of the 8902 antibody are shown in Sequence IDs 1, 2, and 3, and Sequence IDs 4, 5, and 6, respectively. The CDR sequences were assigned according to the definition of IMGT®.

[0276] Example 2 - Epitope binning of anti-MET antibodies This example describes the grouping of anti-MET antibodies against epitope bins based on pair-forming competition patterns measured by biolayer interferometry (BLI). Antibodies belonging to different bins recognize different epitopes on the cMET extracellular domain (ECD).

[0277] Competition among paired antibodies was investigated using biolayer interferometry with an Octet® RH96 instrument (Sartorius). All proteins were diluted in kinetic buffer (Sartorius):phosphate-buffered saline (PBS) in a 1:10 ratio, and the experiment was conducted at 25°C. Human recombinant histidine-tagged cMET (Sinobiological) was captured for 300 seconds on a pre-equilibrated and regenerated anti-penterhistidine (HIS1K) biosensor (Sartorius). Subsequently, a 300 nM anti-MET antibody (primary antibody) was associated with the cMET over 150 seconds, and a 300 nM secondary cMET antibody (secondary antibody) was dissociated over 300 seconds. The sensor was regenerated in 10 mM 1.5 glycine (pH 1.5). Data were analyzed using Octet Biolayer Interferometry Analysis Studio (version 12).

[0278] Competitive analysis of cMET antibodies was performed as a tandem assay using biolayer interferometry: human recombinant biotinylated cMET antigen was captured and, under saturated antigen-binding conditions, conjugated with a primary antibody followed by a secondary antibody. Each antibody pair was defined as a blocker or non-blocker based on lack of response or response greater than 0.1 nm.

[0279] Tables 1 and 2 below show the competition among anti-cMET antibodies 9338 (HC and LC sequences are provided herein in SEQ ID NOs. 19 and 20 for IgG1 type and SEQ ID NOs. 21 and 22 for IgG2 type), 9006 (HC and LC sequences are provided in SEQ ID NOs. 15 and 16 for IgG1 type and SEQ ID NOs. 17 and 18 for IgG2 type), 8902, terisotuzumab (HC and LC sequences are provided in SEQ ID NOs. 23 and 24 for IgG1 type and SEQ ID NOs. 25 and 26 for IgG2 type), and bispecific amivantamab for IgG1 type (HC-1 and HC-2 sequences are provided in SEQ ID NOs. 27 and 28, and LC-1 and LC-2 sequences are provided in SEQ ID NOs. 29 and 30). The response (nm) of the captured antigen-antibody complex bound to the secondary antibody is shown. Dark gray highlights self-blocking. Grouping into epitope bins 1-4 is highlighted in light gray and numbered in the table.

[0280] [Table 1]

[0281] [Table 2]

[0282] Epitope formation is isotype-independent, as confirmed by equivalent epitope binning data for IgG1 and IgG2 antibodies. All antibodies self-compete, as indicated by dark gray. Epitope binning analysis showed that cMET antibodies could be grouped into four distinct epitope bins, highlighted in light gray and numbered bins 1-4. Antibody 9338 and amivantamab bound to similar epitopes (bin 2), while antibodies 8902 (bin 1), 9006 (bin 4), and terisotuzumab (bin 3) each bound to unique, non-competing epitopes.

[0283] Example 3 - Measurement of affinity of human and cynomolgus monkey MET to anti-MET antibody by surface plasmon resonance (SPR)

[0284] The experiment was conducted at 25°C using a ViaCore T200 (Cytiva) in 10 mM HEPES, 150 mM NaCl (pH 7.4), and 0.05% P20 as running buffer.

[0285] Anti-human IgG(Fc) antibody (Human Antibody Capture Kit, Cytiva) was immobilized on two flow cells (reference flow cell and active flow cell) of a CM5 sensor tip by amine coupling according to the manufacturer's instructions. Anti-MET antibody was captured at 200 ng / mL on the active flow cell at 10 μL / min over 60 seconds.

[0286] Binding is measured by injecting human or cynomolgus monkey MET extracellular domains at 50 μl / min in five consecutive infusions at increasingly increasing concentrations (SCK-single-cycle kinetics), followed by 240 seconds of binding and 900 seconds of dissociation.

[0287] The surface of the anti-MET antibody / MET complex is washed by regeneration by injecting a 3M MgCl2 solution into both flow cells at a rate of 20 μl / min over a period of 30 seconds.

[0288] A double reference was used by subtracting both the reference flow cell and the previous blank cycle (including running buffer). Sensorgrams were fitted using the monovalent 1:1 kinetic binding model with Biacore T200 evaluation software (version 3.2).

[0289] Fitting enabled determination of the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (KD = kd / ka) for each complex.

[0290] The following table shows the mean values and corresponding standard errors for each antibody using the human MET extracellular domain (Table 3) and cynomolgus MET extracellular domain (Table 4).

[0291]

Table 3

[0292]

Table 4

[0293] Measurements of affinity and reaction rate showed that all anti-MET antibodies bind to either the human or cynomolgus MET extracellular domain, independent of their isotype being IgG1 or IgG2 and independent of the mutations V205C and S400C carried on their light and heavy chains, respectively. The 8902 antibody showed the best affinity for the human MET extracellular domain compared to the 9006 and 9338 antibodies (similar to terisotuzumab).

