Anti-met antibodies, bispecific antigen-binding molecules that bind to met, and methods of use thereof

Abstract

To provide anti-MET antibodies, bispecific antigen-binding molecules that bind to MET, and methods of use thereof.SOLUTION: Provided herein are antibodies and bispecific antigen-binding molecules that bind to MET and methods of use thereof. The bispecific antigen-binding molecules comprise a first antigen-binding domain and a second antigen-binding domain, where the first and second antigen-binding domains bind to two different (preferably non-overlapping) epitopes of the extracellular domain of human MET. The bispecific antigen-binding molecules are capable of blocking the interaction between human MET and its ligand HGF.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antibodies, bispecific antibodies, and antigen-binding fragments thereof that specifically bind to hepatocyte growth factor receptor (c-Met or MET) and modulate MET signaling, as well as antibody-drug conjugates of such antibodies, and methods of their use.

[0002] Sequence Listing An official copy of the Sequence Listing is being submitted electronically contemporaneously with the present specification via EFS-Web as an ASCII formatted Sequence Listing in the filename 10316WO01_Sequence_Listing_ST25_2017_11_06.TXT, approximately 136 kilobytes in size, created on November 6, 2017. The Sequence Listing contained in this ASCII format document is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Hepatocyte growth factor (HGF), also known as scatter factor [SF], is a heterodimeric paracrine growth factor that exerts its activity through interaction with the HGF receptor (HGFR). HGFR is the product of the c-Met oncogene, also known as MET. MET is a receptor tyrosine kinase consisting of a transmembrane beta chain linked to an extracellular alpha chain via a disulfide bridge. Binding of HGF to MET activates the kinase catalytic activity of MET, leading to phosphorylation of Tyr1234 and Tyr1235 of the beta chain and subsequent activation of downstream signaling pathways.

[0004] Overexpression, activation, or amplification of MET and / or HGF has been shown to be involved in non-small cell lung cancer (NSCLC), gastric cancer, ovarian cancer, pancreatic cancer, thyroid cancer, breast cancer, head and neck cancer, colon cancer, and renal cancer (Sierra and Tsao, Ther. Adv. Med. Oncol. 3(1Suppl):S21-S35, 2011). MET amplification is believed to be a major driver of tumorigenesis in NSCLC and esophagogastric malignancies. Furthermore, mutations resulting in exon 14 deletion of MET have been described as a tumorigenesis driver in a subset of NSCLC. Tumor cell lines with MET gene amplification are highly dependent on MET for growth and survival. Preclinical data implicate MET signaling in resistance to targeted therapy in multiple tumor types, including NSCLC, colorectal cancer, and head and neck squamous cell carcinoma (HNSCC). Both preclinical and recent clinical results show that tumors with these genetic alterations respond to MET inhibitors, identifying MET as a cancer driver. A variety of monovalent MET-blocking antibodies are in clinical development for the treatment of a variety of cancers (see U.S. Patent No. 5,686,292; U.S. Patent No. 5,646,036; U.S. Patent No. 6,099,841; U.S. Patent No. 7,476,724; U.S. Patent No. 9,260,531; and U.S. Patent No. 9,328,173; and U.S. Patent Application Publication No. 2014 / 0349310, and U.S. Patent Application Publication No. 2005 / 0233960). These antibodies include onartuzumab (MetMab) and emibetuzumab (Xiang et al., Clin. Cancer Res. 19(18):5068-78, 2013; and Rosen et al., Clin. Cancer Res. 10 Oct. 2016, doi: 10.1158 / 1078-0432.CCR-16-1418). Some of these antibodies block ligand-dependent MET signaling but are less effective at blocking ligand-independent MET activation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Pat. No. 5,686,292 [Patent Document 2] U.S. Pat. No. 5,646,036 [Patent Document 3] U.S. Patent No. 6,099,841 [Patent Document 4] U.S. Patent No. 7,476,724 [Patent Document 5] U.S. Patent No. 9,260,531 [Patent Document 6] U.S. Patent No. 9,328,173 [Patent Document 7] US Patent Application Publication No. 2014 / 0349310 [Patent Document 8] US Patent Application Publication No. 2005 / 0233960 [Non-patent literature]

[0006] [Non-Patent Document 1] Sierra and Tsao, Ther. Adv. Med. Oncol. 3(Suppl 1):S21-S35, 2011. [Non-Patent Document 2] Xiang et al., Clin. Cancer Res. 19(18):5068-78, 2013 [Non-Patent Document 3] Rosen et al., Clin. Cancer Res. Published October 10, 2016, doi: 10.1158 / 1078-0432.CCR-16-1418 Summary of the Invention [Means for solving the problem]

[0007] There remains a significant unmet medical need for improved anticancer drugs that potently block both ligand-dependent and ligand-independent MET signaling. Provided herein are antibodies, antigen-binding fragments of antibodies, bivalent monospecific antibody combinations, and bispecific antibodies that bind to the human c-Met receptor protein (MET x MET). The antibodies are particularly useful for targeting tumor cells that express MET. The anti-MET antibodies and their antigen-binding portions may be used alone in unmodified form or may be included as part of an antibody-drug conjugate or bispecific antibody.

[0008] Other embodiments will become apparent from review of the following detailed description. In an embodiment of the present invention, for example, the following items are provided: (Item 1) a first antigen-binding domain (D1); a second antigen-binding domain (D2); Including, D1 specifically binds to the first epitope of human MET; A bispecific antigen-binding molecule, wherein D2 specifically binds to a second epitope of human MET. 2. The bispecific antigen-binding molecule of item 1, wherein D1 and D2 do not compete with each other for binding to human MET. (Item 3) 3. The bispecific antigen-binding molecule of item 1 or 2, which is capable of blocking the interaction between human MET and HGF. (Item 4) 4. The bispecific antigen-binding molecule of item 3, which exhibits minimal agonist activity in a cell-based MET activity reporter assay. (Item 5) 4. The bispecific antigen-binding molecule of item 3, which does not exhibit MET agonist activity in a cell-based MET activity reporter assay. (Item 6) 5. The bispecific antigen-binding molecule of item 4, which exhibits a degree of MET agonist activity in a cell-based MET activity reporter assay that is less than 10% of the MET agonist activity of a monovalent antigen-binding molecule containing only D1 or D2. (Item 7) 7. The bispecific antigen-binding molecule of any one of items 1 to 6, which promotes degradation of MET expressed on the cell surface. (Item 8) 8. The bispecific antigen-binding molecule of any one of items 1 to 7, which inhibits the growth of tumors harboring a genetic alteration of MET. (Item 9) 8. The bispecific antigen-binding molecule of any one of items 1 to 7, which promotes tumor regression of tumors harboring a genetic alteration of MET. (Item 10) 10. The bispecific antigen-binding molecule of any one of items 1 to 9, which inhibits the growth of a tumor whose growth is driven by autocrine HGF signaling. (Item 11) 10. The bispecific antigen-binding molecule of any one of items 1 to 9, which promotes tumor regression of tumors whose growth is driven by autocrine HGF signaling. (Item 12) 12. The bispecific antigen-binding molecule according to any one of items 1 to 11, wherein D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 58 and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 138. (Item 13) 13. The bispecific antigen-binding molecule of item 12, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical thereto; HCDR2 comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical thereto; HCDR3 comprises the amino acid sequence of SEQ ID NO: 64 or an amino acid sequence that is at least 95% identical thereto; LCDR1 comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical thereto; LCDR2 comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical thereto; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical thereto. (Item 14) 14. The bispecific antigen-binding molecule of item 13, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 60; HCDR2 comprises the amino acid sequence of SEQ ID NO: 62; HCDR3 comprises the amino acid sequence of SEQ ID NO: 64; LCDR1 comprises the amino acid sequence of SEQ ID NO: 140; LCDR2 comprises the amino acid sequence of SEQ ID NO: 142; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 144. (Item 15) 14. The bispecific antigen-binding molecule of item 13, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence that is at least 95% identical thereto; and an LCVR comprising the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence that is at least 95% identical thereto. (Item 16) 16. The bispecific antigen-binding molecule of item 15, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 58; and an LCVR comprising the amino acid sequence of SEQ ID NO: 138. (Item 17) 17. The bispecific antigen-binding molecule according to any one of items 1 to 16, wherein D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) in the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 82 and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) in the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 138. (Item 18) 18. The bispecific antigen-binding molecule of item 17, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 84 or an amino acid sequence that is at least 95% identical thereto; HCDR2 comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical thereto; HCDR3 comprises the amino acid sequence of SEQ ID NO: 88 or an amino acid sequence that is at least 95% identical thereto; LCDR1 comprises the amino acid sequence of SEQ ID NO: 140 or an amino acid sequence that is at least 95% identical thereto; LCDR2 comprises the amino acid sequence of SEQ ID NO: 142 or an amino acid sequence that is at least 95% identical thereto; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 144 or an amino acid sequence that is at least 95% identical thereto. (Item 19) 19. The bispecific antigen-binding molecule of item 18, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 84; HCDR2 comprises the amino acid sequence of SEQ ID NO: 86; HCDR3 comprises the amino acid sequence of SEQ ID NO: 88; LCDR1 comprises the amino acid sequence of SEQ ID NO: 140; LCDR2 comprises the amino acid sequence of SEQ ID NO: 142; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 144. (Item 20) 19. The bispecific antigen-binding molecule of item 18, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 82, or an amino acid sequence that is at least 95% identical thereto; and an LCVR comprising the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence that is at least 95% identical thereto. (Item 21) 21. The bispecific antigen-binding molecule of item 20, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 82; and an LCVR comprising the amino acid sequence of SEQ ID NO: 138. (Item 22) 2. The bispecific antigen-binding molecule of item 1, wherein the first epitope of human MET comprises amino acids 192 to 204 of SEQ ID NO: 155. (Item 23) 2. The bispecific antigen-binding molecule of item 1, wherein the second epitope of human MET comprises amino acids 305 to 315 and amino acids 421 to 455 of SEQ ID NO: 155. (Item 24) 2. The bispecific antigen-binding molecule according to item 1, wherein the first epitope of human MET comprises amino acids 192 to 204 of SEQ ID NO: 155, and the second epitope of human MET comprises amino acids 305 to 315 and amino acids 421 to 455 of SEQ ID NO: 155. (Item 25) 2. The bispecific antigen-binding molecule of item 1, which is conjugated to a cytotoxin. (Item 26) 26. The bispecific antigen-binding molecule of item 25, wherein the cytotoxin is selected from the group consisting of a biotoxin, a chemotherapeutic agent, and a radioisotope. (Item 27) The cytotoxin is a maytansinoid, an auristatin, a duocarmycin, 225 Ac, 227 26. The bispecific antigen-binding molecule of item 25, selected from the group consisting of Th, and any derivative thereof. (Item 28) 2. The bispecific antigen-binding molecule of item 1, which is conjugated to a cytotoxic agent via a linker. (Item 29) 29. The bispecific antigen-binding molecule of item 28, wherein the cytotoxic agent is a maytansinoid. (Item 30) 30. The bispecific antigen-binding molecule of item 29, comprising the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 18 or 58 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 82. (Item 31) The maytansinoid is [ka] where: [ka] is the bond to the linker. (Item 32) The linker is [ka] where: [ka] represents binding to the bispecific antigen-binding molecule, [ka] 32. The bispecific antigen-binding molecule of item 31, wherein the bond indicated by represents a bond to the maytansinoid. (Item 33) The maytansinoid is [ka] where: [ka] is the bond to the linker. (Item 34) The linker is [ka] where: [ka] represents binding to the bispecific antigen-binding molecule, [ka] 34. The bispecific antigen-binding molecule of item 33, wherein the bond indicated by represents a bond to the maytansinoid. (Item 35) A pharmaceutical composition comprising the bispecific antigen-binding molecule of item 1 and a pharma- ceutical acceptable carrier. (Item 36) 35. A method for treating cancer in a subject suffering from a tumor having a genetic alteration of MET and / or a tumor whose growth is driven by autocrine HGF signaling, comprising administering to the subject a bispecific antigen-binding molecule according to any one of items 1 to 34. (Item 37) 37. The method of claim 36, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), gastric cancer, colorectal cancer, and head and neck cancer. (Item 38) 37. The method of claim 36, further comprising administering to the subject a second anti-cancer therapeutic agent. (Item 39) 1. A method of treating cancer, reducing tumor growth, and / or causing tumor regression in a subject, comprising administering to a subject in need thereof an antibody-drug conjugate (ADC) comprising a bispecific antigen-binding molecule and a cytotoxin, wherein the bispecific antigen-binding molecule: a first antigen-binding domain (D1); a second antigen-binding domain (D2); Including, D1 specifically binds to the first epitope of human MET; D2 specifically binds to a second epitope of human MET. (Item 40) 39. The method of claim 39, wherein D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 18 or 58, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 138. (Item 41) 39. The method of claim 39, wherein D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 82, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 138. (Item 42) 40. The bispecific antigen-binding molecule of item 39, wherein the first epitope of human MET comprises amino acids 192 to 204 of SEQ ID NO: 155. (Item 43) 40. The bispecific antigen-binding molecule of item 39, wherein the second epitope of human MET comprises amino acids 305 to 315 and amino acids 421 to 455 of SEQ ID NO: 155. (Item 44) 40. The bispecific antigen-binding molecule of item 39, wherein the first epitope of human MET comprises amino acids 192 to 204 of SEQ ID NO: 155, and the second epitope of human MET comprises amino acids 305 to 315 and amino acids 421 to 455 of SEQ ID NO: 155. (Item 45) 40. The method of claim 39, wherein the cytotoxin is selected from the group consisting of a biotoxin, a chemotherapeutic agent, and a radioisotope. (Item 46) The cytotoxin is a maytansinoid, an auristatin, a tomaymycin, a duocarmycin, 225 Ac, 227 40. The method according to item 39, wherein the compound is selected from the group consisting of Th, and any derivatives thereof. (Item 47) 40. The method of claim 39, wherein the bispecific antigen binding molecule comprises the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 58 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 82. (Item 48) The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, the cytotoxin comprising: [ka] where: [ka] is a bond to a linker. (Item 49) The linker is [ka] and During the ceremony, [ka] represents binding to the bispecific antigen-binding molecule, [ka] 49. The method of claim 48, wherein the bond indicated by represents the bond to the cytotoxin. (Item 50) The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, the cytotoxin comprising: [ka] where: [ka] is the bond to the linker. (Item 51) The linker is [ka] where: [ka] represents binding to the bispecific antigen-binding molecule, [ka] 43. The method of claim 42, wherein the bond indicated by represents the bond to the cytotoxin. (Item 52) A method for preparing an antibody-drug conjugate comprising: 1 : [ka] and an aqueous diluent. (Item 53) The method of claim 524, wherein the anti-MET antibody comprises the CDRs within the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138. (Item 54) 53. The method of claim 52, wherein the METxMET bispecific antigen binding protein comprises the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO:58 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO:82. (Item 55) 53. The method of claim 52, wherein the METxMET bispecific antigen binding protein comprises the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 18 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 82. (Item 56) Formula A 1 53. The method of claim 52, wherein said compound of formula (I) is present in stoichiometric excess. (Item 57) Formula A 1 The compound of formula A 2 Or A 3 [ka] [ka] 53. The method according to claim 52, comprising administering to said patient a compound of formula (I) or a mixture thereof. (Item 58) Formula A 2 53. The method of claim 52, wherein the compound of formula (1) is stereomerically pure. (Item 59) Formula A 3 53. The method of claim 52, wherein the compound of formula (1) is stereomerically pure. (Item 60) A 1 Or A 2 53. The method of claim 52, wherein said compound of formula (I) is present in a diastereomeric excess of greater than 50%, greater than 70%, greater than 90%, or greater than 95%. (Item 61) Formula A 1 in the presence of silica gel and a diluent to give a compound of formula (a) [ka] with a compound of formula (b) [ka] 53. The method according to claim 52, wherein the compound is prepared by contacting the compound with (Item 62) In the presence of silica gel and a diluent, (i) A compound of formula (a): [ka] with a compound of formula (b): [ka] to synthesize an intermediate, (ii) contacting an anti-MET antibody or a MET x MET bispecific antigen binding protein with the intermediate and an aqueous diluent; An antibody-drug conjugate prepared by the process. [Brief description of the drawings]

[0009] [Figure 1] 1 is a matrix illustrating the components of 272 exemplary MET x MET bispecific antibodies disclosed herein. Each numbered cell of the matrix identifies a unique bispecific antibody comprising a "D1" antigen-binding domain and a "D2" antigen-binding domain, where the D1 antigen-binding domain comprises the CDRs from the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or corresponding anti-MET antibody listed along the Y-axis, and the D2 antigen-binding domain comprises the CDRs from the immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or corresponding anti-MET antibody listed along the X-axis.

[0010] [Diagram 2]FIG. 2 is a schematic diagram of the luciferase-based reporter assay used to assess antibody-induced MET pathway activation or antibody blockade of HGF-induced pathway activation in HEK293T cells containing an SRE-luciferase reporter gene construct.

[0011] [Diagram 3] Figures 3A-B are line graphs showing relative light units (RLU) representing SRE-luciferase expression as a function of antibody concentration in log moles per liter. Filled squares (■) represent parental bivalent monospecific antibody H4H13306P2, filled pyramids (▲) represent parental bivalent monospecific antibody H4H13312P2, filled circles (●) represent monovalent antibodies, filled diamonds (◆) represent isotype controls, and filled inverted pyramids (▼) represent no ligand. Figure 3A shows antibody alone without HGF ligand. Figure 3B shows antibody plus HGF ligand.

[0012] [Figure 4] Figures 4A-B are line graphs showing relative light units (RLU) representing SRE-luciferase expression as a function of antibody concentration in log moles per liter. Filled squares (■) represent anti-MET monovalent antibodies, filled circles (●) represent METxMET bispecific antibodies, and filled diamonds (◆) represent the parent antibody H4H13312P2. Figure 4A shows antibody alone without HGF ligand. Figure 4B shows antibody plus HGF ligand.

[0013] [Diagram 5] 5 is a bar graph showing relative cell growth of MET-amplified gastric cancer SNU5 cells as a function of treatment with human bivalent monospecific anti-MET antibodies 1-18, control antibodies, and anti-MET monovalent antibodies. For comparison purposes, antibody 8 (abscissa) is the parental antibody H4H13306P2 and antibody 11 (abscissa) is the parental antibody H4H13312P2.

[0014] [Figure 6]Figures 6A-B contain bar graphs showing relative cell growth of MET-amplified cells as a function of treatment with METxMET bispecific antibody, control antibody, and anti-MET monovalent antibody. Figure 6A shows the relative growth of SNU5 cells as a function of treatment with control antibody, 0.1, 1, and 10 μg / mL of monovalent antibody, and 0.1, 1, and 10 μg / mL of METxMET bispecific antibody. Figure 6B shows the relative growth of EBC-1 cells as a function of treatment with control antibody and 0.1 and 1 μg / mL of METxMET bispecific antibody.

[0015] [Figure 7] Figures 7A-B show immunoblots of pMET (phosphorylated MET), MET, pErk (phosphorylated Erk), and tubulin (for loading control) extracted from Hs746T cells after treatment with control antibody and MET x MET bispecific antibody (Figure 7A), and expression of MET (and tubulin as a loading control) in Hs746T cells after treatment with MET x MET bispecific antibody for 0, 2, and 6 hours (Figure 7B).

[0016] [Figure 8] FIG. 8 shows immunoblots of pMET, MET, pErk, and tubulin (for loading control) extracted from Hs746T cells following treatment with control antibody, MET x MET bispecific antibody, anti-MET monospecific bivalent parental antibody 1, anti-MET monospecific bivalent parental antibody 2, and the combination of parental antibodies 1 and 2.

[0017] [Figure 9] FIG. 9 shows immunoblots of MET (and tubulin as a loading control) expression in Hs746T cells after treatment with control antibody and MET×MET bispecific antibody for 2, 6, and 18 hours.

[0018] [Figure 10]Figures 10A-B show immunoblots of pMET, MET, pErk, and tubulin (for loading control) extracted from SNU5 cells after treatment with a control antibody and a MET x MET bispecific antibody (Figure 10A), and expression of MET (and tubulin as a loading control) in SNU5 cells after treatment with a control antibody and an anti-MET monovalent antibody (Figure 10B).

[0019] [Figure 11] FIG. 11 shows immunoblots of pMET, MET, pErk, and tubulin (for loading control) extracted from EBC-1 cells following treatment with control antibody and MET×MET bispecific antibody.

[0020] [Figure 12] Figure 12 is a line graph showing the change in EBC-1 tumor volume in cubic millimeters as a function of time in days following implantation of EBC-1 cells in animals treated with a control antibody (black square ■), a MET monovalent antibody (black circle ●), or a MET x MET bispecific antibody (black diamond ◆).

[0021] [Figure 13] Figures 13A-B contain bar graphs showing the relative cell growth of MET-amplified cells as a function of treatment with METxMET bispecific antibody, control antibody, and anti-MET monovalent antibody. Figure 13A shows the relative growth of Hs746T cells as a function of treatment with control antibody, METxMET bispecific antibody, METxMET parent monospecific antibody 1, METxMET parent monospecific antibody 2, and the combination of parent antibodies 1 and 2. Figure 13B shows the relative growth of Hs746T cells as a function of treatment with control antibody, 1, 10, and 25 μg / mL of monovalent antibody, and 1, 10, and 25 μg / mL of METxMET bispecific antibody.

[0022] [Figure 14]FIG. 14 is a bar graph showing relative cell growth of NCI-H596 cells as a function of treatment with control antibody (C), MET×MET bispecific antibody (MM), MET×MET parental monospecific antibody 1 (M1), MET×MET parental monospecific antibody 2 (M2), a combination of parental antibodies 1 and 2 (M1M2), and the MET agonist hepatocyte growth factor (HGF).

[0023] [Figure 15] Figure 15 is a line graph showing the change in Hs746T tumor volume in cubic millimeters as a function of time in days after transplantation of Hs746T cells in animals treated with a control antibody (filled square ■), a MET monovalent antibody (filled circle ●), or a MET x MET bispecific antibody (filled diamond ◆).

[0024] [Figure 16A] Figure 16A is a line graph showing the change in SNU5 tumor volume in cubic millimeters as a function of time in days after implantation of SNU5 cells in animals treated with control antibody (filled square ■), 1 mg / mL MET monovalent antibody (filled circle ●), 10 mg / mL MET monovalent antibody (open circle ○), 1 mg / mL MET x MET bispecific antibody (filled diamond ◆), or 10 mg / mL MET x MET bispecific antibody (open diamond ◇).

[0025] [Figure 16B] Figure 16B is an immunoblot of pMET, MET, and tubulin (loading control) extracted from SNU5 tumors removed from a mouse xenograft model after treatment with a control antibody, 10 mg / kg anti-MET monovalent antibody, and 10 mg / kg MET x MET bispecific antibody.

[0026] [Figure 17]Figure 17 is a line graph showing the change in U87-MG tumor volume in cubic millimeters as a function of time in days following implantation of U87-MG cells in animals treated with a control antibody (black square ■), a MET monovalent antibody (black circle ●), or a MET x MET bispecific antibody (black diamond ◆).

[0027] [Figure 18] Figure 18 is a line graph showing the change in U118-MG tumor volume in cubic millimeters as a function of time in days following implantation of U118-MG cells in animals treated with a control antibody (black square ■), a MET monovalent antibody (black circle ●), or a MET x MET bispecific antibody (white diamond ◇).

[0028] [Figure 19] FIG. 19 is a schematic illustrating the synthesis of maytansinoid 6.

[0029] [Figure 20] FIG. 20 is a schematic illustrating the synthesis of maytansinoid intermediate 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Before describing the invention, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the invention is limited only by the appended claims.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.In this specification, the term "about" when used in connection with a specific recited numerical value means that the value can vary from the recited value by 1% or less.For example, in this specification, the expression "about 100" includes 99 and 101 and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0032] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications and non-patent publications mentioned herein are incorporated by reference in their entirety. MET protein

[0033] As used herein, the terms "MET", "c-Met" and the like refer to (1) an amino acid sequence having the amino acid sequence as set forth in SEQ ID NO: 145 and / or as set forth in NCBI Accession No. NM_001127500.2, which represents the unprocessed preproprotein of isoform "a", (2) an amino acid sequence having the amino acid sequence as set forth in SEQ ID NO: 146 and / or as set forth in NCBI Accession No. NM_000236.2, which represents the unprocessed preproprotein of isoform "b". (3) an amino acid sequence having the amino acid sequence as set forth in SEQ ID NO: 147 and / or as set forth in NCBI Accession No. NM_001311330.1, which represents the unprocessed preproprotein of isoform "c", and / or (3) a mature protein comprising a cytoplasmic alpha subunit (SEQ ID NO: 148) shared by all three isoforms and a transmembrane beta subunit (SEQ ID NO: 149, 150, or 151 for isoforms a, b, and c, respectively). The term "MET" includes both monomeric and multimeric MET molecules. As used herein, the term "monomeric human MET" refers to a MET protein or portion thereof that does not contain or has any multimerization domain and that exists under normal conditions as a single MET molecule without direct physical connection with another MET molecule. An exemplary monomeric MET molecule is a molecule referred to herein as "hMET.mmh" comprising the amino acid sequence of SEQ ID NO: 152 (see, e.g., Example 3 herein). As used herein, the phrase "dimeric human MET" refers to a construct comprising two MET molecules connected to each other via a linker, covalent bond, non-covalent bond, or via a multimerization domain, such as an antibody Fc domain. An exemplary dimeric MET molecule is a molecule referred to herein as "hMET.mFc" comprising the amino acid sequence of SEQ ID NO: 153 (see, e.g., Example 3 herein).

[0034] All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human version of the respective protein, polypeptide, or protein fragment, unless expressly specified as being from a non-human species. Thus, the term "MET" refers to human MET, unless specified as being from a non-human species, e.g., "mouse MET," "monkey MET," etc.

[0035] As used herein, the phrase "cell surface expressed MET" refers to one or more MET proteins, or extracellular domains thereof, that are expressed on the surface of a cell in vitro or in vivo such that at least a portion of the MET protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. "Cell surface expressed MET" may include or consist of a MET protein expressed on the surface of a cell that normally expresses MET protein. Alternatively, "cell surface expressed MET" may include or consist of a MET protein expressed on the surface of a cell that does not normally express human MET on its surface, but has been artificially engineered to express MET on its surface.

[0036] Anti-MET antibodies and antigen-binding fragments thereof According to one embodiment, anti-MET antibodies (e.g., monospecific anti-MET antibodies) are provided. Exemplary anti-MET antibodies according to this embodiment are listed herein in Tables 1 and 2. Table 1 sets forth the amino acid sequence identifiers of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of exemplary anti-MET antibodies, from which bispecific antigen binding molecules (used interchangeably herein with bispecific antigen binding proteins) disclosed herein may be derived. Table 2 sets forth the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary anti-MET antibodies.

[0037] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to MET and stimulate (e.g., activate) the MET signaling pathway in a cell, and the use of such antibodies in therapeutic settings where activation of MET signaling would be beneficial or therapeutically useful. Non-limiting examples of such agonist anti-MET antibodies include the antibody designated herein as "H4H14636D," as well as antibodies and antigen-binding fragments thereof that comprise the heavy and light chain CDRs (SEQ ID NOs: 28, 30, 32, 140, 142, 144) and / or the heavy and light chain variable domains thereof (SEQ ID NOs: 26 / 138).

[0038] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising a heavy chain HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0039] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0040] Provided herein is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising an HCVR and LCVR amino acid sequence pair (HCVR / LCVR), comprising any HCVR amino acid sequence listed in Table 1 paired with any LCVR amino acid sequence listed in Table 1. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 2 / 138, 10 / 138, 18 / 138, 26 / 138, 34 / 138, 42 / 138, 50 / 138, 58 / 138, 66 / 138, 74 / 138, 82 / 138, 90 / 138, 98 / 138, 106 / 138, 114 / 138, 122 / 138 and 130 / 138.

[0041] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0042] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0043] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0044] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0045] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising an HCDR1 and LCDR1 amino acid sequence pair (HCDR1 / LCDR1) that comprises any HCDR1 amino acid sequence listed in Table 1 paired with any LCDR1 amino acid sequence listed in Table 1. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCDR1 / LCDR1 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 4 / 140, 12 / 140, 20 / 140, 28 / 140, 36 / 140, 44 / 140, 52 / 140, 60 / 140, 68 / 140, 76 / 140, 84 / 140, 92 / 140, 100 / 140, 108 / 140, 116 / 140, 124 / 140, and 132 / 140.

[0046] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0047] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising an HCDR2 and LCDR2 amino acid sequence pair (HCDR2 / LCDR2) that comprises any HCDR2 amino acid sequence listed in Table 1 paired with any LCDR2 amino acid sequence listed in Table 1. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR2 / LCDR2 amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCDR2 / LCDR2 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 6 / 142, 14 / 142, 22 / 142, 30 / 142, 38 / 142, 46 / 142, 54 / 142, 62 / 142, 70 / 142, 78 / 142, 86 / 142, 94 / 142, 102 / 142, 110 / 142, 118 / 142, 126 / 142 and 134 / 142.

[0048] Also provided is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0049] Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to MET, comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) that comprises any HCDR3 amino acid sequence listed in Table 1 paired with any LCDR3 amino acid sequence listed in Table 1. According to certain embodiments, the antibody or antigen-binding fragment thereof comprises an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 8 / 144, 16 / 144, 24 / 144, 32 / 144, 40 / 144, 48 / 144, 56 / 144, 64 / 144, 72 / 144, 80 / 144, 88 / 144, 96 / 144, 104 / 144, 112 / 144, 120 / 144, 128 / 144 and 136 / 144.

[0050] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to MET comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-MET antibodies listed in Table 1. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is SEQ ID NO: 4-6-8-140-142-144, 12-14-16-140-142-144, 20-22-24-140-142-144, 28-30-32-140-142-144, 36-38-40-140-142-144, 44-44-48-140-142-144, 52-54-56-140-142-144, 60-62-64-140-142 -144, 68-70-72-140-142-144, 76-78-80-140-142-144, 84-86-88-140-142-144, 92-94-96-140-142-144, 100-102-104-140-142-144, 108-110-112-140-142-144, 116-118-120-140-142-144, 124-126-128-140-142-144 and 132-134-136-140-142-144.

[0051] In a related embodiment, an antibody or antigen-binding fragment thereof that specifically binds to MET comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-MET antibodies listed in Table 1. For example, antibodies or antigen-binding fragments thereof that specifically bind to MET may be selected from the group consisting of SEQ ID NOs: 4-6-8-140-142-144, 12-14-16-140-142-144, 20-22-24-140-142-144, 28-30-32-140-142-144, 36-38-40-140-142-144, 44-44-48-140-142-144, 52-54-56-140-142-144, 60-62-64-140-142-144, 68-70-72-140-142-144, 76-78-80-140-142-144, 84 -The set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within the HCVR / LCVR amino acid sequence pair selected from the group consisting of 86-88-140-142-144, 92-94-96-140-142-144, 100-102-104-140-142-144, 108-110-112-140-142-144, 116-118-120-140-142-144, 124-126-128-140-142-144 and 132-134-136-140-142-144.

[0052] Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in the defined HCVR and / or LCVR amino acid sequences disclosed herein.Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition.In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available to identify CDR sequences within antibodies.

[0053] Also provided herein are nucleic acid molecules encoding anti-MET antibodies or portions thereof. For example, the invention provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0054] Nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1 are also provided; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0055] Nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1 are also provided; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0056] Nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1 are also provided; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0057] Nucleic acid molecules encoding any of the HCDR3 amino acid sequences listed in Table 1 are also provided; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0058] Nucleic acid molecules encoding any of the LCDR1 amino acid sequences listed in Table 1 are also provided; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0059] Nucleic acid molecules encoding any of the LCDR2 amino acid sequences listed in Table 1 are also provided; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR2 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0060] Nucleic acid molecules encoding any of the LCDR3 amino acid sequences listed in Table 1 are also provided; in certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the LCDR3 nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0061] Also provided is a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), and the HCDR1-HCDR2-HCDR3 amino acid sequence set is defined by any of the exemplary anti-MET antibodies listed in Table 1.

[0062] Also provided is a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and the LCDR1-LCDR2-LCDR3 amino acid sequence set is defined by any of the exemplary anti-MET antibodies listed in Table 1.

[0063] Also provided are nucleic acid molecules encoding both an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises the amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments of this aspect, the nucleic acid molecule encodes an HCVR and an LCVR, wherein the HCVR and LCVR are both derived from the same anti-MET antibody listed in Table 1.

[0064] Also provided herein are recombinant expression vectors capable of expressing a polypeptide comprising a heavy or light chain variable region of an anti-MET antibody. For example, a recombinant expression vector comprises any of the nucleic acid molecules described above, i.e., a nucleic acid molecule encoding any of the HCVR, LCVR and / or CDR sequences set out in Table 1. Also included within the scope of this disclosure is a method of producing the antibody or a portion thereof by culturing a host cell into which such a vector has been introduced under conditions allowing the production of the antibody or antibody fragment, and recovering the antibody and antibody fragment thus produced.

[0065] Provided herein are anti-MET antibodies with modified glycosylation patterns. In some embodiments, modifications to remove undesirable glycosylation sites or antibodies lacking fucose moieties present in the oligosaccharide chains may be useful, for example, to increase antibody-dependent cellular cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modifications of galactosylation can be made to modify complement-dependent cytotoxicity (CDC).

[0066] MET×MET bispecific antigen binding molecule The present inventors have observed that certain monospecific anti-MET antigen binding molecules that block HGF binding to MET tend to strongly activate MET signaling (an undesirable outcome for therapeutic molecules). However, the present inventors have surprisingly discovered that bispecific antigen binding molecules that simultaneously bind to two separate epitopes of the MET protein extracellular domain are effective in blocking ligand binding to MET, but cause little agonism of MET signaling.

[0067] Thus, provided herein is a bispecific antigen-binding molecule comprising a first antigen-binding domain (also referred to herein as "D1") and a second antigen-binding domain (also referred to herein as "D2"). Simultaneous binding to two separate MET epitopes by the bispecific antigen-binding molecule results in effective ligand blockade with minimal activation of MET signaling.

[0068] A bispecific antigen-binding molecule comprising a first antigen-binding domain (D1) that specifically binds to a first epitope of human MET and a second antigen-binding domain (D2) that specifically binds to a second epitope of human MET may be referred to herein as a "METxMET bispecific antibody," "METxMET," or other related term. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO: 155. In some embodiments, the second epitope of human MET comprises amino acids 305-315 and amino acids 421-455 of SEQ ID NO: 155. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO: 155 and the second epitope of human MET comprises amino acids 305-315 and amino acids 421-455 of SEQ ID NO: 155.

[0069] In certain embodiments, the D1 and D2 domains of a MET x MET bispecific antibody are non-competitive with each other. Non-competitive between D1 and D2 for binding to MET means that the respective monospecific antigen-binding proteins from which D1 and D2 are derived do not compete with each other for binding to human MET. Exemplary antigen-binding protein competition assays are known in the art, non-limiting examples of which are described elsewhere herein.

[0070] In certain embodiments, as described elsewhere herein, D1 and D2 bind to different (eg, non-overlapping or partially overlapping) epitopes on MET.

[0071] A METxMET bispecific antigen-binding molecule can be constructed using the antigen-binding domains of two separate monospecific anti-MET antibodies. For example, a panel of monoclonal monospecific anti-MET antibodies can be generated using standard methods known in the art. The individual antibodies thus generated can be tested pairwise against each other for cross-competition for MET protein. If two different anti-MET antibodies can bind to MET simultaneously (i.e., do not compete with each other), the antigen-binding domain from a first anti-MET antibody and the antigen-binding domain from a second, non-competing anti-MET antibody can be engineered into a single METxMET bispecific antibody according to the present disclosure.

[0072] According to the present disclosure, a bispecific antigen-binding molecule can be a single multifunctional polypeptide, or it can be a multimeric complex of two or more polypeptides that are covalently or non-covalently associated with each other.As demonstrated by the present disclosure, any antigen-binding construct that has the ability to simultaneously bind to two separate, non-identical epitopes of MET molecule is considered to be a bispecific antigen-binding molecule.Any bispecific antigen-binding molecule described herein or its variants can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques), as known to those skilled in the art.

[0073] Antigen-binding domain The bispecific antigen-binding molecule of the present disclosure comprises two separate antigen-binding domains (D1 and D2). As used herein, the term "antigen-binding domain" refers to any peptide, polypeptide, nucleic acid molecule, scaffold-based molecule, peptide display molecule, or polypeptide-containing construct that can specifically bind to a particular antigen of interest (e.g., human MET). As used herein, terms such as "specifically bind" refer to an antigen-binding domain that binds to a particular antigen with a dissociation constant (K) of 500 pM or less. D) and does not bind to other, unrelated antigens under normal test conditions. An "unrelated antigen" is a protein, peptide, or polypeptide that has less than 95% amino acid identity with one another.

[0074] Exemplary categories of antigen-binding domains that may be used in connection with the present disclosure include antibodies, antigen-binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibodies), receptor molecules that specifically interact with a particular antigen, proteins comprising a ligand-binding portion of a receptor that specifically binds to a particular antigen, antigen-binding scaffolds (such as, for example, DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on naturally occurring repeat proteins [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and aptamers or portions thereof.

[0075] Methods for determining whether two molecules specifically bind to one another are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, as used in the context of the present disclosure, an antigen-binding domain has a K of less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM, or less than about 0.05 pM as measured in a surface plasmon resonance assay. D and comprises a polypeptide that binds to a specific antigen (e.g., a target molecule [T] or an endogenous effector protein [E]) or a portion thereof.

[0076] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIAcore™ system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0077] As used herein, "K D The term "equilibrium dissociation constant" refers to the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise indicated, the K D K values ​​are determined by surface plasmon resonance assay at 25 °C. D Points to a value.

[0078] As indicated above, the "antibody binding domain" (D1 and / or D2) herein may comprise or consist of an antibody or an antigen-binding fragment of an antibody. The term "antibody" as used herein means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., human MET). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (herein referred to as HCVR or VVR). H The heavy chain constant region is made up of three domains: H 1. C H 2 and C H Each light chain comprises a light chain variable region (herein, LCVR or V L The light chain constant region comprises one domain (C L 1) is included. H and V LThe regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs of the antibodies (or antigen-binding portions thereof) provided herein may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0079] The D1 and / or D2 components of the bispecific antigen-binding molecules provided herein may comprise or consist of an antigen-binding fragment of a complete antibody molecule. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., as used herein include any naturally occurring, enzymatically obtainable, synthetic or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from complete antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques, including the manipulation and expression of DNA encoding antibody variable domains and, optionally, antibody constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add or delete amino acids, etc.

[0080] Non-limiting examples of antigen-binding fragments include (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides) or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs) and shark variable IgNAR domains, are also encompassed within the term "antigen-binding fragment" herein.

[0081] An antigen-binding fragment of an antibody will typically contain at least one variable domain. The variable domain may be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L V associated with domain H In an antigen-binding fragment having a domain, H and V L The domains can be positioned relative to each other in any suitable configuration. For example, the variable regions can dimerize into V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of the antibody may contain a monomeric V H or V L It may contain domains.

[0082] In certain embodiments, an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be found in an antigen-binding fragment of an antibody of the present disclosure include: (i) V H -C H 1;(ii)V H -C H 2;(iii)V H -C H 3;(iv)V H -C H 1-C H 2;(v)V H -C H 1-C H 2-C H 3;(vi)V H -C H 2-C H 3;(vii)V H -C L ;(viii)V L -C H 1;(ix)V L -C H 2;(x)V L -C H 3;(xi)V L -C H 1-C H 2;(xii)V L -C H 1-C H 2-C H 3;(xiii)V L -C H 2-C H 3; and (xiv) V L -C LIn any configuration of the variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, which provides a flexible or semi-flexible link between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments may be linked to each other and / or to one or more monomeric V H or V L The variable and constant domains may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above, non-covalently associated (e.g., by disulfide bond(s)) with the domains.

[0083] The bispecific antigen-binding molecules provided herein may comprise or consist of a human antibody and / or a recombinant human antibody, or a fragment thereof. The term "human antibody" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, human antibodies may contain amino acid residues not encoded in human germline immunoglobulin sequences, for example in the CDRs, particularly CDR3 (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0084] The bispecific antigen-binding molecules of the present disclosure may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. The term "recombinant human antibody" is intended herein to include any human antibody prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (discussed further below), antibodies isolated from recombinant combinatorial human antibody libraries (discussed further below), antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), which results in the V H and V L The amino acid sequence of the region is H and V L The sequences are derived from and related to, but may not naturally occur within the human antibody germline repertoire in vivo.

[0085] Methods for making bispecific antibodies are known in the art and may be used to construct the bispecific antigen binding molecules disclosed herein. Exemplary bispecific formats that may be used in the context of the present disclosure include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG and Mab. 2 These include, but are not limited to, bispecific formats (see, e.g., Klein et al., 2012, mAbs 4:6, 1-11 and references cited therein, for a review of the aforementioned formats).