[0294] Example 4 - Measurement of the binding affinity of antibodies to cMET using flow cytometry

[0295] IgG1 or IgG2 anti-cMET antibodies 9006, 8902, and 9338 were tested for binding to the extracellular domains of human and cynomolgus monkey cMET transiently transfected into CHO-S cells by flow cytometry (iQue® Screener PLUS, IntelliCyt, Sartorius). Furthermore, IgG1 or IgG2 reference anti-cMET antibodies terisotuzumab and IgG1 amibantamab were also tested for binding to human or cynomolgus monkey cMET in CHO-S cells. IgG1 or IgG2 isotype control antibodies and pseudotransfected cells were used as negative controls. Antibodies were titrated in double denominations at 3-fold dilutions from 30 μg / ml to 0.5 ng / ml.

[0296] By labeling various populations of transfected CHO-S cells with different intensities using the encoding dyes VL-1 BV421 (Sartorius, 97055) or BL-1 FITC (Sartorius, 90355), it became possible to test more targets per well while still being able to distinguish between different cell populations.

[0297] Antibodies were incubated with CHO-S cells on ice for 30 minutes. After washing, goat anti-human IgG (heavy chain + light chain) (A-21445, Invitrogen) labeled with secondary AF647 fluorescent dye was added, and the cells were incubated on ice for 20 minutes. After washing, antibody binding was detected using a high-throughput flow cytometer iQue Screener PLUS (Sartorius), and the geometric mean of the AF647 signal in each well of both experiments was measured.

[0298] Figures 1A and 1B show the binding curves of IgG1 and IgG2 type anti-cMET antibodies 9006, 8902, and 9338, terisotuzumab, and amivantamab to the extracellular domains of human or cynomolgus monkey cMET expressed on CHO-S cells.

[0299] The assayed antibodies bind to human and cynomolgus monkey cMET proteins presented on cells. Antibodies 8902 and 9338 bind to both human and cynomolgus monkey cMETs with better strength than 9006 and terisotuzumab. However, antibody 9338 also binds to some extent to pseudotransfected CHO-S cells. IgG1 appears to be potenter than IgG2 for antibodies 8902 and 9338, although the binding of IgG1 and IgG2 antibodies is still comparable overall.

[0300] The EC50 (half-maximum effective concentration) values ​​are shown in Table 5 in ng / ml.

[0301] [Table 5]

[0302] Example 5 - Synthesis of antibody-drug conjugates

[0303] Exemplary antibody-drug conjugates (ADCs) were synthesized using either IgG1 or IgG2 type 8902 antibody, 9006 antibody, 9338 antibody, and terisotuzumab antibody, employing the following conjugation method.

[0304] A commercially available linker-payload, MC-VC-PAB-MMAE (CAS: 646502-53-6, Intertim), is used in the synthesis of the exemplified ADC, where the linker is MC-VC-PAB and the payload is MMAE.

[0305] The exemplified ADCs were synthesized using naturally occurring 8902, 9006, 9338, and terisotuzumab antibodies, and the conjugation of the linker-payload to the maleimide group was carried out via naturally occurring interchain disulfide bonds through stochastic conjugation with an average drug-to-antibody ratio of 4. Alternatively, cysteine ​​mutations incorporated into the heavy chain (HC S400C) and light chain (LC V205C) of the peptide backbone were induced in the 8902, 9006, 9338, and terisotuzumab antibodies, and their conjugation was carried out to the maleimide group of the linker-payload, resulting in an average drug-to-antibody ratio of 4.

[0306] 5.1-Procedure for stochastic conjugation of IgG1 and IgG2 onto native antibodies

[0307] 5.1.a-Conjugation Method M1 Conjugation onto IgG1 monoclonal antibody was performed within the range of 6 mg of antibody. To the antibody, 10 mM EDTA solution in 1x PBS (pH 7.4) was added in a 1:1 ratio (v / v), followed by 1 M TCEP hydrochloride solution in 1x PBS (pH 7.4) in a 3x molar excess. The mixture was incubated at +37°C for 2 hours. After reduction, the antibody solution was cooled to room temperature, and a 6x molar excess of 5 mM linker payload solution was added to the mixture. The reaction mixture was incubated at +4°C for 1 hour and 30 minutes. The conjugation was monitored by hydrophobic interaction column chromatography (HIC) using a TSKgel butyl-NPR column (Tosoh Biosciences, 0014947) with mobile phase A (1.5 M ammonium sulfate (NH4) 2SO4, 25 mM dipotassium hydrogen phosphate (K2HPO4), pH 7) and mobile phase B (25 mM dipotassium hydrogen phosphate (K2HPO4), 20% isopropanol, pH 7). All exemplified ADCs synthesized using this method were buffer-exchanged by dialysis (Thermo Fisher, 88254) in 1x PBS (pH 7.4) (Sigma Life Sciences, P3813, 10PAK) over 2 hours at room temperature, purified using 1x PBS (pH 7.4) on a preparative size exclusion chromatography column HiLoad® 26 / 600 Superdex® 200, concentrated using Vivaspin 20, 50KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES (polyethersulfone) filter 25 mm (Whatmann, G896-2502), and stored at +4°C.