[0086] Exemplary antigen-binding domains (D1 and D2) that may be included in the METxMET bispecific antigen-binding molecules provided herein include antigen-binding domains derived from any of the anti-MET antibodies disclosed herein. For example, the present disclosure includes METxMET bispecific antigen-binding molecules that include a D1 or D2 antigen-binding domain that comprises an HCVR that includes an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0087] Also provided herein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain that comprises an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0088] Provided herein are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain that comprises a HCVR and LCVR amino acid sequence pair (HCVR / LCVR) that comprises any of the HCVR amino acid sequences listed in Table 1 paired with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the invention provides METxMET bispecific antigen binding molecules that comprise a D1 or D2 antigen binding domain that comprises a HCVR / LCVR amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1.

[0089] Also provided herein is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain, which comprises a heavy chain CDR1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0090] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, which comprises a heavy chain CDR2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0091] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, which comprises a heavy chain CDR3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0092] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, which comprises a light chain CDR1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0093] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, which comprises a light chain CDR2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0094] Also provided are METxMET bispecific antigen binding molecules comprising a D1 or D2 antigen binding domain, which comprises a light chain CDR3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0095] Also provided is a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain that comprises an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) that comprises any of the HCDR3 amino acid sequences listed in Table 1 paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure also provides an antibody or antigen-binding fragment thereof that comprises an HCDR3 / LCDR3 amino acid sequence pair contained within any of the exemplary anti-MET antibodies listed in Table 1.

[0096] Also provided are METxMET bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within any of the exemplary anti-MET antibodies listed in Table 1.

[0097] In a related embodiment, the disclosure provides a METxMET bispecific antigen binding molecule comprising a D1 or D2 antigen binding domain that comprises a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-MET antibodies listed in Table 1.

[0098] A METxMET bispecific antigen-binding molecule provided herein can comprise a D1 antigen-binding domain derived from any anti-MET antibody in Table 1, and a D2 antigen-binding domain derived from any other anti-MET antibody in Table 1. Non-limiting examples of METxMET bispecific antibodies of the present disclosure are shown in Figure 1. Figure 1 is a matrix illustrating the components of 272 exemplary METxMET bispecific antibodies. Each numbered cell of the matrix (numbered 1-272) identifies a unique bispecific antibody comprising a "D1" antigen-binding domain and a "D2" antigen-binding domain, where the D1 antigen-binding domain comprises CDRs from an immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or corresponding anti-MET antibody listed along the Y-axis, and the D2 antigen-binding domain comprises CDRs from an immunoglobulin variable domain (HCVR / LCVR amino acid sequence pair) or corresponding anti-MET antibody listed along the X-axis. Thus, for example, the METxMET bispecific antigen-binding molecule "number 10" shown in the matrix comprises a D1 antigen-binding domain comprising a HCVR / LCVR pair or 6-CDR set derived from the exemplary anti-MET antibody H4H13290P2, and a D2 antigen-binding domain comprising a HCVR / LCVR pair or 6-CDR set derived from the exemplary anti-MET antibody H4H13321P2. Further examples of METxMET bispecific antibodies provided herein are described in Example 4 herein.

[0099] As a non-limiting illustrative example, the present disclosure includes a METxMET bispecific antigen binding molecule comprising a D1 antigen binding domain and a D2 antigen binding domain, wherein the D1 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:58 / 138, or a set of heavy chain and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO:60-62-64-140-142-144, and the D2 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO:82 / 138, or a set of heavy chain and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO:84-86-88-140-142-144. An exemplary MET x MET bispecific antibody having these sequence characteristics is a bispecific antibody designated H4H14639D, also referred to as bispecific antibody no. 122, which comprises D1 from H4H13306P2 and D2 from H4H13312P2 (see Example 4, Table 5 herein).

[0100] As a further non-limiting illustrative example, the present disclosure includes a METxMET bispecific antigen binding molecule comprising a D1 antigen binding domain and a D2 antigen binding domain, wherein the D1 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 18 / 138, or a set of heavy chain and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 20-22-24-140-142-144, and the D2 antigen binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138, or a set of heavy chain and light chain CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NO: 84-86-88-140-142-144. An exemplary MET x MET bispecific antibody having these sequence characteristics is a bispecific antibody designated H4H14635D, also referred to as bispecific antibody no. 42, which comprises D1 derived from H4H13295P2 and D2 derived from H4H13312P2 (see Example 4, Table 5 herein).

[0101] Multimer-forming components In certain embodiments, the bispecific antigen-binding molecules provided herein may also include one or more multimerizing components. The multimerizing components may function to maintain the association between the antigen-binding domains (D1 and D2). As used herein, a "multimerizing component" is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing component of the same or similar structure or composition. For example, the multimerizing component may be an immunoglobulin C H The multimerization component may be a polypeptide comprising three domains. A non-limiting example of a multimerization component is the Fc portion of an immunoglobulin, such as the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group. In certain embodiments, the multimerization component is an Fc fragment, or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization component is a cysteine ​​residue, or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that include or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0102] In certain embodiments, the bispecific antigen-binding molecule provided herein comprises two multimerization domains M1 and M2, where D1 binds to M1 and D2 binds to M2, and the association of M1 with M2 promotes the physical linkage of D1 and D2 to each other to form a single bispecific antigen-binding molecule. In certain embodiments, M1 and M2 are identical to each other. For example, M1 can be an Fc domain with a specific amino acid sequence, and M2 is an Fc domain with the same amino acid sequence as M1. Alternatively, M1 and M2 can differ from each other at one or more amino acid positions. For example, M1 is ... H M2 may comprise a second Ig C3 domain, H The first IgC may comprise three domains. H 3 domain and second Ig CH The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the target construct to Protein A compared to a reference construct having identical M1 and M2 sequences. In one embodiment, the IgC domain of M1 H The 3 domain binds to protein A and is involved in the IgC domain of M2. H The C3 domain contains mutations that reduce or abolish protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). H Further modifications that may be found within 3 include, for IgG1 Fc domains, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I according to EU); for IgG2 Fc domains, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I according to EU); and for IgG4 Fc domains, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I according to EU).

[0103] The bispecific antigen-binding molecule of the present disclosure may be "isolated". As used herein, an "isolated bispecific antigen-binding molecule" refers to a bispecific antigen-binding molecule that has been identified and separated and / or recovered from at least one component of its natural environment. For example, a bispecific antibody that has been separated or removed from at least one component of an organism or from a tissue or cell in which the antibody is produced is an "isolated bispecific antibody" for the purposes of the present disclosure. An isolated bispecific antigen-binding molecule also includes a molecule in situ in a recombinant cell. An isolated bispecific antigen-binding molecule is a molecule that has been subjected to at least one purification or isolation step. According to certain embodiments, an isolated bispecific antigen-binding molecule may be substantially free of other cellular material and / or chemicals.

[0104] The bispecific antigen-binding molecules or antigen-binding domains thereof (D1 and / or D2) disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antigen-binding proteins or antigen-binding domains are derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present disclosure includes bispecific antigen-binding molecules or antigen-binding domains thereof (D1 and / or D2) described herein that are derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue(s) in the germline sequence from which the antibody is derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations").

[0105] Starting from the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily generate a multitude of bispecific antigen-binding molecules or antigen-binding domains thereof (D1 and / or D2) that contain one or more individual germline mutations or combinations thereof. In certain embodiments, H and / or V L All of the framework and / or CDR residues in the domain are back-mutated to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are back-mutated to the original germline sequence, for example only the mutated residues found in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody is originally derived).

[0106] Furthermore, the bispecific antigen-binding molecules or antigen-binding domains thereof (D1 and / or D2) of the present disclosure can contain any combination of two or more germline mutations in the framework and / or CDR regions, for example, certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once a bispecific antigen-binding molecule or antigen-binding domain thereof (D1 and / or D2) containing one or more germline mutations is obtained, it can be easily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Bispecific antigen-binding molecules or antigen-binding domains thereof (D1 and / or D2) obtained in such a general manner are encompassed within the scope of the present disclosure.

[0107] Modified form The present disclosure also includes anti-MET antibodies and bispecific antigen-binding molecules that comprise variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. Exemplary variants included within this embodiment include variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-MET antibodies and METxMET bispecific antigen-binding molecules that have HCVR, LCVR and / or CDR amino acid sequences that have, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc., conservative amino acid substitutions compared to any of the HCVR, LCVR and / or CDR amino acid sequences shown in Table 1 herein.

[0108] Exemplary variants included within this aspect of the disclosure also include variants that have substantial sequence identity with any of the amino acid sequences of the HCVR, LCVR and / or CDRs disclosed herein. As used herein in the context of amino acid sequences, the term "substantial identity" or "substantially identical" means that two amino acid sequences share at least 95%, 98% or 99% sequence identity when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. 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 are cysteine ​​and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.Alternatively, a conservative replacement is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" replacement is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0109] The sequence similarity of polypeptides, also referred to as the sequence identity between two different amino acid sequences, is typically measured using sequence analysis software. Sequence analysis software matches similar sequences using a measure of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine the sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, using default or recommended parameters. The programs FASTA (e.g., FASTA2 and FASTA3) in GCG version 6.1 provide alignment and percent sequence identity of the best overlap region between the query and search sequences (see Pearson (2000) supra). Another preferred algorithm for comparing the sequences provided herein to a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids. See Acids Res. 25:3389-402.

[0110] Anti-MET Antibodies and MET×MET Bispecific Antigen-Binding Molecules Comprising Fc Modifications According to certain embodiments provided herein, anti-MET antibodies and METxMET bispecific antigen binding molecules are provided that include an Fc domain that includes one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the present disclosure provides an Fc domain that includes one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C H The present invention includes anti-MET antibodies and METxMET bispecific antigen binding molecules that contain mutations in three regions, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes, where the pH ranges from about 5.5 to about 6.0). Such mutations can result in increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F) and 434. In one embodiment, the modifications include a 428L (e.g., M428L) and a 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I) and a 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254 and 256 (e.g., 252Y, 254T and 256E) modification; a 250Q and a 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P).

[0111] For example, the disclosure includes anti-MET antibodies or METxMET bispecific antigen binding molecules comprising an Fc domain that comprises one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434S (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). All possible combinations of the foregoing Fc domain mutations and other mutations in the antibody variable domains disclosed herein are considered to be within the scope of the disclosure.

[0112] Biological characteristics of the antigen-binding molecules provided herein Provided herein are antibodies and antigen-binding fragments thereof that bind with high affinity to monomeric human MET. For example, the disclosure provides antibodies and antigen-binding fragments thereof that bind with high affinity to monomeric human MET, e.g., antibodies having a K of less than about 230 nM as measured by surface plasmon resonance at 25° C. or 37° C. using, for example, the assay format defined in Example 3 herein or a substantially similar assay. D According to certain embodiments, the anti-MET antibodies have a K of less than about 230 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 50 nM, less than about 25 nM, less than about 20 nM, less than about 10 nM, less than about 8 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, or less than about 3 nM at 37° C. as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein or a substantially similar assay. D Anti-MET antibodies are provided that bind to monomeric human MET at

[0113] The present disclosure also includes antibodies and antigen-binding fragments thereof that bind to monomeric human MET (e.g., hMET.mmh) with a dissociation half-life (t1 / 2) of greater than about 1 minute, as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format defined in Example 3 herein or a substantially similar assay. According to certain embodiments, anti-MET antibodies are provided that bind to monomeric human MET with a t1 / 2 of greater than about 1 minute, greater than about 2 minutes, greater than about 4 minutes, greater than about 6 minutes, greater than about 8 minutes, greater than about 10 minutes, greater than about 12 minutes, greater than about 14 minutes, greater than about 16 minutes, greater than about 18 minutes, or greater than about 20 minutes or longer at 37° C., as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein or a substantially similar assay.

[0114] Provided herein are antibodies and antigen-binding fragments thereof that bind to dimeric human MET (e.g., hMET.mFc) with high affinity. For example, the disclosure provides antibodies and antigen-binding fragments thereof that have a K of less than about 3 nM as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format defined in Example 3 herein or a substantially similar assay. D According to certain embodiments, the anti-MET antibodies have a K of less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 0.9 nM, less than about 0.8 nM, less than about 0.7 nM, less than about 0.6 nM, less than about 0.5 nM, less than about 0.4 nM, less than about 0.3 nM, or less than about 0.25 nM at 37° C. as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein or a substantially similar assay. D The present invention provides an anti-MET antibody that binds to dimeric human MET at

[0115] Also provided herein are antibodies and antigen-binding fragments thereof that bind to dimeric human MET (e.g., hMET.mFc) with a dissociation half-life (t1 / 2) of greater than about 4 minutes, as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format defined in Example 3 herein or a substantially similar assay. According to certain embodiments, anti-MET antibodies are provided that bind to dimeric human MET with a t1 / 2 of greater than about 4 minutes, greater than about 5 minutes, greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 105 minutes, or longer, at 37° C., as measured by surface plasmon resonance, e.g., using the assay format defined in Example 3 herein or a substantially similar assay.

[0116] Also provided herein is a MET×MET bispecific antigen binding protein that binds to dimeric human MET (e.g., hMET.mFc) with a dissociation half-life (t1 / 2) of greater than about 10 minutes as measured by surface plasmon resonance at 25° C. or 37° C., e.g., using the assay format defined in Example 5 herein, or a substantially similar assay. According to certain embodiments, there is provided a METxMET bispecific antigen binding protein that binds dimeric human MET with a t1 / 2 of greater than about 10 minutes, greater than about 20 minutes, greater than about 30 minutes, greater than about 40 minutes, greater than about 50 minutes, greater than about 60 minutes, greater than about 70 minutes, greater than about 80 minutes, greater than about 90 minutes, greater than about 100 minutes, greater than about 200 minutes, greater than about 300 minutes, greater than about 400 minutes, greater than about 500 minutes, greater than about 600 minutes, greater than about 700 minutes, greater than about 800 minutes, greater than about 900 minutes, greater than about 1000 minutes, greater than about 1100 minutes or longer at 37°C as measured by surface plasmon resonance, e.g., using the assay format defined in Example 5 herein or a substantially similar assay.

[0117] Also provided herein are anti-MET antibodies and METxMET bispecific antigen binding proteins that block the interaction between HGF and MET, for example, in in vitro ligand binding assays. According to certain embodiments provided herein, METxMET bispecific antigen binding proteins are provided that block the binding of HGF to cells expressing human MET and induce minimal or no MET activation in the absence of HGF signaling. For example, the present disclosure provides METxMET bispecific antigen binding proteins that exhibit a degree of MET agonist activity in a cell-based MET activity reporter assay that is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% of the MET agonist activity observed in a comparable activity reporter assay using a monospecific antibody that includes only D1 or D2.

[0118] The antibodies and antigen-binding proteins of the present disclosure may have one or more of the above biological characteristics, or any combination thereof.The above list of antibody biological characteristics is not intended to be exhaustive.Other biological characteristics of the antibodies provided herein will be apparent to those skilled in the art from review of the present disclosure, including the examples herein.

[0119] Antibody-drug conjugates (ADCs) Provided herein are antibody-drug conjugates (ADCs) comprising an anti-MET antibody or a MET x MET bispecific antigen binding protein conjugated to a therapeutic moiety, such as a cytotoxic agent, a chemotherapeutic agent or a radioisotope.

[0120] Cytotoxic agents include any agent that is detrimental to cell growth, viability or reproduction, including, but not limited to, tubulin interacting agents and DNA damaging agents. Examples of suitable cytotoxic agents and chemotherapeutic agents that can be conjugated to an anti-MET antibody according to this aspect of the disclosure include, for example, 1-(2chloroethyl)-1,2-dimethanesulfonylhydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyn-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitin, aminopterin, anguidine, anthracyclines, anthramycin (AMC), auristatins, bleomycin, busulfan, butyric acid, calicheamicin (e.g., calicheamicin gamma), ... 1), camptothecin, carminomycin, carmustine, cemadotin, cisplatin, colchicine, combretastatin, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, dacarbazine, diacetoxypentyl doxorubicin, dibromomannitol, dihydroxyanthracin dione, disorazole, dolastatin (e.g., dolastatin 10), doxorubicin, duocarmycin, echinomycin, eleutherobin, emetine, epothilone, esperamicin, estramustine, ethidium bromide, etoposide, fluorouracil, geldanamycin, gramicidin D, glucocorticoids, irinotecan, kinesin spindle protein (KSP) inhibitors, leptomycin , leurosine, lidocaine, lomustine (CCNU), maytansinoids, mechlorethamine, melphalan, mercaptopurine, methopterin, methotrexate, mithramycin, mitomycin, mitoxantrone, N8-acetylspermidine, podophyllotoxin, procaine, propranolol, pteridine, puromycin, pyrrolobenzodiazepines (PBDs), rhizoxin, streptozotocin, tallysomycin, taxol, tenoposide, tetracaine, thioepa, chlorambucil, tomaymycin, topotecan, tubulysin, vinblastine, vincristine, vindesine, vinorelbine, and derivatives of any of the foregoing. According to certain embodiments, the cytotoxic agent conjugated to the anti-MET antibody is a maytansinoid, such as DM1 or DM4, a tomaymycin derivative, or a dolastatin derivative. According to certain embodiments, the cytotoxic agent conjugated to the anti-MET antibody is an auristatin, such as MMAE, MMAF, or a derivative thereof.Other cytotoxic agents known in the art are considered to be within the scope of the present disclosure, including, for example, protein toxins such as ricin, C. difficile toxin, Pseudomonas exotoxin, ricin, diphtheria toxin, botulinum toxin, bryodin, saporin, pokeweed toxin (i.e., phytolaccatoxin and phytolaccigenin), and others such as those set forth in Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469.

[0121] In certain embodiments, the cytotoxic agent is a maytansinoid, such as a derivative of maytansine.Suitable maytansinoids include DM1, DM4, or their derivatives, stereoisomers, or isotopologues.Suitable maytansinoids also include, but are not limited to, those disclosed in WO2014 / 145090A1, WO2015 / 031396A1, US2016 / 0375147A1, and US2017 / 0209591A1, the entirety of which is incorporated herein by reference.

[0122] In some embodiments, the maytansinoid has the following structure: [ka] where A is an optionally substituted arylene or heteroarylene.

[0123] In some embodiments, the maytansinoid has the following structure: [ka] where A is an optionally substituted arylene or heteroarylene.

[0124] In some embodiments, the maytansinoid has the following structure: [ka] wherein n is an integer from 1 to 12; 1 is alkyl.

[0125] In some embodiments, the maytansinoid is [ka] [ka] [ka] [ka] It is.

[0126] In some embodiments, the maytansinoid is [ka] It is.

[0127] In some embodiments, the maytansinoid is [ka] It is.

[0128] Also provided herein are antibody-radionuclide conjugates (ARC) comprising an anti-MET antibody conjugated to one or more radionuclides. Exemplary radionuclides that can be used in the context of this aspect of the disclosure include, for example, 225 Ac, 212 Bi, 213 Bi, 131 I, 186 Re, 227 Th, 222 Rn, 223 Ra, 224 Ra and 90 These include, but are not limited to, Y.

[0129] In certain embodiments provided herein, ADCs are provided that include an anti-MET antibody or a METxMET bispecific antigen binding protein conjugated to a cytotoxic agent (e.g., any of the cytotoxic agents disclosed above) via a linker molecule. A linker is any group or moiety that links, connects, or binds an antibody or antigen binding protein described herein to a therapeutic moiety, such as a cytotoxic agent. Suitable linkers can be found, for example, in Antibody-Drug Conjugates and Immunotoxins; edited by Phillips, GL, Springer Verlag: New York, 2013; Antibody-Drug Conjugates; edited by Ducry, L.; Humana Press, 2013; Antibody-Drug Conjugates; edited by Wang, J., Shen, W.-C., and Zaro, JL; Springer International Publishing, 2015, the contents of each of which are incorporated herein by reference in their entirety. In general, suitable binder linkers for the antibody conjugates described herein are those that are stable enough to take advantage of the circulating half-life of the antibody, and at the same time can release their payload after antigen-mediated internalization of the conjugate. The linker can be cleavable or non-cleavable. Cleavable linkers include linkers that are cleaved by intracellular metabolism, such as hydrolysis, reduction, or enzymatic cleavage, following internalization. Non-cleavable linkers include linkers that release the attached payload via lysosomal degradation of the antibody following internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolytically unstable linkers, enzymatically cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers.Suitable linkers also include, but are not limited to, those that are or contain a peptide, a glucuronide, a succinimide-thioether, a polyethylene glycol (PEG) unit, a hydrazone, a mal-caproyl unit, a dipeptide unit, a valine-citrulline unit, and a para-aminobenzyl (PAB) unit.

[0130] Any linker molecule or linker technology known in the art may be used to create or construct the ADCs of the present disclosure. In certain embodiments, the linker is a cleavable linker. According to other embodiments, the linker is a non-cleavable linker. Exemplary linkers that can be used in the context of the present disclosure include, for example, linkers comprising or consisting of MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), a dipeptide moiety in a protease cleavable linker, ala-phe (alanine-phenylalanine), a dipeptide moiety in a protease cleavable linker, PAB (p-aminobenzyloxycarbonyl), SPP (N-succinimidyl 4-(2-pyridylthio)pentanoate), SMCC (N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate), SIAB (N-succinimidyl (4-iodo-acetyl)aminobenzoate), and variants and combinations thereof. Additional examples of linkers that can be used in the context of the present disclosure are provided, for example, in US 7,754,681 and Ducry, Bioconjugate Chem. 2010, 21:5-13, and references cited therein, the contents of which are incorporated herein by reference in their entireties.

[0131] In certain embodiments, the linker is stable under physiological conditions. In certain embodiments, the linker is cleavable, for example, in the presence of an enzyme or at a certain pH range or value, and can release at least the payload portion. In some embodiments, the linker comprises an enzyme-cleavable moiety. Exemplary enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester bonds, hydrazones, and disulfide linkages. In some embodiments, the linker comprises a cathepsin-cleavable linker.

[0132] In some embodiments, the linker comprises a non-cleavable moiety.

[0133] Suitable linkers also include, but are not limited to, those that are chemically bound to a single binding agent, such as two cysteine ​​residues of an antibody. Such linkers may serve to mimic disulfide bonds in antibodies that are disrupted as a result of the conjugation process.

[0134] In some embodiments, the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non-proteinogenic, and L- or D-α-amino acids. In some embodiments, the linker comprises alanine, valine, glycine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, derivatives thereof, or combinations thereof. In certain embodiments, one or more side chains of the amino acid are linked to the side chain groups described below. In some embodiments, the linker comprises valine and citrulline. In some embodiments, the linker comprises lysine, valine, and citrulline. In some embodiments, the linker comprises lysine, valine, and alanine. In some embodiments, the linker comprises valine and alanine.

[0135] In some embodiments, the linker comprises a self-immolative group. The self-immolative group can be any such group known to those skilled in the art. In certain embodiments, the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). Those skilled in the art will recognize that the self-immolative group can undergo a chemical reaction that releases the remaining atoms of the linker from the payload.

[0136] In some embodiments, the linker is [ka] where: [ka] is the attachment to an antibody or antigen-binding protein (e.g., via a lysine residue), [ka] is a bond to a cytotoxic agent (e.g., DM1). In some embodiments, the linker is [ka] where: [ka] is the attachment to an antibody or antigen-binding protein (e.g., via a lysine residue), [ka] is a bond to a cytotoxic agent (e.g., DM1). In certain embodiments, the linker is [ka] It is.

[0137] In certain embodiments, the linker is [ka] It is.

[0138] In some embodiments, the linker is maleimidylmethyl-4-trans-cyclohexanecarboxysuccinate: [ka] It comes from.

[0139] In some embodiments, the linker is [ka] where: [ka] is the attachment to an antibody or antigen-binding protein (e.g., via a lysine residue), [ka] is a cytotoxic agent (e.g., a compound having the formula: [ka] The bond is to a compound having the formula:

[0140] The disclosure includes ADCs in which a linker connects an anti-MET antibody or a MET x MET bispecific antigen binding protein to a drug or cytotoxin by a bond at a specific amino acid within the antibody or antigen binding molecule. Exemplary amino acid bonds that can be used in the context of this embodiment include, for example, lysine (see, e.g., US 5,208,020; US 2010 / 0129314; Hollander et al., Bioconjugate Chem. 2008, 19:358-361; WO2005 / 089808; US5,714,586; US2013 / 0101546; and US2012 / 0585592), cysteine ​​(see, e.g., US2007 / 0258987; WO2013 / 055993; WO2013 / 055990; WO2013 / 053873; WO2013 / 053872; WO2011 / 130598; US2013 / 0101546; and US7,750,116), selenocysteine ​​(see, e.g., WO2008 / 122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formylglycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51 and Rabuka et al., Nat. Protocols, 2012, 10:1052-1067), unnatural amino acids (see, e.g., WO2013 / 068874 and WO2012 / 166559), and acidic amino acids (see, e.g., WO2012 / 05982). Linkers can also be conjugated to the antigen-binding protein by binding to carbohydrate (see, e.g., US2008 / 0305497, WO2014 / 065661 and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO2013 / 085925, WO2010 / 010324, WO2011 / 018611 and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313).Site-specific conjugation techniques can also be used for direct conjugation to specific residues of an antibody or antigen-binding protein (see, e.g., Schumacher et al., J Clin Immunol (2016) 36(Suppl 1):100). Site-specific conjugation techniques include, but are not limited to, transglutaminase-mediated glutamine conjugation (see, e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0141] According to certain embodiments, the disclosure provides ADCs in which an anti-MET antibody or a METxMET bispecific antigen binding protein as described herein is conjugated to a linker-drug composition as set forth in International Patent Publication WO2014 / 145090, the disclosure of which is hereby incorporated by reference in its entirety (e.g., compound "7", also referred to herein as "M0026" and depicted below). [ka]

[0142] Also provided herein are antibody-drug conjugates comprising the monospecific anti-MET antibodies and METxMET bispecific antibodies disclosed herein, wherein the anti-MET antibody or METxMET bispecific antibody is conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is a maytansinoid. In certain embodiments, the maytansinoid has the following formula: [ka] wherein n is an integer from 1 to 12; 1 is alkyl. In certain embodiments, the maytansinoid is [ka] In certain embodiments, the cytotoxic agent is a maytansinoid, and the maytansinoid is covalently attached to the antibody via a non-cleavable linker. In certain embodiments, the cytotoxic agent is a maytansinoid, and the maytansinoid is covalently attached to the antibody via a cleavable linker.

[0143] In one embodiment, the antibody comprises: [ka] It is conjugated to During the ceremony, [ka] is the binding to the antibody.

[0144] In one embodiment, the antibody comprises: [ka] It is conjugated to During the ceremony, [ka] is the binding to the antibody.

[0145] In one embodiment, the antibody comprises: [ka] is conjugated to [ka] is the binding to the antibody.

[0146] In one embodiment, the antibody comprises: [ka] It is conjugated to During the ceremony, [ka] is the binding to the antibody.

[0147] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n having During the ceremony, Ab is an anti-MET antibody or a MET x MET bispecific antigen binding protein described herein; L is a linker; Pay is a cytotoxic agent; n is an integer from 1 to 10.

[0148] In some embodiments, the Ab is an anti-MET antibody that comprises the CDRs within the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 82 / 138. In some embodiments, the Ab is an anti-MET antibody that comprises the HCVR amino acid sequence of SEQ ID NO: 82 and the LCVR amino acid sequence of SEQ ID NO: 138.

[0149] In some embodiments, the Ab is a METxMET bispecific antigen binding protein comprising the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 58 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 82. In some aspects, the METxMET bispecific antigen binding protein further comprises the CDRs within the LCVR amino acid sequence of SEQ ID NO: 138. In some embodiments, the Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 58 and the D2-HCVR amino acid sequence of SEQ ID NO: 82. In some aspects, the METxMET bispecific antigen binding protein further comprises the LCVR amino acid sequence of SEQ ID NO: 138.

[0150] In some embodiments, the Ab is a METxMET bispecific antigen binding protein comprising the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 18 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 82. In some aspects, the METxMET bispecific antigen binding protein further comprises the CDRs within the LCVR amino acid sequence of SEQ ID NO: 138. In some embodiments, the Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 18 and the D2-HCVR amino acid sequence of SEQ ID NO: 82.

[0151] In some embodiments, L is a cleavable linker. In some embodiments, L is a non-cleavable linker. In some embodiments, L comprises a dipeptide. In some embodiments, L comprises a PAB moiety.

[0152] In some embodiments, L has the structure: [ka] The moiety includes

[0153] In some embodiments, L has the structure: [ka] The moiety includes

[0154] In some embodiments, L has the structure: [ka] The moiety includes

[0155] In some embodiments, L has the structure: [ka] The moiety includes

[0156] In some embodiments, Pay is a maytansinoid.

[0157] In some embodiments, Pay is [ka] where R 1 is alkyl.

[0158] In some embodiments, Pay is [ka] It is.

[0159] In some embodiments, Pay is [ka] It is.

[0160] In some embodiments, n is an integer from 2 to 5.

[0161] In some embodiments, -L-Pay is [ka] and During the ceremony, [ka] is the binding to the antibody.

[0162] In some embodiments, -L-Pay is [ka] where: [ka] is the binding to the antibody.

[0163] In some embodiments, -L-Pay is [ka] where: [ka] is the binding to the antibody.

[0164] In some embodiments, -L-Pay is [ka] and During the ceremony, [ka] is the binding to the antibody.

[0165] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is an anti-MET antibody comprising the HCVR amino acid sequence of SEQ ID NO:82 and the LCVR amino acid sequence of SEQ ID NO:138; L-Pay is [ka] where: [ka] is the bond to the antibody; and n is an integer from 2 to 5.

[0166] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is an anti-MET antibody comprising the HCVR amino acid sequence of SEQ ID NO:82 and the LCVR amino acid sequence of SEQ ID NO:138; L-Pay is [ka] where: [ka] is the bond to the antibody; and n is an integer from 2 to 5.

[0167] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is an anti-MET antibody comprising the HCVR amino acid sequence of SEQ ID NO:82 and the LCVR amino acid sequence of SEQ ID NO:138; L-Pay is [ka] where: [ka] is the bond to the antibody; and n is an integer from 2 to 5.

[0168] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is an anti-MET antibody comprising the HCVR amino acid sequence of SEQ ID NO:82 and the LCVR amino acid sequence of SEQ ID NO:138; L-Pay is [ka] where: [ka] is the bond to the antibody; and n is an integer from 2 to 5.

[0169] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO:58 and the D2-HCVR amino acid sequence of SEQ ID NO:82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0170] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO:58 and the D2-HCVR amino acid sequence of SEQ ID NO:82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0171] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO:58 and the D2-HCVR amino acid sequence of SEQ ID NO:82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0172] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO:58 and the D2-HCVR amino acid sequence of SEQ ID NO:82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0173] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 18 and the D2-HCVR amino acid sequence of SEQ ID NO: 82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0174] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 18 and the D2-HCVR amino acid sequence of SEQ ID NO: 82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0175] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 18 and the D2-HCVR amino acid sequence of SEQ ID NO: 82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0176] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein Ab is a METxMET bispecific antigen binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 18 and the D2-HCVR amino acid sequence of SEQ ID NO: 82; L-Pay is [ka] where: [ka] is a bond to an antigen-binding protein; and n is an integer from 2 to 5.

[0177] The antibody drug conjugates described herein can be prepared using conjugation conditions known to those of skill in the art (see, e.g., Doronina et al., Nature Biotechnology, 2003, vol. 21, no. 7, p. 778, which is incorporated herein by reference in its entirety). In some embodiments, an antibody drug conjugate of an anti-MET antibody or METxMET bispecific antigen binding protein is prepared by contacting an anti-MET antibody or METxMET bispecific antigen binding protein described herein with a compound comprising a desired linker and a cytotoxic agent, where the linker has a moiety that is reactive with the antibody or antigen binding protein, e.g., at a desired residue of the antibody or antigen binding protein.

[0178] In some embodiments, a process for preparing an antibody-drug conjugate is provided, comprising administering to a patient an anti-MET antibody or a MET×MET bispecific antigen binding protein described herein, the antibody being represented by the following formula A: 1 : [ka] and an aqueous diluent.

[0179] In some embodiments, the compound of formula A 1 In some embodiments, the compound of formula A is present in stoichiometric excess. 1 is present in 5-6 fold stoichiometric excess. In some embodiments, the aqueous diluent comprises HEPES. In some embodiments, the aqueous diluent comprises DMA.

[0180] In some embodiments, the compound of formula A 1The compound of formula A 2 Or A 3 : [ka] [ka] It is a compound of the formula:

[0181] In some embodiments, the compound of formula A 2 The compound is stereomerically pure A 3 In some embodiments, the compound of formula A 1 The compound of formula A 1 Or A 2 wherein A 1 Or A 2 The compounds of formula (I) are present in a diastereomeric excess of greater than 50%. In certain embodiments, the diastereomeric excess is greater than 70%. In certain embodiments, the diastereomeric excess is greater than 90%. In certain embodiments, the diastereomeric excess is greater than 95%.

[0182] The term "diastereomeric excess" refers to the difference between the molar fraction of a desired single diastereomer compared to the remaining diastereomers in a composition. Diastereomeric excess is calculated as follows: (amount of single diastereomer)-(amount of other diastereomer) / 1. For example, a composition containing 90% of 1 and 10% of 2, 3, 4, or a mixture thereof has a diastereomeric excess of 80% [(90-10) / 1]. A composition containing 95% of 1 and 5% of 2, 3, 4, or a mixture thereof has a diastereomeric excess of 90% [(95-5) / 1]. A composition containing 99% of 1 and 1% of 2, 3, 4, or a mixture thereof has a diastereomeric excess of 98% [(99-1) / 1]. Diastereomeric excess can be calculated similarly for any one of 1, 2, 3, or 4.

[0183] In some embodiments, the compound of formula A 1is reacted in the presence of silica gel and a diluent to produce a compound of formula (a): [ka] with a compound of formula (b) [ka] In some embodiments, the diluent comprises an organic solvent and water.

[0184] As used herein, in the presence of silica gel and a diluent, (i) A compound of formula (a): [ka] with a compound of formula (b): [ka] to synthesize an intermediate, (ii) contacting an anti-MET antibody or a MET x MET bispecific antigen binding protein described herein with the intermediate and an aqueous diluent; Products prepared by the processes are also provided.

[0185] In some embodiments, a process for preparing an antibody-drug conjugate comprises agonizing an anti-MET antibody or a MET x MET bispecific antigen binding protein described herein, the antibody being represented by the following formula B: [ka] Provided herein is a process that includes contacting a compound having the formula: where LG is a leaving group, and an aqueous diluent.

[0186] In some embodiments, the compound of formula B is present in stoichiometric excess. In some embodiments, the compound of formula B is present in 5-6 fold stoichiometric excess. In some embodiments, the aqueous diluent comprises HEPES. In some embodiments, the aqueous diluent comprises DMA. In some embodiments, -C(O)-LG is an ester, e.g., NHS or a trifluorophenyl ester.

[0187] In some embodiments, the compound of formula B has the formula B 1 : [ka] It is a compound of the formula:

[0188] In some embodiments, the compound of formula B 1 is a compound of formula C: [ka] is prepared by contacting with a peptide coupling reagent, N-hydroxysuccinimide (NHS), and an organic diluent. Suitable peptide coupling reagents include those that activate or render reactive a carboxylic acid moiety for reaction with a nucleophile. In certain embodiments, the peptide coupling reagent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC). In some embodiments, the organic solvent is dichloromethane.

[0189] In some embodiments, the compound of formula C is a compound of formula D: [ka] is prepared by contacting with adipic acid, a peptide coupling agent, and an organic solvent. In certain embodiments, the peptide coupling agent is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ). In certain embodiments, the organic solvent comprises dichloromethane. Compound D can be prepared as described in WO2014 / 145090.

[0190] Epitope mapping and related techniques The epitope to which the antibody and antigen binding domain bind can consist of a single contiguous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids of the MET protein. Alternatively, the relevant epitope can consist of multiple non-contiguous amino acids (or amino acid sequences) of MET. In some embodiments, the epitope is located at or near the ligand binding domain of MET. In other embodiments, the epitope is located outside the ligand binding domain of MET, e.g., at a location on the surface of MET where the antibody, when bound to such an epitope, does not interfere with HGF binding to MET.

[0191] As described elsewhere herein, the individual antigen-binding domains (D1 and D2) of a METxMET bispecific antigen-binding molecule may bind to different epitopes, or non-overlapping epitopes, or partially overlapping epitopes relative to each other. As used herein, "partially overlapping epitopes" means that the first and second epitopes share less than 5, less than 4, less than 3, or only 1 common amino acid as determined by any epitope mapping methodology known in the art (e.g., X-ray crystallography, alanine scanning mutagenesis, hydrogen / deuterium exchange [HDX], domain swapping, etc.). The D1 and D2 domains may be non-competitive with each other. For example, in certain embodiments, the binding of the D1 domain of a particular METxMET bispecific antigen-binding molecule to its epitope on MET does not inhibit (or only minimally inhibits) the binding of the D2 domain of a METxMET bispecific antigen-binding molecule to its epitope on MET. Due to the non-overlapping (or at most partially overlapping) nature of the epitopes of the D1 and D2 components, a METxMET bispecific antigen-binding molecule can bind to a single MET molecule on the cell surface.

[0192] Various techniques known to those of skill in the art can be used to determine the epitope on MET with which the antibodies and antigen-binding domains of the present disclosure interact. Exemplary techniques that can be used to determine the epitope or binding domain of a particular antibody or antigen-binding domain include, for example, point mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection, and peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids in a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves deuterium labeling of the protein of interest, followed by binding of an antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water to allow hydrogen-deuterium exchange at all residues except those protected by the antibody (which remain deuterium-labeled). After dissociation of the antibody, the target protein is subjected to protease cleavage and analysis by mass spectrometry, which reveals the deuterium-labeled residues that correspond to the specific amino acids with which the antibody interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A. X-ray crystallography can also be used to identify amino acids within a polypeptide with which an antibody interacts.

[0193] Further provided herein are anti-MET antibodies (including bispecific antibodies) that bind to the same epitope as any of the specific exemplary antibodies or antigen-binding domains described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Table 1 herein). Similarly, provided herein are anti-MET antibodies that compete with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences set forth in Table 1 herein) for binding to MET. In some embodiments, the human MET epitope to which the anti-MET antibody binds comprises amino acids 192-204, amino acids 305-315, and / or amino acids 421-455 of SEQ ID NO:155. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO:155, and the second epitope of human MET comprises amino acids 305-315 and amino acids 421-455 of SEQ ID NO:155.

[0194] By using routine methods known in the art and exemplified herein, it can be easily determined whether an antibody binds to the same epitope as a reference anti-MET antibody or competes for binding with it. For example, to determine whether a test antibody binds to the same epitope as a reference anti-MET antibody provided herein, the reference antibody is bound to a MET protein. The ability of the test antibody to bind to a MET molecule is then evaluated. If the test antibody can bind to MET after saturation binding with the reference anti-MET antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-MET antibody. On the other hand, if the test antibody cannot bind to a MET molecule after saturation binding with the reference anti-MET antibody, the test antibody may bind to the same epitope as that bound by the reference anti-MET antibody. Additional routine experimental methods (e.g., peptide mutation and binding analysis) can then be performed to confirm whether the observed lack of binding of the test antibody is indeed due to binding to the same epitope as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. This type of experiment can be performed using ELISA, RIA, Biacore, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art. According to certain embodiments, two antibodies bind to the same (or overlapping) epitope if, for example, 1, 5, 10, 20 or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90% or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, two antibodies are considered to bind to the same epitope if essentially every amino acid mutation in the antigen that reduces or eliminates the binding of one antibody reduces or eliminates the binding of the other. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduce or eliminate the binding of the other.

[0195] To determine whether an antibody competes for binding (or cross-competes for binding) with a reference anti-MET antibody, the binding procedure described above is performed in two orientations: in the first orientation, the reference antibody is allowed to bind to the MET protein under saturating conditions, followed by assessment of binding of the test antibody to the MET molecule. In the second orientation, the test antibody is allowed to bind to the MET molecule under saturating conditions, followed by assessment of binding of the reference antibody to the MET molecule. If in both orientations, only the first (saturating) antibody is able to bind to the MET molecule, it is concluded that the test and reference antibodies compete for binding to MET. As will be appreciated by those skilled in the art, an antibody that competes for binding with a reference antibody will not necessarily bind to the same epitope as the reference antibody, but may sterically block binding of the reference antibody by binding to an overlapping or adjacent epitope.

[0196] Preparation of human antibodies The anti-MET antibodies and METxMET bispecific antibodies provided herein can be fully human antibodies. Methods for generating monoclonal antibodies, including fully human monoclonal antibodies, are known in the art. Any such known methods can be used in the context of the present disclosure to generate human antibodies that specifically bind to human MET.