[0308] 5.1.b-Conjugation Method M2 Conjugation on IgG1 or IgG2 monoclonal antibodies was performed within the range of 6 mg of antibody. To the antibody, 10 mM EDTA solution in 1x PBS (pH 7.4) was added in a 1 / 1 (v / v) ratio, followed by 1 M TCEP hydrochloride solution in 1x PBS (pH 7.4) in a 6-fold molar excess. The mixture was incubated at +37°C for 2 hours. After reduction, the antibody solution was cooled to room temperature, and a 10-fold molar excess of 5 mM linker payload solution was added to the mixture. The reaction mixture was incubated at +4°C for 2 hours. The conjugation was monitored by hydrophobic interaction column chromatography using a TSKgel butyl-NPR column (Tosoh Biosciences, 0014947) with mobile phase A (1.5 M ammonium sulfate (NH4) 2SO4, 25 mM dipotassium hydrogen phosphate (K2HPO4), pH 7) and mobile phase B (25 mM dipotassium hydrogen phosphate (K2HPO4), 20% isopropanol, pH 7). All exemplified ADCs synthesized using this method were buffer-exchanged by dialysis (Thermo Fisher, 88254) in 1x PBS (pH 7.4) (Sigma Life Sciences, P3813, 10PAK) over 2 hours at room temperature, purified using 1x PBS (pH 7.4) on a preparative size exclusion chromatography column HiLoad® 26 / 600 Superdex® 200, concentrated using Vivaspin 20, 50KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES (polyethersulfone) filter 25 mm (Whatmann, G896-2502), and stored at +4°C.

[0309] 5.1.c-Conjugation Method M3

[0310] Conjugation on IgG2 monoclonal antibody was performed within the range of 13 mg of antibody. To this antibody, 10 mM EDTA solution in 1x PBS (pH 7.4) was added in a 1 / 1 (v / v) ratio, followed by 1 M TCEP hydrochloride solution in 1x PBS (pH 7.4) in a 50-fold molar excess. The mixture was incubated at +37°C for 2 hours. After reduction, the antibody solution was cooled to room temperature, and a 25-fold molar excess 5 mM linker payload solution was added to the mixture. The reaction mixture was incubated at +4°C for 1 hour and 30 minutes. The conjugations were monitored by hydrophobic interaction column chromatography using a TSKgel butyl-NPR column (Tosoh Biosciences, 0014947) with mobile phase A (1.5 M ammonium sulfate (NH4) 2SO4, 25 mM dipotassium hydrogen phosphate (K2HPO4), adjusted to pH 7) and mobile phase B (25 mM dipotassium hydrogen phosphate (K2HPO4), 20% isopropanol, adjusted to pH 7). All exemplified ADCs synthesized using this method were conjugated to protein A resin (GE Healthcare), a reductivable protein, in a ratio of 10 mg antibody to 1 ml resin. This step was carried out by adding 1x PBS (pH 7.4) to obtain a solvent of only 5% in the slurry and by mixing for 30 minutes in a Bio-Rad sized disposable column. In a vacuum manifold, a 5x50 column volume of resin was washed with 5% DMSO / PBS solution, followed by washing with 1x the column volume of PBS (pH 7.4) to remove excess linker-payload. The ADC was then eluted with IgG elution buffer, and the buffer was replaced by dialysis (Thermo Fisher, 88254) in 1x PBS (pH 7.4) (Sigma Life Sciences, P3818, 10PAK) at room temperature for 2 hours.All exemplified ADCs obtained by this method were purified using 1x PBS (pH 7.4) on a preparative size exclusion chromatography column HiLoad® 26 / 600 Superdex® 200, concentrated using Vivaspin 20, 50KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES (polyethersulfone) filter 25 mm (Whatmann, G896-2502), and stored at +4°C.

[0311] 5.2-HC S400C LC V205C antibody site-specific cysteine ​​conjugation procedure

[0312] 5.2. Conjugation method on a-IgG1 monoclonal antibody M4

[0313] Conjugation was performed within the range of 10 mg of antibody. To this antibody, 10 mM EDTA solution in 1x PBS (pH 7.4) was added in a 1 / 1 (v / v) ratio, followed by 1 M TCEP hydrochloride solution in 1x PBS (pH 7.4) in a 10-fold molar excess. The mixture was stirred at +37°C for 2 hours. After reduction, the antibody was cooled to room temperature, and the buffer was replaced by dialysfiltration using vivaspin 20, 50 kD, and PES (polyethersulfone) (Sartorius Stedim, VS2031). To the recovered and reduced antibody, 1x PBS (pH 7.4) was added to reach the initial antibody volume, and then the monoclonal antibody was reoxidized at room temperature for 1 hour and 45 minutes using 10 mM DHAA solution in 20-fold molar excess DMSO / PBS (1 / 1). After removing excess DHAA by dialysfiltration using Vivaspin 20, 50 KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), 1x PBS (pH 7.4) was added to the solution to reach the initial antibody volume. To the mixture, DMSO, which was no more than 20% of the final reaction volume, and a 10-fold molar excess of 5 mM linker-payload solution were added. The reaction mixture was stirred at 500 rpm for 2 hours at room temperature. Reverse-phase chromatography was used to monitor conjugation using an Agilent PLRP-S column, 4000 Å, 5 μm, 4.6 × 50 mm (buffer A: water, 0.1% trifluoroacetic acid; buffer B: acetonitrile, 0.1% trifluoroacetic acid; column held at +80°C; flow rate 1.5 ml / min). All exemplified ADCs obtained by this method were purified using 1x PBS (pH 7.4) on a preparative size exclusion chromatography column HiLoad® 26 / 600 Superdex® 200, concentrated using Vivaspin 20, 50KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES (polyethersulfone) filter 25 mm (Whatmann, G896-2502), and stored at +4°C.