[0197] For example, using VELOCIMMUNE™ technology, or any other similar known method for generating fully human monoclonal antibodies, high affinity chimeric antibodies against MET are first isolated, having human variable regions and mouse constant regions. As in the experimental section below, the antibodies are characterized and selected for desired characteristics including affinity, ligand blocking activity, selectivity, epitope, etc. If necessary, the mouse constant region is replaced with a desired human constant region, e.g., wild-type or modified IgG1 or IgG4, to generate a fully human anti-MET antibody. The constant region selected can vary depending on the particular application, but the high affinity antigen binding and target specificity characteristics reside in the variable region. In certain cases, fully human anti-MET antibodies are isolated directly from antigen-positive B cells.

[0198] biological equivalent The anti-MET antibodies and antibody fragments provided herein include proteins having amino acid sequences that vary from the amino acid sequences of the described antibodies but retain the ability to bind to human MET. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit essentially the same biological activity as the described antibody. Similarly, DNA sequences encoding the anti-MET antibodies of the present disclosure include sequences that contain one or more additions, deletions, or substitutions of nucleotides compared to the disclosed sequences, but encode anti-MET antibodies or antibody fragments that are essentially biologically equivalent to the anti-MET antibodies or antibody fragments of the present disclosure. Examples of such variant amino acid and DNA sequences are discussed above.

[0199] Two antigen binding proteins or antibodies are considered to be bioequivalent if they are pharmaceutical equivalents or pharmaceutical substitutes that do not show significant differences in their rate and extent of absorption when administered, for example, at the same molar dose, under similar experimental conditions, either in single doses or multiple doses. In cases where some antibodies are equivalent in their extent of absorption but not in their rate of absorption, they may still be considered to be bioequivalent because such differences in absorption rates are intentional, reflected in the label, not essential to achieving effective body drug concentrations, for example in chronic use, and considered medically irrelevant for the particular drug product being tested.

[0200] In one embodiment, two antigen binding proteins are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency.

[0201] In one embodiment, two antigen binding proteins are bioequivalent if a patient can be such switched between the reference product and the biological product one or more times without a predicted increased risk of adverse effects, including clinically significant changes in immunogenicity or diminished efficacy, compared to ongoing therapy without switching between the reference product and the biological product.

[0202] In one embodiment, two antigen binding proteins are biologically equivalent if they both act through a common mechanism(s) of action for the condition(s) of use, to the extent that such mechanism is known.

[0203] Bioequivalence can be demonstrated by in vivo and in vitro methods. Measurements of bioequivalence include, for example, (a) in vivo tests in humans or other mammals that measure the concentration of an antibody or its metabolites in blood, plasma, serum or other biological fluids as a function of time; (b) in vitro tests that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo tests in humans or other mammals that measure the relevant acute pharmacological effects of the antibody (or its target) as a function of time; and (d) tests in well-controlled clinical trials that establish the safety, efficacy or bioavailability or bioequivalence of the antibody.

[0204] Biologically equivalent variants of the anti-MET antibodies provided herein can be constructed, for example, by making various substitutions of residues or sequences or by deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine ​​residues that are not essential for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bridges in the renaturation. In other contexts, biologically equivalent antibodies can include anti-MET antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, for example, mutations that eliminate or remove glycosylation.

[0205] Species selectivity and species cross-reactivity According to certain embodiments, the present disclosure provides anti-MET antibodies (and antigen-binding molecules comprising anti-MET antigen-binding domains) that bind to human MET but not to MET from other species. The present disclosure also includes anti-MET antibodies (and antigen-binding molecules comprising anti-MET antigen-binding domains) that bind to human MET and MET from one or more non-human species. For example, anti-MET antibodies and antigen-binding molecules can bind to human MET and may or may not bind to one or more of mouse, rat, guinea pig, hamster, gerbil, pig, cat, dog, rabbit, goat, sheep, cow, horse, camel, cynomologous, marmoset, rhesus monkey, or chimpanzee MET. According to certain exemplary embodiments, anti-MET antibodies and antigen-binding molecules that specifically bind to human MET and cynomolgus monkey (e.g., Macaca fascicularis) MET are provided. Other anti-MET antibodies and antigen-binding molecules bind to human MET but do not bind, or only weakly bind, to cynomolgus monkey MET. multispecific antibodies

[0206] As described elsewhere herein, the present disclosure provides bispecific antigen-binding molecules comprising two different antigen-binding domains, where a first antigen-binding domain (D1) binds a first epitope on MET and a second antigen-binding domain (D2) binds a second epitope on MET. In certain embodiments, the first and second epitopes on MET bound by the D1 and D2 domains are different, non-overlapping, or partially overlapping. According to this aspect, the D1 domain may comprise any of the amino acid sequences of the HCVR / LCVR or CDR as depicted in Table 1 herein, and the D2 domain may comprise any other of the amino acid sequences of the HCVR / LCVR or CDR as depicted in Table 1 herein (as long as the binding specificity of the D1 domain is different from the binding specificity of the D2 domain and / or the antigen-binding protein from which D1 was derived does not compete with the antigen-binding protein from which D2 was derived for binding to MET). In some embodiments, the human MET epitope to which the anti-MET antibody binds comprises amino acids 192-204, amino acids 305-315, and / or amino acids 421-455 of SEQ ID NO: 155. In some embodiments, the first epitope of human MET comprises amino acids 192-204 of SEQ ID NO: 155, and the second epitope of human MET comprises amino acids 305-315 and amino acids 421-455 of SEQ ID NO: 155.

[0207] According to another aspect of the present disclosure, there is also provided a conventional bispecific antibody, in which one arm of the bispecific antibody binds to human MET and the other arm of the bispecific antibody is specific for a second antigen other than MET. The MET-binding arm can comprise any of the HCVR / LCVR or CDR amino acid sequences depicted in Table 1 herein. In certain embodiments, the MET-binding arm binds to human MET and blocks HGF binding to MET. In other embodiments, the MET-binding arm binds to human MET but does not block HGF binding to MET.

[0208] An exemplary bispecific antibody format that can be used in the context of the present disclosure is a first immunoglobulin (Ig) C H 3 domain and second Ig C H The use of a first and a second Ig C H In one embodiment, the first Ig C domain is a IgG1 domain that is a IgG2 domain that is a IgG3 domain that is a IgG4 domain that is a IgG4 domain that is a IgG5 ... H The 3 domain binds to protein A and the second Ig C H The C3 domain contains mutations that reduce or abolish Protein A binding, such as the H95R modification (according to the IMGT exon numbering; H435R according to the EU numbering). H 3 may further comprise a Y96F modification (by IMGT; Y436F by EU). H Further modifications that may be found within 3 include, for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU); for IgG2 antibodies, N44S, K52N and V82I (by IMGT; N384S, K392N and V422I by EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU). Variations in the bispecific antibody formats described above are considered to be within the scope of this disclosure.

[0209] Other exemplary bispecific formats that can be used in the context of the present disclosure include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG and Mab. 2 These include, but are not limited to, bispecific formats (see, e.g., Klein et al., 2012, mAbs 4:6, 1-11 and references cited therein, for a review of the aforementioned formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that self-assemble into multimeric complexes with defined composition, valency and geometry (see, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0210] Therapeutic Formulations and Administration Provided herein are pharmaceutical compositions comprising the anti-MET antibodies or METxMET bispecific antigen binding molecules of the invention. The pharmaceutical compositions may be formulated with suitable carriers, excipients and other agents that provide improved transport, delivery, tolerance, etc.

[0211] Therapeutic Uses of Antibodies Provided herein are methods that include administering to a subject in need thereof a therapeutic composition comprising an anti-MET antibody or a METxMET bispecific antigen-binding molecule (e.g., an anti-MET comprising any of the HCVR / LCVR or CDR sequences set forth in Table 1 herein, or a METxMET bispecific antigen-binding molecule comprising any of the D1 and D2 components as set forth in Table 5 herein). The therapeutic composition may comprise any of the anti-MET antibodies or METxMET bispecific antigen-binding molecules disclosed herein and a pharma- ceutically acceptable carrier or diluent.

[0212] The anti-MET antibodies and MET x MET bispecific antigen binding molecules are particularly useful for the treatment, prevention and / or amelioration of any disease or disorder associated with or mediated by MET expression, signaling or activity, or which can be treated by blocking the interaction between MET and HGF, or otherwise inhibiting MET activity and / or signaling, and / or promoting receptor internalization, and / or reducing the number of cell surface receptors.

[0213] For example, the anti-MET antibodies and METxMET bispecific antigen binding molecules of the disclosure are useful for treating tumors that express (or overexpress) MET. For example, anti-MET antibodies or METxMET bispecific antigen binding molecules can be used to treat primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and adnexa, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and specialized sensory organs such as the eye. In certain embodiments, anti-MET antibodies and METxMET bispecific antigen binding molecules are used to treat one or more of the following cancers: acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer (e.g., gastric cancer with MET amplification), glioblastoma. , head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), Kaposi's sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lung cancer (e.g., non-small cell lung cancer [NSCLC]), lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, synovial sarcoma, thyroid cancer, and Wilms' tumor.

[0214] Within the context of the methods of treatment described herein, the anti-MET antibodies and METxMET bispecific antigen binding molecules can be administered as monotherapy (i.e., as the only therapeutic agent) or in combination with one or more additional therapeutic agents, examples of which are described elsewhere herein.

[0215] Combination Therapies and Formulations Compositions and therapeutic formulations comprising any of the anti-MET antibodies and METxMET bispecific antigen-binding molecules described herein in combination with one or more additional therapeutically active components, as well as methods of treatment comprising administering such combinations to a subject in need thereof, are provided.

[0216] Anti-MET antibodies and MET x MET bispecific antigen binding molecules can be used in combination with other antibodies to treat or inhibit MET, including MET antagonists (e.g., anti-MET antibodies [e.g., onartuzumab, emibetuzumab, and H4H14639D] or small molecule inhibitors of MET), EGFR antagonists (e.g., anti-EGFR antibodies [e.g., cetuximab or panitumumab] or small molecule inhibitors of EGFR [e.g., gefitinib or erlotinib]), antagonists of another EGFR family member, such as Her2 / ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 [e.g., trastuzumab or T-DM1 {KADCYLA®}], anti-ErbB3 or anti-ErbB4 antibodies, or antibodies that inhibit ErbB2, ErbB3 or ErbB4 activity). small molecule inhibitors of EGFRvIII), EGFRvIII antagonists (e.g., anti-EGFRvIII antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-α inhibitors (e.g., anti-PDGFR-α antibodies), PDGFR-β inhibitors (e.g., anti-PDGFR-β antibodies or small molecule kinase inhibitors such as, e.g., imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, -B, -C or -D antibodies, aptamers, siRNA, etc.), VEGF antagonists (e.g., aflibercept, etc., VEGF traps, e.g., US 7,087,see US 2009 / 0142354, such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-MSLN antibodies), CA9 antagonists (e.g., , anti-CA9 antibody), uroplakin antagonist (e.g., anti-uroplakin [e.g., anti-UPK3A] antibody), MUC16 antagonist (e.g., anti-MUC16 antibody), Tn antigen antagonist (e.g., anti-Tn antibody), CLEC12A antagonist (e.g., anti-CLEC12A antibody), TNFRSF17 antagonist (e.g., anti-TNFRSF17 antibody), LGR5 antagonist (e.g., anti-LGR5 antibody), monovalent CD20 antagonist (e.g., monovalent anti-CD20 antibody such as rituximab), CD20xCD3 bispecific antibody, PD-1 blocker (e.g., anti-PD-1 antibody, e.g., pembrolizumab or nivolumab), and the like. Other agents that may be beneficially administered in combination with the antibodies provided herein include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors.

[0217] Illustratively, a PD-1 inhibitor, such as an anti-PD-1 antibody, can be combined with an anti-Met antibody-drug as described herein. Target patient populations specifically include patients with tumors that overexpress c-Met mutations, such as patients with c-Met-expressing non-small cell lung cancer.

[0218] Provided herein are compositions and therapeutic formulations comprising any of the anti-MET antibodies and MET x MET bispecific antigen binding molecules described herein in combination with one or more chemotherapeutic agents. Exemplary chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide (Cytoxan™); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and trimethylolmelamine. e) including ethyleneimine and methylamelamine; chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, etc., nitrogen mustards; carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc., nitrosurea;Aclacinomycin, actinomycin, austramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, etc., antibiotics; methotrexate and and 5-fluorouracil (5-FU), etc.; folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate, etc.; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.; pyrimidine analogues, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.; androgens, such as calusterone, dromostanol propionate, epithiostanol, mepitiostane, testolactone, etc.; anti-adrenal drugs, such as aminoglutethimide, mitotane, trilostane, etc.; folic acid supplements, such as floric acid, etc.; aceglatone; aldophosphamide glycosides glycoside);aminolevulinic acid;amsacrine;bestrabucil;bisantrene;edatrexate;defofamine;demecolcine;diaziquone;elfornithine;elliptinium acetate;ethoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;mitoguazone;mitoxantrone;mopidamol;nitracrine;pentostatin;phenamet;Pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; schizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (Taxol™, Bristol-Myers; Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere™; Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antihormones that act to regulate or inhibit hormone action in tumors, such as, for example, pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0219] Anti-MET antibodies and METxMET bispecific antigen binding molecules can also be administered in combination and / or co-formulated with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN-gamma and / or NSAIDs.

[0220] The additional therapeutically active component(s), e.g., any of the agents listed above or derivatives thereof, can be administered immediately prior to, concurrently with, or immediately following administration of the anti-MET antibody or METxMET bispecific antigen-binding molecule; (for purposes of this disclosure, such administration regimes will be considered administration of the antibody "in combination with" the additional therapeutically active component.) The present disclosure includes pharmaceutical compositions in which an anti-MET antibody or METxMET bispecific antigen-binding molecule is co-formulated with one or more of the additional therapeutically active component(s) described elsewhere herein.

[0221] Dosing regimen According to certain embodiments, multiple doses of an anti-MET antibody or METxMET bispecific antigen-binding molecule (or a pharmaceutical composition comprising a combination of an anti-MET antibody or METxMET bispecific antigen-binding molecule and any of the additional therapeutically active agents mentioned herein) can be administered to a subject over a defined time course. The method according to this aspect comprises sequentially administering to a subject multiple doses of an anti-MET antibody or METxMET bispecific antigen-binding molecule provided herein. As used herein, "sequentially administering" means that each dose of antibody is administered to a subject at different time points, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present disclosure includes methods comprising sequentially administering to a patient a single initial dose of an anti-MET antibody or METxMET bispecific antigen-binding molecule, followed by one or more secondary doses of the anti-MET antibody or METxMET bispecific antigen-binding molecule, and optionally followed by one or more tertiary doses of the anti-MET antibody or METxMET bispecific antigen-binding molecule.

[0222] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time sequence of administration of anti-MET antibody or METxMET bispecific antigen-binding molecule. Thus, an "initial dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"); a "secondary dose" is a dose administered after the initial dose; and a "tertiary dose" is a dose administered after the secondary dose. The initial, secondary and tertiary doses can all contain the same amount of anti-MET antibody or METxMET bispecific antigen-binding molecule, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of antibody contained in the initial, secondary and / or tertiary doses varies from each other during the course of treatment (e.g., adjusted upwards or downwards as appropriate). In certain embodiments, two or more (e.g., 2, 3, 4 or 5) doses are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0223] Diagnostic Uses of Antibodies The anti-MET antibodies or METxMET bispecific antigen binding molecules of the present disclosure can also be used to detect and / or measure MET or MET-expressing cells in a sample, e.g., for diagnostic purposes. For example, an anti-MET antibody or fragment thereof can be used to diagnose a condition or disease characterized by aberrant expression of MET (e.g., overexpression, underexpression, lack of expression, etc.). An exemplary diagnostic assay for MET can include, for example, contacting a sample obtained from a patient with an anti-MET antibody or METxMET bispecific antigen binding molecule of the present invention, where the antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-MET antibody or METxMET bispecific antigen binding molecule can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S or 125 MET can be a radioisotope, such as I; a fluorescent or chemiluminescent moiety, such as fluorescein or rhodamine; or an enzyme, such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure MET in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immuno-PET (e.g., 89 Zr, 64 Cu, etc.), and fluorescence activated cell sorting (FACS).

[0224] Samples that can be used in the MET diagnostic assay of the present disclosure include any tissue or fluid sample that can be obtained from a patient. In general, the level of MET in a particular sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal MET levels or activity) will be measured to first establish a baseline or standard level of MET. This baseline level of MET can then be compared to the level of MET measured in a sample obtained from an individual suspected of having a MET-related disease or condition. EXAMPLES

[0225] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions provided herein, and are not intended to limit the scope of what the inventors consider to be their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0226] Example 1 Generation of anti-MET antibodies Anti-MET antibodies were obtained by immunizing genetically engineered mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions with an immunogen containing recombinant human MET extracellular domain fused to human Fc (R&D Systems, catalog #358-MT, Minneapolis, Minn.). The mice used for immunization express a "universal light chain," i.e., antibodies produced in these mice have different heavy chain variable regions but essentially identical light chain variable domains.

[0227] Antibody immune responses were monitored by MET-specific immunoassays. Once the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to preserve their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines that produce MET-specific antibodies. Using this technique, several anti-MET chimeric antibodies (i.e., antibodies that have human variable domains and mouse constant domains) were obtained. In addition, several fully human anti-MET antibodies were isolated directly from antigen-positive B cells without fusion to myeloma cells, as described in US2007 / 0280945A1.

[0228] The particular biological properties of exemplary anti-MET antibodies generated according to the methods of this example, and bispecific antibodies constructed therefrom, are described in detail in the Examples below.

[0229] Example 2 Heavy and light chain variable region amino acid and nucleic acid sequences Table 1 sets forth the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs of selected anti-MET antibodies described herein. (As noted above, all of the antibodies produced in Example 1 have identical light chain variable regions and therefore also have identical light chain CDR sequences.) The corresponding nucleic acid sequence identifiers are set forth in Table 2. [Table 1] [Table 2]

[0230] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H4H", etc.), followed by a numerical identifier (e.g., "13290", "13291", "13295", etc.), followed by the "P2" suffix, as shown in Tables 1 and 2. Thus, according to this nomenclature, antibodies may be referred to herein as, for example, "H4H13290P2", "H4H13291P2", "H4H13295P2", etc. The prefixes in the antibody names used herein indicate the particular Fc region isotype of the antibody. In particular, an "H4H" antibody has a human IgG4 Fc (all variable regions are fully human, as indicated by the first "H" in the antibody name). As will be appreciated by those of skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG4 Fc can be converted to an antibody having a human IgG1 Fc, etc.), but in any event, the variable domains (including the CDRs) (which are indicated by the numerical identifiers shown in Tables 1 and 2) will remain the same and the binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0231] Example 3 Surface plasmon resonance derived binding affinity and kinetic constants of a human monoclonal anti-MET (monospecific) antibody. The binding affinity and kinetic constants of human anti-MET antibodies were determined by surface plasmon resonance (Biacore 4000 or T-200) at 37°C. The anti-Met antibodies tested in this example were bivalent monospecific binders of MET. The antibody expressed as human IgG4 (referred to as "H4H") was captured on a CM4 or CM5 Biacore sensor surface derivatized by amine coupling with a mouse monoclonal anti-human Fc antibody (GE, BR-1008-39). Various concentrations of soluble monomeric (human (h)Met.mmh; SEQ ID NO: 152; macaca fascicularis (mf)Met.mmh; SEQ ID NO: 154) or dimeric (hMet.mFc; SEQ ID NO: 153) Met protein were injected over the captured surface of anti-MET antibodies at flow rates of 30 or 50 μL / min. Association of hMET.mmh or hMET.mFc to captured monoclonal antibodies was monitored for 4 or 5 min, and dissociation of hMET.mmh or hMET.mFc in HBS-ET (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20) or PBS-P (0.01 M sodium phosphate pH 7.4, 0.15 M NaCl, 0.05% v / v surfactant P20) running buffer was monitored for 10 min.

[0232] Kinetic binding rate constant (k a ) and the dissociation rate constant (k d ) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

number

[0233] The binding kinetic parameters of the monospecific anti-Met antibodies to the monomeric and dimeric Met proteins are shown in Table 3 below. [Table 3-1] [Table 3-2]

[0234] As shown in Table 3, several antibodies demonstrated high affinity binding to human and monkey MET proteins.

[0235] Example 4 Anti-Met antibodies bind to distinct epitopes on the MET receptor. To assess whether the two anti-Met antibodies could compete with each other for binding to their respective epitopes on MET, a binding competition assay was performed using real-time label-free biolayer interferometry (BLI) on an OCTET® HTX biosensor (ForteBio Corp., Menlo Park, CA).

[0236] Briefly, approximately 0.25 nM of human MET extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hMet.mmh) was first captured on an anti-pentaHis antibody-coated OCTET® biosensor (ForteBio Corp., #18-5079) by immersing the biosensor in a well containing a 20 μg / mL solution of hMET.mmh for 5 minutes. The antigen-captured biosensor was then saturated with a first anti-MET monoclonal antibody (hereafter referred to as mAb-1) by immersing the biosensor in a well containing a 50 μg / mL solution of mAb-1 for 5 minutes. The biosensor was then immersed in a well containing a 50 μg / mL solution of a second anti-MET monoclonal antibody (hereafter referred to as mAb-2) for 3 minutes. All biosensors were washed in OCTET® HEPES-buffered saline-EDTA polysorbate 20 (HBS-EP) buffer between each step of the experiment. Real-time binding responses were monitored during the course of the experiment and the binding responses at the end of each step were recorded. The binding responses of mAb-2 to anti-MET pre-complexed with mAb-1 were compared and the 50% inhibition threshold was used to determine the competitive / non-competitive behavior of the different anti-MET monoclonal antibodies. Table 4 clearly defines the relationship of bidirectional competing antibodies, independent of the order of binding. [Table 4]

[0237] Example 5 Construction of a bispecific antibody with two distinct antigen-binding domains specific for different epitopes of MET This example describes the construction of a bispecific antibody comprising two different antigen-binding domains (D1 and D2), where D1 and D2 are derived from different anti-MET antibodies and, as a result, bind to separate epitopes in the MET extracellular domain.

[0238] The individual anti-MET antigen-binding domains used to construct the bispecific antibodies of this example were derived from the various bivalent monospecific anti-MET antibodies described herein in Examples 1-3. All anti-MET antibodies described herein contain the same ("common") light chain (comprising the light chain variable region [LCVR] amino acid sequence of SEQ ID NO: 138 and the light chain CDRs [LCDR1, LCDR2, and LCDR3] amino acid sequences of SEQ ID NOs: 140, 142, and 144). Additionally, all bispecific antibodies exemplified in this example contain a "D2" arm derived from the exemplary anti-MET antibody H4H13312P2. Thus, both antigen-binding domains (D1 and D2) of all bispecific antibodies described in this example contain this common light chain variable region, and all D2 binding arms contain heavy chain variable regions derived from H4H13312P2, although the bispecific antibodies differ from each other with respect to their D1 heavy chain variable region (HCVR) and heavy chain CDRs (HCDRs). The components of the bispecific antibody of this example are summarized in Table 5. [Table 5]

[0239] Example 6 Surface plasmon resonance derived binding affinity and kinetic constants of a MET*MET human bispecific monoclonal antibody The binding affinity and kinetic constants of the METxMET human bispecific antibody constructed according to Example 4 herein were determined by surface plasmon resonance (Biacore 4000 or T-200) at 37°C. The bispecific antibody expressed as human IgG4 (referred to as "H4H") was captured on a CM4 or CM5 Biacore sensor surface derivatized by amine coupling with a mouse monoclonal anti-human Fc antibody (GE, BR-1008-39). Various concentrations of soluble monomeric MET protein (hMet.mmh; SEQ ID NO: 152) Met protein were injected over the anti-METxMET bispecific antibody captured surface at a flow rate of 30 or 50 μL / min. Association of analytes to captured bispecific antibodies was monitored for 4 or 5 min, and dissociation of analytes in HBS-ET (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20) or PBS-P (0.01 M sodium phosphate pH 7.4, 0.15 M NaCl, 0.05% v / v surfactant P20) running buffer was monitored for 10 min.

[0240] Kinetic binding rate constant (k a ) and the dissociation rate constant (k d ) was determined as described in Example 3.

[0241] The binding kinetic parameters of the bispecific anti-Met antibodies to the monomeric Met protein (hMET.mmh) are shown in Table 6. [Table 6]

[0242] As shown in Table 6, the bispecific "METxMET" antibodies described herein exhibited T1 / 2 values ​​of greater than 1155 minutes.

[0243] As shown in Table 7, the off-rate of bispecific antibody H4H14639D is significantly lower than the off-rates of each of its parent antibodies, H4H13306P2 and H4H13312P2. [Table 7]

[0244] Example 7 Anti-Met antibodies block HGF-mediated Met activation in an SRE-luciferase reporter bioassay The ability of anti-MET antibodies to block hepatocyte growth factor (HGF)-mediated MET activation was examined in a luciferase-based reporter assay. The growth factor HGF binds to the extracellular domain of its receptor c-Met (MET), causing rapid homodimerization and activating several downstream signaling cascades. The anti-MET antibodies tested in this example were either bivalent monospecific binders of MET, or anti-MET "bispecifics" in which each arm of the bispecific antibody binds to a different, distinct epitope on MET.

[0245] An engineered cell-based luciferase reporter assay (Figure 2) was used to determine the ability of anti-MET antibodies to activate MET signaling (Figure 3, panel A; Table 8, columns 3 and 4) and block ligand-mediated activation of MET (Figure 3, panel B; Table 8 columns 1 and 2). Briefly, the CIGNAL™ Lenti SRE Reporter (luc) kit (SABiosciences, Hilden, DE) was used to generate HEK293 / SRE-Luc cells. HEK293 (human embryonic kidney) cells were selected because they endogenously express c-Met. HEK293 / SRE-Luc cells stably integrated a serum response element (SRE)-dependent luciferase (Luc) reporter (see Dinter et al., PLoS ONE, vol. 10(2):e0117774, 2015). HEK293 / SRE-Luc cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), penicillin / streptomycin / glutamine, and 1 μg / ml puromycin.

[0246] Next, 2.0×10 51000 x 1000 cells were seeded in luciferase assay medium in 96-well plates and incubated at 37 °C for 24 h at 5% CO. 2 The cells were incubated overnight at 37°C in 5% CO. Dose-response curves for hepatocyte growth factor (HGF) were generated by adding serial dilutions of HGF (0.01 pM-1.0 nM) to the cells and recording the luciferase signal after 4-6 h of incubation at 37°C in the absence of antibody. To generate antibody inhibition curves, cells were preincubated with serial dilutions of anti-human MET antibody (1.1 pM-200 nM) for 1 h at 37°C. HGF at concentrations of 73 pM or 100 pM was then added for an additional 4-6 h before recording the signal. Separately, the ability of the antibodies to activate c-Met in the absence of ligand was also assessed.

[0247] Luciferase activity was detected using the ONE-Glo™ Luciferase Assay System (Promega, Madison, WI) and light emitted was measured in a Victor or Envision luminometer (Perkin Elmer, Shelton, CT) and expressed as relative light units (RLU). EC50 / IC50 values ​​were determined from a four-parameter logistic equation over a 12-point response curve using GRAPHPAD PRISM®. Percent HGF blockade and fold MET activation (mAb alone) were reported for the highest antibody dose. Results are shown in Table 8. [Table 8-1] [Table 8-2]

[0248] As summarized in Table 8, most antibodies inhibited activation of the SRE reporter with IC50 values ​​ranging from less than 2.0 pM to approximately 1.0 nM. Several exemplary monospecific bivalent anti-MET antibodies, such as H4H13306P2 and H4H13309P2, were potent inhibitors of SRE-luc activation with percent inhibition values ​​of 67% and 77%, respectively. Anti-MET bispecific antibodies (MET x MET) showed greater inhibition of SRE-luc activation overall. For example, MET x MET bispecific antibody H4H14639D showed 95 percent inhibition. Additionally, several blocking antibodies showed weak activation in the absence of ligand, with activation fold response values ​​ranging from 0.8 to 14.4 above baseline levels.

[0249] As also shown in FIG. 3, the bivalent monospecific antibodies H41413306P2 and H4H13312P2 each activate the Met pathway in the absence of HGF ligand (Panel A) and block HGF activation of Met (Panel B).

[0250] The effect of bispecific METxMET antibodies (e.g., H4H14639D) on HGF-dependent and HGF-independent MET activation was also assessed using the HEK293 / SRE-Luc system. SRE-driven luciferase activity was measured in HEK293T cells treated with MET antibody H4H14639D (METxMET bispecific antibody), monovalent anti-MET antibodies, and H4H14639D parent antibody H4H13312P2 at various concentrations to ascertain the level of HGF-independent MET agonism. While the parent anti-MET monospecific bivalent antibody exhibited MET agonist activity, neither the monovalent antibody nor the METxMET bispecific antibody exhibited MET agonist activity (Figure 4, Panel A).

[0251] SRE-driven luciferase activity was measured in HEK293T cells treated with MET antibody H4H14639D (MET x MET bispecific antibody), monovalent anti-MET antibody, and H4H14639D parent antibody H4H13312P2 at various concentrations to ascertain the level of inhibition or blockade of HGF-dependent MET agonism. Although the parent anti-MET monospecific bivalent antibody showed some HGF blocking activity, both the monovalent antibody and the MET x MET bispecific antibody showed greater HGF blockade (Figure 4, Panel B).

[0252] The METxMET bispecific antibody blocks HGF signaling and exhibits low MET agonist activity.

[0253] Example 8 Anti-Met antibodies inhibit the growth of MET-amplified cells Selected anti-Met antibodies were then tested for their ability to inhibit the growth of MET-amplified SNU5 cells. 3 Human gastric carcinoma (SNU5) cells were seeded in complete growth medium in the presence of anti-MET antibodies at concentrations ranging from 1.5 pM to 100 nM. SNU5 complete growth medium contained Iscove's modified Dulbecco's medium, 10% FBS, and penicillin / streptomycin / glutamine. Cells were incubated for 5 days and viable cell numbers were determined using the CELLTITER-GLO® Luminescent Cell Viability Assay Kit (Promega, Madison, WI) according to the manufacturer's instructions.

[0254] As summarized in Table 9, several anti-MET antibodies, such as H4H13312P2 and H4H13325P2, blocked SNU5 growth by more than 50%, with overall IC50s ranging from 44 pM to 780 pM.

[0255] FIG. 5 shows the relative cell growth of SNU5 cells treated with various anti-MET bivalent monospecific antibodies (i.e., conventional antibodies). A subset of conventional MET antibodies inhibits the growth of SNU5 MET-amplified gastric cancer cells (FIG. 5). SNU5 cells in 96-well plates were treated with 10 μg / ml of each antibody, and cell growth was determined after 5 days by reduction of ALAMARBLUE® reagent (Thermo Fisher Scientific, Waltham, MA). Monovalent MET antibodies (FIG. 5, second row) were generated using the heavy and light chain variable sequences of MetMab set forth in U.S. Pat. No. 7,892,550 B2, which is incorporated herein by reference in its entirety. Conventional antibody 8 is H4H13306P2 and conventional antibody 11 is H4H13312P2, which were used to construct the MET×MET bispecific antibody H4H14639D.

[0256] In another growth assay, the blocking activity of the MET x MET bispecific antibody (i.e., H4H14639D) was evaluated in both SNU5 and non-small cell lung cancer (NSCLC) cell lines EBC-1, which exhibit an amplified Met gene and overexpress MET (Lutterbach et al., Cancer Res. 67(5):2081-2088, 2007). Complete growth medium for EBC-1 cells contained MEM Earle's salts, 10% fetal bovine serum (FBS), penicillin / streptomycin / glutamine, and non-essential amino acids for MEM. H4H14369D showed the greatest percent inhibition of MET activity by SRE-luciferase readout. In this experiment, 3.0 × 10 3 SNU5 or EBC-1 cells were seeded in complete growth medium in the presence of H4H14639D at concentrations ranging from 15 pM to 100 nM. Cells were incubated at 37 °C for 24 h at 5% CO. 2The cells were incubated at 37°C for 3 days in 5% formaldehyde. Cells were then fixed with 4% formaldehyde and stained with 3 μg / ml Hoechst 33342 to label nuclei. Images were acquired with an IMAGEXPRESS® Micro XL (Molecular Devices, Sunnyvale, CA) and nuclear counts were determined by METAXPRESS® Image Analysis software (Molecular Devices, Sunnyvale, CA). Background nuclear counts from cells treated with 40 nM digitonin were subtracted from all wells and viability was expressed as a percentage of untreated controls. IC50 values ​​were determined from a four-parameter logistic equation over a 10-point response curve (GRAPHPAD PRISM®). IC50 values ​​and percent cell kill are shown in Table 9. [Table 9]

[0257] As summarized in Table 10 below, the METxMET bispecific antibody H4H14639D inhibited the growth of EBC-1 and SNU5 cells by 37 and 40 percent, respectively, with IC50s of 0.82 nM and 0.3 nM. [Table 10]

[0258] SNU5 cells (stomach) in 96-well plates were treated with 0.1 μg / mL, 1 μg / mL, or 10 μg / mL of control antibody, monovalent MET antibody, or MET x MET bispecific antibody. After 5 days, cell growth was determined by reduction of ALAMARBLUE® reagent (Thermo Fisher Scientific, Waltham, Mass.). The MET x MET bispecific antibody significantly reduced the relative cell growth of SNU5 cells compared to the control and monovalent antibodies (Figure 6, Panel A).

[0259] Similarly, the effect of MET x MET bispecific antibody on the growth of EBC-1 cells was evaluated. 2,500 EBC-1 cells were seeded in 96-well plates and cultured in Dulbecco's medium supplemented with 10% FBS. Cells were treated with 0.1 μg / mL or 1 μg / mL of control antibody or MET x MET bispecific antibody, followed by incubation at 37°C, 5% CO. 2 The cells were incubated with 500 mM NaCl. After 5 days, relative cell growth was determined by measuring the reduction of the indicator dye ALAMARBLUE® to its highly fluorescent form in a SPECTRAMAX® M3 plate reader (Molecular Devices, LLC, Sunnyvale, Calif.). The results are shown in Table 11 and FIG. 6, panel B. The MET×MET bispecific antibody (H4H14639D) significantly reduced the relative cell growth of EBC-1 cells compared to the control antibody (FIG. 6, panel B).

[0260] Several anti-MET antibodies, both bivalent monospecific and MET x MET bivalent antibodies, are potent inhibitors of SRE-Luc activation and inhibit the growth of Met-amplified and MET-overexpressing cell lines. [Table 11]

[0261] Example 9 METxMET bispecific antibodies induce moderate and transient MET pathway activity in NCI-H596 NSCLC cells The effect of a MET × MET bispecific antibody on the MET pathway in human lung adenosquamous carcinoma cells was evaluated in vitro.

[0262] 250,000 NCI-H596 cells were seeded in 12-well plates and cultured in RPMI medium supplemented with 10% FBS. The cells were dually treated with 50 ng / ml hepatocyte growth factor (HGF) or 10 μg / ml MET x MET bispecific antibody H4H14639D. The cells were then incubated at 37 °C for 2 h at 5% CO 2The cells were incubated at 37°C for 0, 2, 6, or 18 hours, after which cell lysates were prepared, protein content was normalized, and immunoblot analysis was performed. MET and ERK phosphorylation were quantified by the ImageJ image processing program (T. Collins, BioTechniques, 43:S25-S30, 2007). Phosphorylation levels were normalized to tubulin loading control and expressed as fold change compared to control treatment. The results are summarized in Table 12. [Table 12]

[0263] HGF treatment of NCI-H596 cells induced strong activation of MET and ERK that peaked at 2 h and persisted after 18 h. Moderate MET and ERK phosphorylation was detected by H4H14636D bispecific antibody treatment, which returned to baseline levels by 18 h or 6 h, respectively.

[0264] Example 10 A METxMET bispecific antibody induces MET degradation and inhibits pathway activity more potently than monospecific antibodies in Hs746T gastric cancer cells The effect of the METxMET bispecific antibody on MET activity in human gastric cancer cells was evaluated in vitro. 250,000 Hs746T human gastric cancer cells (H. Smith, J. Nat'l. Cancer Inst. 62(2):225-230, 1979) were seeded in 12-well plates and cultured in modified Dulbecco's medium supplemented with 10% FBS. Cells were treated with (1) hFc control molecule at 5 μg / ml, (2) parental bivalent monospecific anti-MET antibody H4H13306P2 at 5 μg / ml, (3) parental bivalent monospecific anti-MET antibody H4H13312P2 at 5 μg / ml, (4) a combination of H4H13306P2 at 2.5 μg / ml and H4H13312P2 at 2.5 μg / ml, or (5) MET×MET bispecific antibody H4H14639D at 5 μg / ml. Cells were then incubated at 37 °C for 24 h at 5% CO. 2The cells were incubated at 37°C for 18 h. After 18 h, cell lysates were prepared, normalized for protein content, and immunoblot analysis was performed. MET expression, MET phosphorylation, and ERK phosphorylation were quantified by the ImageJ image processing program (T. Collins, BioTechniques, vol. 43:S25-S30, 2007). The results are summarized in Table 13 and Figure 7, panel A, which shows the raw data of the immunoblot. Figure 7, panel B, shows MET protein expression in cells treated with 10 μg / ml of MET x MET bispecific antibody for 0, 2, or 6 h. Total MET levels in Hs747T cells were decreased over time upon treatment with MET x MET bispecific antibody. Similar results were obtained with the MET-amplified human papillary adenocarcinoma NCI-H820 cell line (Bean et al., "MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to getfitnib or erlotinib," Proc. Natl. Acad. Sci. 2007 Dec. 26, 104(52):20932-20937). [Table 13]

[0265] The bispecific antibody H4H14639D induced MET degradation more potently than its parent conventional antibodies, and phosphorylation of both MET and ERK was inhibited by treatment with H4H14636D more effectively than the parent antibodies or the parent antibody combination.

[0266] Hs746T gastric cancer cells were treated for 18 hours with control antibody, METxMET bispecific antibody H4H14639D, anti-MET parental antibody H4H13306P2, anti-MET parental antibody H4H13312P2, and the combination of parental antibodies 1 and 2 at 10 μg / ml of each antibody, or the combination of parental antibodies at 5 μg / ml of each. MET expression (MET) and pathway activation (pMET and pErk) were determined by immunoblotting with the indicated antibodies (Figure 8). In Hs746T gastric cancer cells, the METxMET bispecific antibody inhibited MET pathway activation more effectively than its parental antibodies.

[0267] Example 11 MET×MET bispecific antibodies induce MET degradation more potently than monospecific antibodies in NCI-H596 lung cancer cells The effect of METxMET bispecific antibody and parental bivalent monospecific anti-MET antibody on expression levels of hepatocyte growth factor receptor (HGFR or MET) in human lung adenosquamous carcinoma cells was evaluated. 250,000 NCI-H596 human lung adenosquamous carcinoma cells were seeded in 12-well plates and cultured in RPMI medium supplemented with 10% FBS. Cells were treated with (1) 5 μg / ml hFc control molecule, (2) 5 μg / ml parental bivalent monospecific anti-MET antibody H4H13306P2, (3) 5 μg / ml parental bivalent monospecific anti-MET antibody H4H13312P2, (4) a combination of 2.5 μg / ml H4H13306P2 and 2.5 μg / ml H4H13312P2, or (5) 5 μg / ml METxMET bispecific antibody H4H14639D. Cells were then incubated at 37 °C for 3 h at 5% CO. 2 The cells were incubated at 37°C for 18 hours. After 18 hours, cell lysates were prepared, protein content was normalized, and immunoblot analysis was performed. MET expression was quantified by the ImageJ image processing program (T. Collins, BioTechniques, 43:S25-S30, 2007). The results are summarized in Table 14. [Table 14]

[0268] NCI-H596 (MET exon 14 skipping mutation) lung cancer cells were also treated with 10 μg / ml of control or MET×MET bispecific antibody for 2, 6, or 18 hours. MET expression was determined by immunoblotting (FIG. 9), which shows MET×MET bispecific antibody-induced degradation of MET with increasing treatment time.

[0269] In NCI-H596 lung cancer cells, the bispecific antibody H4H14636D induces MET degradation more potently than its parent conventional antibody.