[0314] 5.2. Conjugation method on β-IgG2 monoclonal antibody M5 Conjugation was performed within the range of 10 mg of antibody. To the monoclonal antibody, 10 mM EDTA solution in 1x PBS (pH 7.4) was added in a 1 / 1 (v / v) ratio, followed by 1 M TCEP hydrochloride solution in 1x PBS (pH 7.4) in a 10-fold molar excess. The mixture was stirred at +37°C for 2 hours. After reduction, the antibody was cooled to room temperature, and the buffer was replaced by dialysfiltration using vivaspin 20, 50 kD, and PES (polyethersulfone) (Sartorius Stedim, VS2031). To the recovered and reduced antibody, 1x PBS (pH 7.4) was added to reach the initial antibody volume, and then the antibody was reoxidized at room temperature for 3 hours with 10 mM DHAA solution in 40-fold molar excess DMSO / PBS (1 / 1). After removing excess DHAA by diafiltration using Vivaspin 20, 50 kD, PES (polyethersulfone) (Sartorius Stedim, VS2031), 1x PBS (pH 7.4) was added to the solution to reach the initial antibody volume. Then, DMSO, which was the solvent not exceeding 20% ​​of the final reaction volume, and a 10-fold molar excess of 5 mM linker-payload solution were added to the mixture. The reaction mixture was stirred at 500 rpm for 2 hours at room temperature. Reverse-phase chromatography was used to monitor conjugation using an Agilent PLRP-S column, 4000 Å, 5 μm, 4.6 × 50 mm (buffer A: water, 0.1% trifluoroacetic acid; buffer B: acetonitrile, 0.1% trifluoroacetic acid; column held at +80°C; flow rate 1.5 ml / min). The ADCs exemplified by this method were purified using 1x PBS (pH 7.4) on a preparative size exclusion chromatography column HiLoad® 26 / 600 Superdex® 200, concentrated using Vivaspin 20, 50KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES (polyethersulfone) filter 25 mm (Whatmann, G896-2502), and stored at +4°C.

[0315] 5.2. Conjugation method on c-IgG2 monoclonal antibody M6 Conjugation was performed within the range of 10 mg of antibody. To the monoclonal antibody, 10 mM EDTA solution in 1x PBS (pH 7.4) was added in a 1 / 1 (v / v) ratio, followed by 1 M TCEP hydrochloride solution in 1x PBS (pH 7.4) in a 10-fold molar excess. The mixture was stirred at +37°C for 2 hours. After reduction, the antibody was cooled to room temperature, and the buffer was replaced by diafiltration using vivaspin 20, 50 kD, and PES (polyethersulfone) (Sartorius Stedim, VS2031). To the recovered and reduced antibody, 1x PBS (pH 7.4) was added to reach the initial antibody volume, and then the antibody was reoxidized at room temperature for 1 hour with 10 mM DHAA solution in 60-fold molar excess DMSO / PBS (1 / 1). After removing excess DHAA by dialysfiltration using Vivaspin 20, 50 KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), 1x PBS (pH 7.4) was added to the solution to reach the initial antibody volume. To the mixture, DMSO, which was no more than 20% of the final reaction volume, and a 10-fold molar excess of 5 mM linker-payload solution were added. The reaction mixture was stirred at 500 rpm for 2 hours at room temperature. Reverse-phase chromatography was used to monitor conjugation using an Agilent PLRP-S column, 4000 Å, 5 μm, 4.6 × 50 mm (buffer A: water, 0.1% trifluoroacetic acid; buffer B: acetonitrile, 0.1% trifluoroacetic acid; column held at +80°C; flow rate 1.5 ml / min). All exemplified ADCs obtained by this method were purified using 1x PBS (pH 7.4) on a preparative size exclusion chromatography column HiLoad® 26 / 600 Superdex® 200, concentrated using Vivaspin 20, 50KD, PES (polyethersulfone) (Sartorius Stedim, VS2031), sterile filtered through a 0.2 μm sterile PES (polyethersulfone) filter 25 mm (Whatmann, G896-2502), and stored at +4°C.

[0316] 5.3-Characterization by ADC Analysis All synthesized ADCs were characterized by analytical size exclusion chromatography (Superdex200Increase5 / 150GL, GE Healthcare, 28990945) to determine monomer ratios, and by liquid chromatography-mass spectrometry to determine drug-antibody ratios.

[0317] The drug-to-antibody ratio (DAR) of exemplary ADCs was determined by liquid chromatography-mass spectrometry (LC-MS) (80% A phase (water / 0.1% trifluoroacetic acid), 20% B phase (acetonitrile / 0.1% trifluoroacetic acid)).

[0318] ADCs were analyzed either in their intact state using a deglycosylation process with PNGase F enzyme (New England Biolaboratories®, P0705L), or after reduction with 5 mM (final concentration) dithiothreitol DTT (Thermo Scientific, Rockford, Illinois, 20291).

[0319] The ADC was mounted on a Bioresolve RP mAb polyphenyl column, 450 Å, 2.7 μm, 2.1 × 150 mm (Waters, Saint-Quentin-en-Yvelines, France, 186008946). For analysis in both intact and reduced states, a desalting step was performed at a flow rate of 0.6 mL / min with 20% B for 1.5 minutes. Elution was performed at a flow rate of 0.6 mL / min with a gradient from 1.5 minutes with 20% B to 16.5 minutes with 50% B. Washing was performed at a flow rate of 0.6 mL / min with 100% B from 16.8 minutes to 18.8 minutes. Finally, a conditioning step was performed at a flow rate of 0.6 mL / min with 20% B from 19.2 minutes for 1.8 minutes (total operation time = 21 minutes).

[0320] In this method, mobile phase A was ultrapure water obtained using a Mili-Q® system, and mobile phase B was mass spectrometry grade acetonitrile (Biosolve, France, Deuce, 0001204101BS) supplemented with 0.1% formic acid (Fischer Chemical: A117-50-50ML). The column temperature was set to +80°C.

[0321] LC-MS analysis was performed using a Waters UPLC H-class biochromatography system-Xevo G2 XS Q-TOF ESI mass spectrometer (Waters, Manchester, UK). Electrospray-ionization time-of-flight mass spectra of the analytes were acquired using UNIFI® software (Waters, Manchester, UK). The extracted intensities for the m / z spectra were then decoded using the Max Entropy (MaxEnt1) method of MassLynx® software to determine the mass of each intact antibody species or antibody fragment reduced according to treatment. Finally, the drug-antibody ratio was determined from the decoded spectra by summing the peak areas of the integrated mass spectra (total ion current values) of a given species (monoclonal antibody or associated fragment) that was both unconjugated and conjugated. For the determination of the drug-antibody ratio, the ratios of each identified species were calculated from the peak intensity values ​​from the decoded spectra. The obtained ratios were multiplied by the number of attached drugs. Based on the total result, a complete ADC * The final mean antibody-drug ratio for 2 was estimated.

[0322] Size exclusion chromatography (SEC) was performed for quality control of each ADC by measuring the monomer ratio of the conjugate. The analysis was performed on an analytical column Superdex200Increase5 / 150GL (GE Healthcare, 28990945) under homogeneous solvent conditions of 100% PBS (pH 7.4) (Sigma Life Sciences, P3813, 10PAK) at a flow rate of 0.45 ml / min for 12 minutes. The percentage of aggregates in the conjugate sample was quantified based on the absorbance of the peak area at 280 nm. This calculation was based on the ratio obtained by dividing the high molecular weight eluate at 280 nm by the sum of the absorbance peak areas of the high molecular weight and monomer eluates at the same wavelength, and multiplying the result by 100.

[0323] 5.4-Results

[0324] Characterization of exemplary ADCs is summarized in Table 6 (coupling method, LC-MS method, drug-antibody ratio, aggregation status after conjugation (aggregates %), ADC stability (aggregates %), stability after 1 week at +37°C, and yield). The mean drug-antibody ratio was determined using the LC-MS method described above, and the aggregate ratio was measured by size exclusion chromatography (SEC) during ADC quality control and after stability testing (168 hours incubation in PBS buffer at +37°C).

[0325] [Table 6]

[0326] All the exemplified ADCs in Table 6 above showed efficient conjugation, with the exception of 9006IgG2, which was linked via stochastic cysteine ​​conjugation at a drug-antibody ratio of 2.3. The majority of the ADCs demonstrated good stability after incubation in PBS buffer at +37°C for one week. Some variability in stability was detected in conjugates containing IgG2 of antibody 9006, IgG2 of antibody 9338, IgG1 of antibody 8902, and IgG1 of antibody 9338, likely due to the conjugation site. The results led to the conclusion that MMAE was successfully conjugated to the exemplified antibodies, and that site-specific cysteine ​​conjugation at the mutated site V205C S400C stabilized the ADC conjugates compared to stochastic conjugation.

[0327] Example 6 - Activity of anti-MET antibody and anti-MET ADC

[0328] Cell lines EBC-1, SNU-5 (both amplified by MET), and H1650 (not amplified by MET, referred to here as H1650 3D) were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide. The cells were seeded in 96-well clear-bottom plates and exposed to anti-MET monoclonal antibody or anti-MET ADC for 120 hours.

[0329] The effect of anti-MET monoclonal antibodies or anti-MET ADCs on cell viability was evaluated by quantifying intracellular ATP levels using 75 μL of CellTiter-Glo (CTG) reagent (Promega) per well after a 5-day incubation. All conditions were tested using either triple or double decans. Luminescence was quantified using a multi-purpose plate reader.

[0330] IC50 (median inhibitory concentration) was calculated using standard four-parameter curve fitting. IC50 is defined as the concentration of the compound at which the CTG signal decreases to 50% of the signal measured for the control. Two independent experiments were performed.