[0270] Example 12 A METxMET bispecific antibody induces MET degradation and inhibits pathway activity more potently than monospecific antibodies in SNU5 gastric cancer cells The effects of bivalent monospecific anti-MET antibodies and several MET x MET bispecific antibodies on the expression levels of hepatocyte growth factor receptor (HGFR or MET) in gastric cancer cells were evaluated. Human gastric cancer SNU5 cells were seeded in Iscove's medium containing 20% ​​FBS plus penicillin-streptomycin single lutamine. 24 h after seeding, cells were treated with control hFc, anti-MET parent bivalent monospecific antibody H4H13312P2, or MET x MET bispecific antibodies (H4H14634D, H4H14635D, H4H14636D, H4H14637D, H4H14638D, H4H14639D, H4H14640D, H4H14641D) for 18 h. Cell lysates were then prepared and analyzed by Western blotting. Immunoblots were probed for MET and tubulin. MET protein expression levels were quantified and normalized to tubulin loading control. The results are shown in Table 15 and Figure 10, panel B. [Table 15]

[0271] SNU5 cancer cells were treated with 10 μg / ml of control antibody, MET×MET bispecific antibody, or monovalent MET antibody for 18 hours as described above. MET expression (FIG. 10, panels A and B) and pathway activation (i.e., pMET and pERK; panel A) were determined by immunoblotting with the indicated antibodies. Immunoblots are shown in FIG. 10.

[0272] Treatment of SNU5 cells with METxMET bispecific antibody induced degradation of MET more potently than treatment with bivalent monospecific anti-MET antibody (H4H13312P2) (Figure 10, Panel B), monovalent MET antibody, or control hFc. Treatment of SNU5 cells with METxMET bispecific antibody inhibited downstream effectors of the MET pathway. Similar results were obtained for the MET-amplified non-small cell lung cancer adenocarcinoma cell line NCI-H1993 (Kubo et al., "MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors," Int. J. Cancer, 2009 Apr. 15;124(8):1778-1784).

[0273] (Example 13) A MET x MET bispecific antibody induces MET degradation, inhibits pathway activity, and inhibits tumor growth more potently than monospecific antibodies in EBC-1 cells As described above, MET-amplified human lung squamous cell carcinoma EBC-1 cells (Lutterbach et al., "Lung cancer cell lines harboring MET gene amplification are dependent on Met for growth and survival," Cancer Res. 2007 Mar. 1;67(5):2081-8) were treated with control antibody or 10 μg / ml of MET x MET bispecific antibody for 18 hours. MET expression and MET pathway activation confirmed by pMET and pErk expression were determined by immunoblotting with the indicated antibodies. Immunoblots are shown in FIG. 11.

[0274] Treatment of EBC-1 cells harboring MET gene amplification with a METxMET bispecific antibody induced more potent degradation of MET than treatment with a control antibody. Treatment of EBC-1 cells with a METxMET bispecific antibody inhibited downstream effectors of the MET pathway.

[0275] In another experiment, 5 million EBC-1 cells were implanted subcutaneously into the flank of CB-17 SCID mice. 3 Upon reaching tumor volume 1, mice were randomized into groups of six and treated twice weekly with 25 mg / kg control antibody or 25 mg / kg MET x MET bispecific antibody H4H14639D. Tumor growth was monitored for 30 days post-implantation, and tumor volumes (mm) were recorded for each experimental group over time. 3 ) was measured. The results are shown in Table 16 and Figure 12, which demonstrate that the METxMET bispecific antibody significantly inhibits the growth of EBC-1 tumors. [Table 16]

[0276] Example 14 MET×MET bispecific antibodies inhibit the in vitro growth of Hs746T gastric cancer cells more potently than monospecific antibodies The effect of the METxMET bispecific antibody on the growth of human gastric cancer cells was evaluated in vitro. 2,500 Hs746T human gastric cancer cells (H. Smith, J. Nat'l. Cancer Inst. 62(2):225-230, 1979) were seeded in 96-well plates and cultured in modified Dulbecco's medium supplemented with 10% FBS. Cells were treated with (1) 5 μg / ml of each of the individual bivalent monospecific anti-MET antibodies (H4H13306P2 or H4H13312P2), (2) a combination of two bivalent monospecific anti-MET parent antibodies (H4H13306P2 and H4H13312P2) at 2.5 μg / ml each, or (3) 5 μg / ml of a bispecific antibody (H4H14639D) containing one binding arm from H4H13306P2 and the other binding arm from H4H13312P2. Cells were then incubated at 37 °C for 2 h at 37 °C for 3 h at 37 °C in 5% CO. 2 , and incubated at 37°C. After 5 days, relative cell growth was determined by measuring the reduction of the indicator dye ALAMAR BLUE® (ThermoFischer Scientific, Waltham, MA) to its highly fluorescent form in a SPECTRAMAX® M3 plate reader (Molecular Devices, Sunnyvale, CA). Increasing fluorescence correlates with cell growth. Table 17 shows the relative Hs746T cell growth for each antibody treatment normalized to control (no treatment) Hs746T cell growth. Bispecific antibody H4H14639D inhibits Hs746T cell proliferation more potently than its parent monospecific antibodies individually or in combination. [Table 17]

[0277] Hs746T gastric cancer cells were treated with control antibody, METxMET bispecific antibody H4H14639D, anti-MET parental antibody H4H13306P2, anti-MET parental antibody H4H13312P2, and the combination of parental antibodies 1 and 2 at 2 μg / ml of each antibody. After 5 days, cell growth was determined by reduction of ALAMAR BLUE® reagent (FIG. 13, Panel A). In Hs746T gastric cancer cells, the METxMET bispecific antibody inhibited cell growth more than the parental antibodies alone or in combination, and inhibited MET pathway activation more effectively than its parental antibodies.

[0278] Hs746T gastric cancer cells in 96-well plates were treated with 25 μg / mL of control antibody, 1 μg / mL, 10 μg / mL, or 25 μg / mL of monovalent MET antibody, or 1 μg / mL, 10 μg / mL, or 25 μg / mL of MET×MET bispecific antibody. After 5 days, Hs746T gastric cancer cell growth was determined by reduction of ALAMARBLUE® reagent ( FIG. 13 , Panel B). MET×MET bispecific antibody potently inhibits the growth of MET-amplified cells.

[0279] Example 15 MET x MET bispecific antibodies do not induce growth of NCI-H596 lung cancer cells in vitro The effect of MET x MET bispecific antibodies on the growth of human non-small cell lung cancer (NSCLC) cells (NCI-H596) was evaluated in vitro. 10,000 NCI-H596 lung adenosquamous carcinoma cells (Nair et al., J. Nat'l. Cancer Inst. 86(5):378-383, 1994) were seeded on top of a 0.66% agar layer in medium supplemented with 1% fetal bovine serum (FBS) in 96-well plates. Cells were cultured in RPMI 1640 medium supplemented with 1% FBS containing 0.3% agarose. Cells were treated with (1) 5 μg / ml of each parental bivalent monospecific anti-MET antibody (H4H13306P2 or H4H13312P2), (2) a combination of two parental bivalent monospecific anti-MET antibodies (H4H13306P2 and H4H13312P2) at 2.5 μg / ml each, (3) a bispecific antibody (H4H14639D) containing one binding arm from H4H13306P2 and the other binding arm from H4H13312P2 at 5 μg / ml, or (4) 100 ng / mL hepatocyte growth factor (HGF). Cells were then incubated at 37 °C for 2 h at 37 °C for 3 h at 37 °C for 1 h at 37 °C. 2 The plates were incubated at 37°C for 2 weeks. After 2 weeks, relative cell growth was determined by measuring the reduction of the indicator dye ALAMAR BLUE® (Thermo Fischer Scientific, Waltham, MA) to its highly fluorescent form in a SPECTRAMAX® M3 plate reader (Molecular Devices, Sunnyvale, CA). Increasing fluorescence correlates with cell growth. Table 18 and Figure 14 show the relative NCI-H596 cell growth for each antibody treatment normalized to control (no treatment) NCI-H596 cell growth. Treatment of NCI-H596 lung cancer cells with HGF resulted in strong growth induction in soft agar. The MET x MET (MM in Figure 14) bispecific antibody H4H14639D did not significantly alter growth compared to control treated cells. Moderate induction of cell growth was observed with each parental bivalent monospecific antibody H4H13306P2 (M1) or H4H13312P2 (M2) individually or in combination (H4H13306P2 and H4H13312P2) (M1M2). [Table 18]

[0280] Example 16 MET×MET bispecific antibodies inhibit the in vitro growth of SNU5 gastric cancer cells more potently than monospecific antibodies The effect of the MET x MET bispecific antibody on the growth of human gastric cancer cells was evaluated in vitro. 2,500 SNU5 human gastric cancer cells (Ku and Park, Cancer Research 2013). Res. Treat. 37(1):1-19, 2005) were seeded in 96-well plates and cultured in Iscove's modified Dulbecco's medium supplemented with 20% FBS. Cells were treated with (1) 5 μg / ml of individual bivalent monospecific anti-MET antibodies (H4H13306P2 or H4H13312P2), (2) a combination of two bivalent monospecific anti-MET antibodies (H4H13306P2 and H4H13312P2) at 2.5 μg / ml each, or (3) 5 μg / ml of a bispecific antibody (H4H14639D) containing one binding arm from H4H13306P2 and the other binding arm from H4H13312P2. Cells were then incubated at 37 °C for 24 h at 37 °C for 3 h at 37 °C. 2 The plates were incubated at 37° C. After 5 days, the relative cell growth was measured using a SPECTRAMAX® M3 plate reader (Molecular The fluorescence was determined by measuring the reduction of the indicator dye ALAMAR BLUE® (Thermo Fischer Scientific, Waltham, Mass.) to its highly fluorescent form in a 100-μg / mL ELISA Kit (Thermo Fischer Scientific, Sunnyvale, Calif.). Increasing fluorescence correlates with cell growth. Table 19 shows the relative SNU5 cell growth for each antibody treatment normalized to control (no treatment) SNU5 cell growth. Bispecific antibody H4H14639D inhibits SNU5 cell proliferation more potently than its parent monospecific antibody. [Table 19]

[0281] (Example 17) MET x MET bispecific antibody induces regression of Hs746T tumor xenografts The effect of the MET x MET bispecific antibody on human gastric cancer tumors was evaluated in an immunodeficient mouse model. Three million Hs746T human gastric cancer cells were implanted subcutaneously into the flank of CB-17 SCID mice (Bancroft et al., J. Immunol. 137(1):4-9, 1986). When tumor volumes reached approximately 200 mm 3 Once tumor size reached 100%, mice were randomized into groups of 6 and treated twice weekly with 25 mg / kg control antibody or 25 mg / kg METxMET bispecific antibody (H4H14639D). Tumor growth was monitored for the control group for 16 days post-implantation, when control-treated tumors reached the size limit of the protocol. Tumor growth was monitored for 30 days post-implantation for the H4H14639-treated group.

[0282] Treatment of tumors with the METxMET bispecific antibody induced a regression in tumor size over a 21 day period compared to the start of treatment. Control treated tumors showed an average increase in volume of approximately 12-fold over 16 days of growth (Table 20). Tumor volume over time showing Hs746T tumor regression by METxMET bispecific antibody is shown in Figure 15. [Table 20]

[0283] (Example 18) MET x MET bispecific antibody induces regression of SNU5 tumor xenografts The effect of the MET x MET bispecific antibody on human gastric cancer tumors was evaluated in an immunodeficient mouse model. Ten million SNU5 human gastric cancer cells were implanted subcutaneously into the flank of CB-17 SCID mice. Tumor volumes were approximately 500 mm 3Upon reaching tumor size, mice were randomized into groups of 5 and treated twice weekly with control antibody at 10 mg / kg or METxMET bispecific antibody (H4H14639D) at either 1 mg / kg or 10 mg / kg. Tumor growth was monitored for 81 days post-implantation, when control-treated tumors reached the size limit of the protocol.

[0284] Tumors from mice treated with 1 mg / kg or 10 mg / kg METxMET antibody demonstrated a mean reduction in size of about 95% or about 98%, respectively. Control-treated tumors showed a mean increase in volume of about 12-fold from the start of treatment (Table 21). [Table 21]

[0285] Subcutaneously implanted SNU5 tumors were treated twice weekly with control antibody, 1 mg / kg or 10 mg / kg monovalent MET antibody, or 1 mg / kg or 10 mg / kg METxMET bispecific antibody. Strong and sustained regression (i.e., reduction in tumor volume) of MET-amplified SNU5 tumors was observed over time in mice treated with METxMET bispecific antibody (Figure 16, Panel A). Proteins were extracted from tumors at the end of the study, and pathway activation, as indicated by MET expression and MET phosphorylation (pMET expression), was determined by immunoblotting. METxMET-treated mice (tumors) showed reduced MET and pMET expression compared to controls (Figure 16, Panel B). METxMET bispecific antibody is a potent inhibitor of tumors with MET amplification.

[0286] (Example 19) MET x MET bispecific antibody induces regression of U87-MG tumor xenografts The effect of the METxMET bispecific antibody on human glioblastoma tumors was evaluated in an immunodeficient mouse model. Five million U87-MG human glioblastoma cells (Vordermark and Brown, Int. J. Radiation Biol. 56(4):1184-1193, 2003) were implanted subcutaneously into the flank of CB-17 SCID mice. The U87-MG glioblastoma xenograft model is driven by autocrine HGF signaling. Tumor volumes of approximately 100 mm 3 Upon reaching tumor mass, mice were randomized into groups of six and treated with control antibody or METxMET bispecific antibody (H4H14639D). 25 mg / kg of antibody (control or METxMET) was administered to each mouse twice weekly. Tumor growth was monitored for 29 days after implantation, when control-treated tumors reached the size limit of the protocol.

[0287] Tumors in mice treated with METxMET antibodies demonstrated a mean reduction in size of about 38%, while control-treated tumors showed a mean increase in volume of about 19-fold over 29 days of growth (Table 22). Tumor volume over time showing U87-MG tumor regression by METxMET bispecific antibodies is shown in Figure 17. [Table 22]

[0288] (Example 20) MET × MET bispecific antibody inhibits the growth of U118-MG tumor xenografts The effect of the METxMET bispecific antibody on human glioblastoma tumors was evaluated in an immunodeficient mouse model. The U118-MG glioblastoma xenograft model is driven by autocrine HGF signaling. Five million U118-MG human glioblastoma cells (Olopade et al., Cancer Research, 52:2523-2529, 1992) were implanted subcutaneously into the flank of CB-17 SCID mice. Tumor volumes were approximately 100 mm 3Upon reaching tumor mass, mice were randomly divided into groups of six and treated with control antibody or METxMET bispecific antibody (H4H14639D). 25 mg / kg of antibody (control or METxMET) was administered to each mouse twice a week. Tumor growth was monitored for 72 days after implantation.

[0289] The MET antibody inhibited tumor growth by 99% over a 72 day period (Table 23). [Table 23]

[0290] In a separate experiment, U118-MG glioblastoma tumors implanted subcutaneously in mice were treated twice weekly with 25 mg / kg of control antibody, monovalent MET antibody, or MET x MET bispecific antibody. Tumor volume (mm 3 ) was measured for each experimental group over time. The results are shown in Figure 18, which demonstrates that the METxMET bispecific antibody inhibits the growth of U118-MG tumors.

[0291] Example 21 Maytansinoid synthesis Maytansine 3-N-methyl-L-alanine-N-Me-beta-alanine-carbamyl-(p-amino)benzyl-citrulline-valine-adipoyl-succinate (compound 1 in FIG. 20) was synthesized from compound 2 (FIG. 19) as described below.

[0292] Maytansine 3-N-methyl-L-alanine-Fmoc-N-Me-beta-alanine (compound 3, Figure 19). Des-acetyl-maytansine (compound 2, Figure 19, 0.433 g, 0.666 mmol), Fmoc-N-Me-beta-Ala (0.434 g, 1.33 mmol), and HATU (0.757 g, 1.99 mmol) were weighed into a dry flask, dissolved in anhydrous DMF (9 mL), and treated with 4-methylmorpholine (0.300 mL, 2.73 mmol). The flask was sealed with a rubber septum, purged with argon, and the reaction was stirred at ambient temperature. After 3 days, the mixture was evaporated to an oil, dissolved in acetonitrile and water, and purified by flash chromatography on a 275 g C18 silica column (30-90% acetonitrile in water over 20 min, 0.05% acetic acid in both phases). Lyophilization of the product fractions afforded the title compound as a white solid. The crude was purified on an 80 g silica gel column (EtOAc to 5:5:1 EtOAc:DCM:MeOH over 17 min). Pure fractions were combined, evaporated and dried under vacuum overnight to afford the title compound as a white solid (0.424 g, 66%). MS (ESI, positive): C 51 H 61 ClN 4 O 12 Calculated value: 956.4; Found values: 956.9 (M+H), 979.0 (M+Na), 939.0 (MH 2 O+H).

[0293] N-tert-butoxycarbonyl-N-methyl-beta-alanine succinate ester (compound 4, FIG. 19). The title compound was prepared from commercially available Boc-N-Me-beta-Ala-OH by methods well known in the art (see Widdison et al., J. Med. Chem, 2006, 49(14), 4401). 1 H NMR (300 MHz, CDCl 3 ): δ 3.62 (bm, 2H), 2.88 (m, 9H), 1.47 (s, 9H).

[0294] Maytansine 3-N-methyl-L-alanine-Boc-N-Me-beta-alanine (compound 5, FIG. 19). Method A: The product of the previous step (compound 4, FIG. 19, 0.453 g, 1.51 mmol) and des-acetyl-maytansine (compound 2, FIG. 19, 0.304 g, 0.468 mmol) were dissolved in 3:1 acetonitrile:water (8 mL) and diluted with 1 M NaHCO 3 The reaction was treated with aqueous solution (0.5 mL) and stirred at ambient temperature for 18 hours. When the reaction was complete as determined by TLC, it was then stirred with brine for 10 minutes and extracted three times with ethyl acetate (EtOAc). The combined organic layers were then washed with Na 2 SO 4 The mixture was dried at rt, filtered, and the filtrate was concentrated and dried under vacuum to a golden syrup which was purified by flash column chromatography on a 20 g silica gel cartridge (0-10% MeOH in EtOAc over 15 min) to give the title compound as a white solid (0.084 g, 43%). MS (ESI, positive): 41 H 59 ClN 4 O 12 Calculated value: 834.4; Found values: 835.2 (M+H), 857.2 (M+Na), 817.4 (MH 2 O+H).

[0295] Method B: Boc-N-Me-beta-Ala-OH (0.294 g, 1.45 mmol) was dissolved in anhydrous DMF (5 mL) and treated with pentafluorophenyl diphenylphosphinate (FDPP, 0.555 g, 1.44 mmol), and the reaction was stirred at ambient temperature for 30 min. The mixture was then transferred to a larger flask containing a mixture of des-acetyl-maytansine (compound 2, FIG. 19, 0.462 g, 0.711 mmol) and diisopropylethylamine (DIEA, 0.250 mL, 1.44 mmol) in anhydrous DMF (7 mL), the flask was sealed with a rubber septum, purged with argon, and the reaction was stirred again at ambient temperature. After 24 h, the reaction was concentrated in vacuo to an oil, dissolved in ethyl acetate (EtOAc, 2 mL) and purified on a 40 g silica gel cartridge (EtOAc to 5:5:1 EtOAc / DCM / MeOH over 15 min) to give the title compound as a pale yellow solid (0.468 g, 79%). MS (ESI, positive): C 41 H 59 ClN 4 O 12 Calculated value, 834.4; Found value, 857.2 (M+Na), 817.2 (MH 2 O+H).

[0296] Maytansine 3-N-methyl-L-alanine-N-Me-beta-alanine (compound 6, Figure 19). Method A: Maytansine N-Me-L-Ala-Boc-N-Me-beta-Ala (compound 5, Figure 19, 0.464 g, 0.555 mmol) was dissolved in a 3:1:1 mixture of acetonitrile / water / trifluoroacetic acid (7 mL), the flask was sealed with a rubber septum, purged with argon and the reaction was stirred at ambient temperature for 24 hours then capped and stored at -20°C for 3 days. The crude reaction mixture was warmed to ambient temperature for 2 h, briefly concentrated under vacuum, and purified on a 100 g C18 RediSep Gold column (20-80% acetonitrile in water over 25 min, 0.1% TFA in both solvents), and the combined pure fractions were partially evaporated at ambient temperature, frozen in a dry ice bath, and lyophilized to give the title compound as a pale yellow solid (0.295 g, 63%). MS (ESI, positive): C 36 H51 ClN 4 O 10 Calculated value, 734.3; found values ​​735.7 (M+H), 1471.3 (2M+H).