[0331] The IC50 data and arithmetic mean values ​​for each experiment are shown in Tables 7 and 8 for all antibodies and ADCs tested. Curves for some antibodies and ADCs are shown in Figures 2A and 2B.

[0332] Treatment of MET-amplified EBC-1 and SNU-5 cell lines with naked anti-MET antibodies against IgG1 and IgG2 showed a dose-dependent effect on cell viability (Figures 2A and 2B), and the IgG1 antibody 8902 was one of the most potent antibodies compared to other naked IgG1 antibodies.

[0333] When examining the activity of ADCs containing IgG1 or IgG2 antibodies in cell lines amplified by such METs, all ADCs were potenter than the payload MMAE, and the ADC containing IgG1 of antibody 8902 was one of the most potent ADCs compared to ADCs containing other antibodies (IC50 of 0.025 nM in EBC-1 and 0.013 nM in SNU-5 for 8902IgG1 ADC, and IC50 of 0.025 nM in EBC-1 and 0.015 nM in SNU-5 for 8902IgG2 ADC) (Table 7).

[0334] In the H1650 cell line, which is not amplified by MET, neither naked IgG1 nor IgG2 antibodies showed a significant dose-dependent effect (Figures 2A and 2B). Although ADCs were less potent than payload MMAEs, ADCs showed a potent cell-dependent effect on cell viability. In particular, ADCs containing 8902IgG1 antibody or IgG2 antibody showed potent activity in this model, with IC50 values ​​of 33.85 nM and 44.2 nM, respectively (Table 7).

[0335] In conclusion, either IgG1 or IgG2 type 8902 antibody exhibits potent activity against cell viability in MET-amplified cell lines. ADCs containing either IgG1 or IgG2 type 8902 antibody exhibit a potent effect on cell viability in either MET-amplified or non-MET-amplified cell lines.

[0336] Overall, these results demonstrate a potent effect of 8902 antibody and ADCs containing 8902 antibody on cell viability.

[0337] [Table 7] TIFF2026518691000022.tif218150

[0338] Example 7 - In vivo efficacy of 8902MET naked antibody in SNU5, an in vivo model amplified by MET.

[0339] The inventors determined the in vivo therapeutic effect of 8902 antibody, formulated in phosphate-buffered saline (PBS) and targeting the MET protein, in the human gastric cancer cell line SNU-5, which is amplified by MET after intravenous (IV) administration.

[0340] SNU-5 cells obtained from ATCC were cultured in IMDM supplemented with 20% fetal bovine serum and 0.5% penicillin-streptomycin. The cells were resuspended in RPMI without phenol red and cultured in 5 × 10⁶ solutions. 6 0.2 ml containing individual cells was subcutaneously inoculated into the right flank of female SCID mice provided by Charles River. Implantation of SNU-5 tumor cells was performed 24 to 72 hours after whole-body irradiation with gamma rays (1.44 Gray, Cobalt-60, BioMep, France).

[0341] Animals were randomized based on their individual tumor volume. Randomization was based on a numerical value of 100-200 mm².3 The study was conducted when the animals reached a certain stage. 60 out of 84 animals were randomized into 10 groups of 6 animals each. Homogeneity between groups was tested by analysis of variance (ANOVA). 8902IgG2 Met antibody (15 mg / kg) was administered intravenously once in PBS.

[0342] As shown in Figure 3A, a single dose of anti-MET8902IgG2 antibody at 15 mg / kg induced potent tumor regression in the SNU-5 human gastric cancer model. Importantly, no significant effect on the body weight of the treated animals was observed (Figure 3B), which indicates good tolerability of this antibody at an effective dose in mice.

Claims

1. An anti-MET antibody or its antigen-binding site, wherein the antibody is as follows: a. Antibodies in which H-CDR1, H-CDR2, and H-CDR3 contain the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively; b. Antibodies in which L-CDR1, L-CDR2, and L-CDR3 contain the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively; c. An antibody in which VH is at least 90% identical to the amino acid sequence of SEQ ID NO: 7; d. An antibody in which VL is at least 90% identical to the amino acid sequence of SEQ ID NO: 8; e. VH is an antibody containing the amino acid sequence of SEQ ID NO: 7; f. Antibody VL contains the amino acid sequence of SEQ ID NO: 8; g. An antibody having an HC having at least 90% identical amino acid sequence to SEQ ID NO: 11 or 13; h. In antibodies, LC is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 14; i. In antibodies, the heavy chain (HC) contains the amino acid sequence of SEQ ID NO: 11; j. An antibody having an LC containing the amino acid sequence of SEQ ID NO: 12; k. An antibody in which the heavy chain (LC) contains the amino acid sequence of SEQ ID NO: 13; and l. An antibody containing the amino acid sequence of SEQ ID NO: 14 in LC, An anti-MET antibody or its antigen-binding site, selected from the group consisting of the following.

2. The anti-MET antibody or antigen-binding site according to claim 1, wherein H-CDR1, H-CDR2, H-CDR3 and L-CDR1, L-CDR2, L-CDR3 of the antibody each contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively.

3. The anti-MET antibody or antigen-binding site according to claim 2, wherein the antibody has a heavy chain variable region (VH) that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 7, and a light chain variable region (VL) that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

8.

4. The anti-MET antibody or antigen-binding site according to claim 2, wherein the antibody has a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

8.

5. The anti-MET antibody or antigen-binding site according to claim 2, wherein the heavy chain (HC) of the antibody is at least 90% identical to the amino acid sequence of SEQ ID NO: 11 or 13, and the light chain (LC) of the antibody is at least 90% identical to the amino acid sequence of SEQ ID NO: 12 or 14.

6. The anti-MET antibody or antigen-binding site thereof according to claim 2, wherein the heavy chain (HC) of the antibody contains the amino acid sequence of SEQ ID NO: 11, and the light chain (LC) of the antibody contains the amino acid sequence of SEQ ID NO:

12.

7. The anti-MET antibody or antigen-binding site thereof according to claim 2, wherein the heavy chain (HC) of the antibody contains the amino acid sequence of SEQ ID NO: 13, and the light chain (LC) of the antibody contains the amino acid sequence of SEQ ID NO:

14.

8. The antibody or antigen binding site according to claim 1, wherein the antibody is isotype IgG.

9. The antibody or antigen binding site according to any one of claims 8, wherein the antibody is isotype IgG1 or IgG2.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain or its antigen-binding site, a nucleotide sequence encoding a light chain or its antigen-binding site, or both, of an anti-MET antibody according to any one of claims 1 to 9.

11. An isolated nucleic acid molecule according to claim 10, comprising the nucleotide sequence of sequence number 9 or 10.

12. A vector comprising an isolated nucleic acid molecule according to claim 10 or 11, further comprising an expression control sequence.

13. A host cell comprising a nucleotide sequence encoding a heavy chain or its antigen-binding site and / or a nucleotide sequence encoding a light chain or its antigen-binding site of an anti-MET antibody according to any one of claims 1 to 9.

14. A host cell comprising an isolated nucleic acid molecular sequence according to any one of claims 10 to 11.

15. A non-human transformed animal or plant that expresses the nucleotide sequence, comprising a nucleotide sequence encoding a heavy chain or an antigen-binding site thereof, and / or a nucleotide sequence encoding a light chain or an antigen-binding site thereof, of an anti-MET antibody according to any one of claims 1 to 9.

16. A non-human transformed animal or plant comprising an isolated nucleic acid molecular sequence according to any one of claims 10 to 11.

17. A bispecific binding molecule having binding specificity for the anti-MET antibody or its antigen-binding site as described in claim 1.

18. The bispecificity conjugation molecule according to claim 17, wherein the bispecificity conjugation molecule includes an antigen-binding site of an antibody, and its H-CDR1, H-CDR2, H-CDR3 and L-CDR1, L-CDR2, L-CDR3 each contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

19. An antibody-drug conjugate (ADC) represented by the formula Ab-(L-D)p, wherein Ab is the anti-Met antibody or its antigen-binding site as described in claim 1, D is the payload or drug portion, L is a linker that covalently binds Ab to D, and p is an integer from 1 to 16.

20. The ADC according to claim 19, wherein Ab is an anti-Met antibody or its antigen-binding site, and H-CDR1, H-CDR2, H-CDR3 and L-CDR1, L-CDR2, L-CDR3 each contain the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, and 6.

21. The ADC according to claim 19 or 20, wherein the drug portion is selected from Eg5 inhibitors, V-ATPase inhibitors, HSP90 inhibitors, IAP inhibitors, mTor inhibitors, microtubule stabilizers, microtubule destabilizers, auristatin, drastatin, mytansinoids, methionine aminopeptidase (MetAP) inhibitors, protein nuclear export inhibitor CRM1, DPPIV inhibitors, mitochondrial phosphate transfer inhibitors, protein synthesis inhibitors, kinase inhibitors, CDK2 inhibitors, CDK9 inhibitors, proteasome inhibitors, kinesin inhibitors, HDAC inhibitors, DNA damaging agents, DNA alkylating agents, DNA intercalators, DNA minor groove binders, RNA polymerase inhibitors, amanitin, spliceosome inhibitors, topoisomerase inhibitors, DHFR inhibitors, and apoptosis inducers.

22. A composition comprising an anti-MET antibody or antigen binding site according to any one of claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, or an ADC according to any one of claims 19 to 21.

23. A pharmaceutical composition comprising the composition described in claim 22 and a pharmaceutically acceptable excipient.

24. A method for producing an antibody or an antigen-binding site according to any one of claims 1 to 9, comprising: providing a host cell according to any one of claims 13 to 14; culturing the host cell under conditions suitable for the expression of the antibody or site; and isolating the obtained antibody or site.

25. A method for treating a patient having a MET-mediated disorder, comprising administering to the patient an anti-MET antibody or its antigen-binding site according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, a composition according to claim 22, or a pharmaceutical composition according to claim 23.

26. A method for treating a patient who has cancer or is suspected of having cancer, comprising administering to the patient an anti-MET antibody or antigen-binding site thereof according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, a composition according to claim 22, or a pharmaceutical composition according to claim 23.

27. A method for reducing or suppressing tumor growth in a patient, comprising administering to the patient an anti-MET antibody or antigen-binding site thereof according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, a composition according to claim 22, or a pharmaceutical composition according to claim 23.

28. A method for reducing or delaying the expansion of a cancer cell population in a patient, comprising administering to the patient an anti-MET antibody or antigen-binding site thereof according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, a composition according to claim 22, or a pharmaceutical composition according to claim 23.

29. The method according to any one of claims 25 to 28, wherein the cancer is dependent on MET activation and / or MET expression.

30. Cancers include melanoma, uveal melanoma, renal cancer, thyroid cancer, mesothelioma, hepatocellular carcinoma, lung cancer including non-small cell lung cancer and small cell lung cancer, gastric cancer including gastric cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bile duct cancer, head and neck cancer including oral cancer, cervical cancer and endometrial cancer, bladder cancer and urothelial carcinoma, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, brain tumor, cranial nerve tumor, brainstem tumor, brainstem nerve tumor, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem glioma, brainstem nerve tumor The method according to any one of claims 25 to 29, wherein the cancer is a glioma, brainstem glioma, brainstem glioma, cholangiocarcinoma, head and neck cancer including oral cancer, cervical cancer and cervical canal cancer, bladder cancer and urothelial carcinoma, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, adrenocortical carcinoma, brain cancer, splenic cancer, kidney cancer, thymoma, multiple myeloma, plasmacytic myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancy derived from T cells or B cells, hematological cancer, myeloid leukemia, or myeloma.

31. The method according to claim 30, wherein the cancer is lung cancer, pancreatic cancer, or gastric cancer.

32. The method according to claim 31, wherein the cancer is a blood cancer.

33. The method according to claim 32, wherein the hematological cancer is chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, non-Hodgkin lymphoma, or myelodysplastic syndrome (MDS).

34. The method according to any one of claims 25 to 28, further comprising administering at least one additional therapeutic procedure.

35. The method according to claim 34, wherein the additional treatment is a chemotherapeutic agent, an antitumor agent, an anti-angiogenic agent, a different anti-cancer antibody, a tyrosine kinase inhibitor, a MET pathway inhibitor, and / or radiotherapy.

36. Use of an anti-MET antibody or antigen-binding site thereof according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, the composition according to claim 22, or the pharmaceutical composition according to claim 23, for the treatment of a patient having a MET-mediated disorder.

37. Use of an anti-MET antibody or antigen-binding site thereof according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, the composition according to claim 22, or the pharmaceutical composition according to claim 23, for the treatment of a patient having cancer or a patient suspected of having cancer.

38. Use of an anti-MET antibody or antigen-binding site thereof according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, the composition according to claim 22, or the pharmaceutical composition according to claim 23, for the purpose of reducing or suppressing the growth of a patient's tumor.

39. Use of an anti-MET antibody or antigen-binding site thereof according to claims 1 to 9, a bispecific binding molecule according to any one of claims 17 to 18, an ADC according to any one of claims 19 to 21, the composition according to claim 22, or the pharmaceutical composition according to claim 23, for the purpose of reducing or delaying the expansion of a cancer cell population in a patient.

40. The use according to any one of claims 36 to 39, wherein the cancer is dependent on MET activation and / or MET expression.

41. Cancers include melanoma, uveal melanoma, renal cancer, thyroid cancer, mesothelioma, hepatocellular carcinoma, lung cancer including non-small cell lung cancer and small cell lung cancer, gastric cancer including gastric cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bile duct cancer, head and neck cancer (including oral cancer, cervical cancer and uterine canal cancer), bladder cancer and urothelial carcinoma, uterine cancer, ovarian cancer, and breast cancer. The use according to any one of claims 36 to 40, wherein the cancer is a bile duct cancer, head and neck cancer including oral cancer, cervical cancer and cervical canal cancer, bladder cancer and urothelial carcinoma, uterine cancer, ovarian cancer, breast cancer, prostate cancer, sarcoma, testicular cancer, glioblastoma, adrenocortical carcinoma, brain cancer, spleen cancer, kidney cancer, thymoma, multiple myeloma, plasmacytic myeloma, leukemia, lymphoma, acute myeloid leukemia, bone marrow cancer, chronic lymphocytic leukemia, lymphoblastic leukemia including acute lymphoblastic leukemia, follicular lymphoma, lymphoid malignancy of T cell or B cell origin, hematological cancer, myeloid leukemia, or myeloma.

42. The use according to claim 41, wherein the cancer is lung cancer, pancreatic cancer, or gastric cancer.

43. The use according to claim 41, wherein the cancer is a blood cancer.

44. The use according to claim 43, wherein the hematological cancer is chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), Hodgkin lymphoma, non-Hodgkin lymphoma, or myelodysplastic syndrome (MDS).

45. The use according to any one of claims 36 to 39, further comprising administering at least one additional therapeutic procedure.

46. The use according to claim 45, wherein the additional treatment is a chemotherapeutic agent, an antitumor agent, an anti-angiogenic agent, a different anti-cancer antibody, a tyrosine kinase inhibitor, a MET pathway inhibitor, and / or radiotherapy.

47. Use of an anti-MET antibody or its antigen-binding site according to claims 1 to 9 for detecting and / or measuring the level of MET in a sample from a patient.

48. A kit comprising an anti-MET antibody or its antigen-binding site as described in claims 1 to 9.

Images