[0297] Method B: Maytansine N-Me-L-Ala-Fmoc-beta-Ala (compound 3, Figure 19, 0.422 g, 0.441 mmol) was dissolved in 5% piperidine in DMF (6.00 mL, 3.04 mmol), the reaction flask was sealed with a rubber septum, purged with argon, and the mixture was stirred at ambient temperature. After 3 h the reaction was complete by LCMS so it was concentrated under vacuum, sealed, and stored at -20 °C overnight. The crude product was warmed to ambient temperature, treated with acetonitrile and 10% aqueous acetic acid (3 mL each), and purified by flash chromatography on a 275 g C18 silica column (10-90% acetonitrile in water over 20 min, 0.05% acetic acid in both solvents). Lyophilization of the product fractions afforded the title compound as a white solid. The solid was triturated three times with dry diethyl ether, filtered, the solid rinsed through the frit with DCM, and the filtrate evaporated and dried under vacuum to give the title compound as a white solid (0.311 g, 89%). MS (ESI, positive): C 36 H 51 ClN 4 O 10 Calculated for 734.3; found 735.0 (M+H).

[0298] Maytansine 3-N-methyl-L-alanine-N-Me-beta-alanine-carbamyl-(p-amino)benzyl-citrulline-valine-Fmoc (compound 7, FIG. 20). Step 1: The product of the previous step (compound 6, FIG. 19, 0.310 g, 0.390 mmol), 1-hydroxy-7-azabenzotriazole (HOAT, 0.164 g, 1.20 mmol), sodium bicarbonate (0.138 g, 1.64 mmol), and Fmoc-valine-citrulline-(p-amino)benzyl-(p-nitrophenyl)carbonate (0.595 g, 0.776 mmol, prepared by methods known in the art, see Gangwar et al., U.S. Pat. No. 7,714,016 B2) were dissolved in anhydrous DMF (10 mL), the reaction flask was sealed with a rubber septum, purged with argon, and the mixture was stirred at ambient temperature. After 24 hours, the reaction was partially evaporated under vacuum to approximately 2-3 mL, treated with 10% aqueous acetic acid and water (approximately 1 mL each), dissolved in acetonitrile (approximately 6 mL), and purified by flash chromatography on a 275 g C18 silica column (30-90% acetonitrile in water over 20 min, 0.05% acetic acid in both solvents). Partial evaporation, freezing, and lyophilization afforded the title compound as a white solid (0.362 g, 68%). MS (ESI, positive): C 70 H 88 ClN 9 O 17 Calculated value: 1361.6; Found values: 1362.1 (M+H), 1384.1 (M+Na), 1344.1 (MH 2 O+H).

[0299] Step 2: The product of the previous step (0.360 g, 0.264 mmol) was dissolved in 5% piperidine in DMF (7 mL), the reaction flask was sealed with a rubber septum, purged with argon, and the mixture was stirred at ambient temperature. After 3 h, the reaction was evaporated under vacuum, the residue was treated with 10% aqueous acetic acid (2 mL), dissolved in acetonitrile (4 mL), and purified by flash chromatography on a 275 g C18 silica column (10-70% acetonitrile in water over 20 min, 0.05% acetic acid in both solvents). Pure fractions were combined, stored at -20 °C overnight, partially evaporated under vacuum at 25-30 °C, frozen on dry ice, and lyophilized for 6 days to give the title compound as a pale yellow solid (0.303 g, 95%). MS (ESI, positive): C 15 H 78 ClN 9 O 15 Calculated for 1139.5; found 1140.1 (M+H), 1162.0 (M+Na).

[0300] Maytansine 3-N-methyl-L-alanine-N-Me-beta-alanine-carbamyl-(p-amino)benzyl-citrulline-valine-adipic acid (compound 8, Figure 20). The product of the preceding step (compound 7, Figure 20, 0.205 g, 0.171 mmol), adipic acid (0.258 g, 1.77 mmol), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 0.215 g, 0.869 mmol) were dissolved in dry DCM (10 mL) and anhydrous methanol (5 mL), the reaction flask was sealed with a rubber septum, purged with argon, and the mixture was stirred at ambient temperature. After 21 h, the reaction was evaporated under vacuum and the residue was dissolved in a few mL of acetonitrile / water and purified by flash chromatography on a 150 g C18 silica column (20-80% acetonitrile in water over 17 min, 0.05% acetic acid in both solvents). Partial evaporation of the pure fractions, freezing, and lyophilization for 18 h gave the title compound as a white solid (0.140 g, 65%). MS (ESI, positive): C 61 H 86 ClN 9 O18 Calculated for 1267.6; found 1268.9 (M+H), 1290.9 (M+Na).

[0301] Maytansine 3-N-methyl-L-alanine-N-Me-beta-alanine-carbamyl-(p-amino)benzyl-citrulline-valine-adipoyl-succinate (compound 1, Figure 20). The product of the preceding step (compound 8, Figure 20, 0.061 g, 0.048 mmol), N-hydroxysuccinimide (0.063 g, 0.55 mmol), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC-HCl, 0.071 g, 0.37 mmol) were dissolved in dry DCM (7 mL), the reaction flask was sealed with a rubber septum, purged with argon, and the mixture was stirred at ambient temperature. After 5 days, the reaction was evaporated under vacuum and the residue was dissolved in a few mL of acetonitrile / water and purified by flash chromatography on a 100 g C18 silica column (30-90% acetonitrile in water over 15 min, 0.05% acetic acid in both solvents). Partial evaporation of the purest product fractions, freezing, and lyophilization for 18 h gave the title compound as a white solid (0.044 g, 67%). MS (ESI, positive): C 65 H 89 ClN 10 O 20 Calculated value: 1364.6; Found values: 1365.7 (M+H), 1387.7 (M+Na), 1347.7 (MH 2 O+H). 1 H-NMR (500 MHz; CDCl 3 ): δ 7.56 (d, J = 8.3 Hz, 2H), 7.20 (d, J = 8.7 Hz, 1H), 6.80 (s, 1H), 6.71 (m, 1H), 6.62 (d, J = 10.0 Hz, 1H), 6.39 (dd, J = 15.1, 11.3 Hz, 1H), 5.68 (dd, J = 15.3, 9.1 Hz, 1H), 5.38-5.32 (m, 1H), 5.03 (t, J = 15.1 Hz, 1H), 4.88 (d, J = 12.3 Hz, 1H), 4.73 (d, J = 11.3 Hz, 1H), 4.61 (dd, J = 9.1, 3.6 Hz, 1H), 4.26 (d, J = 7.0 Hz, 1H), 4.17 (t, J = 7.1 Hz, 1H), 3.95 (s, 3H), 3.61 (d, J = 11.7 Hz, 1H), 3.57 (d, J = 12.4 Hz, 1H), 3.46 (d, J = 9.1 Hz, 2H), 3.33 (s, 3H), 3.27 (t, J = 6.9 Hz, 1H), 3.17-3.07 (m, 5H), 2.97 (dd, J = 16.6, 9.9 Hz, 1H), 2.88 (d, J = 11.7 Hz, 3H), 2.84 (s, 4H), 2.77 (s, 2H), 2.66 (s, 2H), 2.62 (t, J = 4.8 Hz, 2H), 2.56 (d, J = 13.1 Hz, 1H), 2.32 (t, J = 6.6 Hz, 2H), 2.15 (d, J = 14.0 Hz, 1H), 2.10 (q, J = 6.8 Hz, 1H), 1.92 (s, 4H), 1.75 (m, 5H), 1.61 (s, 3H), 1.52 (s, 3H), 1.27 (d, J = 6.3 Hz, 3H), 1.22 (dt, J = 12.7, 6.3 Hz, 6H), 0.95 (t, J = 5.9 Hz, 7H), 0.78 (s, 3H).

[0302] DM1 was synthesized as a single diastereomer based on the procedures described in WO2015 / 031396 (e.g., Example 2, paragraph

[0106] ), the entirety of which is incorporated herein by reference.

[0303] Example 22 Antibody conjugation and characterization of the conjugates Antibody Conjugation

[0304] Antibodies (H4H14639D, H4H13312P, H4H14635D, and isotype control; 10-20 mg / ml) in 50 mM HEPES, 150 mM NaCl, pH 8.0, and 10-15% (v / v) DMA were conjugated with a 5-6 fold excess of SMCC-DM1 diastereomers (maytansinoid A), prepared as described in Example 21, or maytansine 3-N-methyl-L-alanine-N-Me-beta-alanine-carbamyl-(p-amino)benzyl-citrulline-valine-adipoyl-succinate (compound 1, Figure 20) (maytansinoid B), for 2 h at ambient temperature. Conjugates were purified by size exclusion chromatography or exhaustive ultrafiltration and sterile filtered. Protein concentrations were determined by UV spectroscopy. All conjugates used were verified to be >90% monomeric by size exclusion HPLC and <1% unconjugated linker payload by RP-HPLC. All conjugated antibodies were analyzed for linker payload loading values ​​by UV and / or by mass difference of native vs. conjugated according to Hamblett et al. (American Association for Cancer Research. 2004 Oct. 15; Vol. 10(20): 7063-70). Payload to antibody ratios are reported in Table 24. [Table 24]

[0305] Characterization of conjugates by liquid chromatography-mass spectrometry To determine the loading of the linker payload on the antibody, the conjugates were deglycosylated and analyzed by LC-MS.

[0306] For the assay, 50 μg of conjugate was diluted in milli-Q water to a final concentration of 1 mg / mL. 10 μL of PNGase F solution [PNGase F solution was prepared by adding 150 μL of PNGase F stock (New England Biolabs, Cat. No. P0704L) and 850 μL of milli-Q water and mixed thoroughly] was added to the diluted conjugate solution, which was then incubated overnight at 37 °C. A 5 μL injection of each sample was made into an LC-MS (Waters Synat G2-Si) and eluted with a gradient mobile phase of 20-40% over 25 min at 0.1 mL / min (mobile phase A:H 2 Mobile phase A: 0.1% v / v FA in O; mobile phase B: 0.1% v / v FA in acetonitrile). LC separation was achieved on a Waters Acquity BEH C4 column (1.0 × 50 mm, 1.7 μM) at 80 °C.

[0307] Mass spectrometry spectra were deconvoluted using Masslynx software and the drug-to-antibody ratio (DAR) was calculated using the following formula: 1. Relative percentage (%) of drug (Dn) by distribution peak intensity (PI):

number

number

[0308] Example 23 Surface plasmon resonance derived binding affinity and kinetic constants of conjugated human monoclonal anti-MET (monospecific and bispecific) antibodies Equilibrium dissociation constants (K ) of MET binding to anti-MET antibodies conjugated with either MCC-DM1 diastereomers (maytansinoid A) or maytansine 3-N-methyl-L-alanine-N-Me-beta-alanine-carbamyl-(p-amino)benzyl-citrulline-valine-adipoyl-succinate (compound 1, FIG. 20) (maytansinoid B). D The binding affinity (IL-1 value) was determined using a real-time surface plasmon resonance biosensor assay on a Biacore 2000 instrument. The Biacore sensor surface was coated with anti-MET To capture the ADC and parent unmodified antibody expressed containing human constant regions, it was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE Healthcare, #BR-1008-39). Biacore binding studies were performed in HEPES-buffered saline (HBS)-EP running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20). Human MET expressing a C-terminal myc-myc-hexahistidine tag (hMET-mmh) was prepared in-house. Different concentrations (3-fold dilutions) of hMET-mmh prepared in HBS-EP running buffer (ranging from 30 nM to 1.1 nM) were injected over the anti-MET ADC or antibody capture surface at a flow rate of 40 μL / min. The association of hMET-mmh to the captured ADC and monoclonal antibody, respectively, was monitored for 4 min. Subsequently, hMET-mmh dissociation was monitored for 6 min in HBS-EP running buffer. Anti-human Fc surfaces were stained with 20 mM H 3 PO 4 The antibody was regenerated by a brief injection of 100 mM NaCl. All binding kinetic experiments were carried out at 25°C.

[0309] Kinetic binding rate constant (k a ) and the dissociation rate constant (k dThe binding-dissociation equilibrium constant (K) was determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c curve-fitting software. All sensorgrams were double-referenced by subtracting the buffer-injected sensorgram signal from the corresponding analyte sensorgram, thereby removing artifacts caused by dissociation of the antibody from the capture surface. D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rate constants as follows:

number

[0310] The binding kinetic parameters for anti-Met monospecific and bispecific antibodies conjugated with maytansinoid A or maytansinoid B are shown below in Table 25, with some experiments performed in duplicate. [Table 25]

[0311] (Example 24) In vitro efficacy of anti-MET antibody drug conjugates (ADCs) To determine the relative cell killing potency of the anti-MET antibody drug conjugates (ADCs) described herein, cell killing assays were performed on multiple cell lines expressing various levels of endogenous MET. EBC-1 (Riken Cell Bank; #RBRC-RCB1965), MKN-45 (JCRB; #JCRB0254), NCI-H1993 (ATCC; #CRL-5909), and J.RT3 (ATCC; #TIB-153) cell lines were maintained in RPMI + 10% FBS + 1× penicillin / streptomycin / L-glutamine (P / S / G), and SNU-5 (ATCC; #CRL-5973) was maintained in RPMI + 10% FBS + 1× penicillin / streptomycin / L-glutamine (P / S / G). Hs746t (ATCC; #HTB-135) and HEK293 (ATCC; #003041) were maintained in DME + 10% FBS + 1x P / S / G, and MDA-MB-231 (ATCC; #HTB-26) were maintained in Liebowitz L-15 + 10% FBS + 1x P / S / G + 1x non-essential amino acids (NEAAs). 2 Cells were maintained without ATP, U87MG (ATCC; #HTB-14) were maintained in MEM Earle's salts + 15% FBS + 1x P / S / G + 1x NEAA, T47D (ATCC; #HTB-133) were maintained in RPM1 1640 + 10% FBS + 1x P / S / G + 10 mM HEPES + 1 mM sodium pyruvate + 10 μg / ml bovine insulin, and A549 (ATCC; #CCL-185) were maintained in Kaighn's nutrient mixture F-12 (HAM F-12K) + 10% FBS + 1x P / S / G.

[0312] First, the relative binding of anti-MET antibodies was assessed across a panel of cell lines by flow cytometry using unconjugated H4H14635D, H4H14639D and H4H13312P2 antibodies. 6of cells were incubated with 10 μg / ml of H4H14635D, H4H14639D, H4H13312P2, or isotype control antibody (REGN1945) in PBS+2% FBS (FACS buffer) for 30 minutes on ice. After one wash with FACS buffer, cells were incubated with 10 μg / ml of Alexa647-conjugated anti-human secondary antibody (Jackson ImmunoResearch, #109-606-170) for 30 minutes on ice. After one further wash with FACS buffer, samples were fixed with Cytofix (BD Biosciences, #554655), filtered with FACS buffer, and run on an iQue flow cytometer (Intelicyte). Mean fluorescence intensity (MFI) data was determined using FlowJo software (FlowJo LLC). FACS binding is expressed as fold MFI binding above isotype control levels, and the results are summarized in Table 26. The relative binding of the three anti-Met antibodies was comparable in each cell line and ranged from 447-fold to 7-fold above the isotype control. No detectable binding of any of the three anti-MET antibodies tested was observed in T47D, HEK293, or J.RT3 cells.

[0313] To measure the in vitro cytotoxicity of anti-MET ADCs, nuclei counts were assessed after 3 or 6 days of treatment with the ADCs. Briefly, cells were seeded in complete growth medium in 96-well collagen-coated plates (Greiner, VWR; #82050-812) at 750–3000 cells / well and incubated at 37 °C for 3 h at 5% CO. 2 For cell viability curves, serially diluted ADC, unconjugated antibody, or free payload were added to the cells at final concentrations ranging from 100 nM to 0.01 nM (based on toxin concentration) and incubated at 37 °C in 5% CO. 2 The cells were then incubated in 4% formaldehyde at 37°C for 3-6 days. The cells were then fixed with 4% formaldehyde and treated with 3μg / ml Hoechst 33342 nuclear stain (Invitrogen, #H3570). ImageXpress micro Images were acquired on a 350 nm XL (Molecular Devices, Sunnyvale, CA) and nuclei number was determined by MetaXpress image analysis software (Molecular Devices, Sunnyvale, CA). Background nuclei number from cells treated with 40 nM digitonin was subtracted from all wells and viability was expressed as a percentage of untreated controls. IC 50 Values ​​were determined from a four-parameter logistic equation over a 10-point response curve (GraphPad The untreated condition for each dose-response curve was also included in the analysis and is expressed as the lowest dose. IC 50 The values ​​and percent cell kill are shown in Tables 27 and 28.

[0314] As summarized in Table 27, the anti-MET antibody-drug conjugate H4H14639D-maytansinoid A enhanced cell viability with ICs ranging from 0.35 nM to 0.96 nM in Met-amplified EBC-1, SNU-5, MKN-45, NCI-H1993, and Hs746t cell backgrounds. 50 H4H14639D-maytansinoid A also specifically reduced the cytotoxicity of 13.91 nM. The percentage of cells killed (% maximum kill) ranged from 73% to 100%. 50 H4H14639D-maytansinoid A specifically killed 84% of A549 cells with an IC value of 1. 50 Values ​​were greater than 37 nM in low expressing (MDA-MB-231 and U87MG) and non-expressing (T47D, HEK293, and J.RT3) cell lines. A similarly conjugated isotype control antibody had an IC of greater than 35 nM. 50 The methyl disulfide version of DM1 (MeS-DM1) killed all cell lines tested with IC values ​​ranging from 0.07 nM to 2.86 nM. 50 Killed by value.

[0315] In a separate experiment, three anti-Met antibodies (H4H14639D, H4H14635D, and H4H13312P2) were conjugated to maytansinoid payloads of maytansinoid A or maytansinoid B, and in vitro cytotoxicity was evaluated in EBC-1, Hs746t, A549, and T47D cells after 6 days of treatment. As summarized in Table 28, all anti-Met antibody-drug conjugates showed low IC of 10 pM in EBC-1 cells, 0.82 nM in Hs746t cells, and 3.5 nM in A549 cells. 50 Anti-Met antibody potently and specifically reduced cell viability in Met-positive cells at 100% CI, with the percentage of cells killed being greater than 95% in EBC-1 cells, greater than 86% in Hs746t cells, and greater than 72% in A549 cells. T47D cells (Met-negative) were ADCs were not specifically killed. A similarly conjugated isotype control antibody had IC values ​​of >5 nM in EBC-1 cells, >33 nM in Hs746t cells, and >90 nM in A549 and T47D cells in all cell lines tested. 50 Unconjugated H4H14639D reduced cell viability in EBC-1, Hs746t, and A549 cells, but by a lower percentage than the conjugated antibody. Unconjugated H4H14635D and H4H13312P2 had little to no effect on viability in any of the cell lines tested. The methyl disulfide version of DM1 (MeS-DM1) inhibited all lines tested with IC ranging from 0.12 nM to 1.39 nM. 50 In contrast, M24, the payload released from maytansinoid B, killed cells with an IC50 of >100 nM. [Table 26] [Table 27] [Table 28]

[0316] (Example 25) In vivo efficacy against gastric cancer cells Three million Hs746T gastric cancer cells were implanted subcutaneously into the flank of CB-17 SCID mice. Tumor volumes were approximately 150 mm 3 Upon reaching tumor mass, mice were randomized into groups of six and treated with control antibodies REGN1945-maytansinoid B or REGN1945-maytansinoid A at 10 mg / kg, or H4H14639D-maytansinoid A or H4H14639D-maytansinoid B at 3 or 10 mg / kg. All antibodies were administered once a week for three doses. Tumor growth was monitored for 37 days after implantation.

[0317] The effect of H4H14639D-maytansinoid A or H4H14639D-maytansinoid B on the growth of human tumor xenografts in immunodeficient mice was evaluated and the results are shown in Table 29. Tumors treated with control antibody REGN1945-maytansinoid B or REGN1945-maytansinoid A grew to reach the size limit of the protocol within 20 days. Tumors treated with 3 mg / kg H4H14639D-maytansinoid A grew to reach the size limit of the protocol within 27 days. Growth of tumors treated with 3 mg / kg H4H14639D-maytansinoid B was inhibited for the duration of the experiment. Treatment of tumors with 10 mg / kg H4H14639D-maytansinoid A or H4H14639D-maytansinoid B induced a regression of tumor size compared to the start of treatment. [Table 29]

[0318] (Example 26) In vivo efficacy against lung cancer cells Five million EBC1 lung cancer cells were implanted subcutaneously into the flank of CB-17 SCID mice. Tumor volumes were approximately 170 mm. 3Upon reaching tumor mass, mice were randomized into groups of six and treated with 15 mg / kg of control antibody REGN1945-maytansinoid B, or 2.5, 5, 10, or 15 mg / kg of H4H14639D-maytansinoid B. Antibodies were administered twice weekly. Tumor growth was monitored for 73 days after implantation.

[0319] The effect of H4H14639D on the growth of human tumor xenografts in immunodeficient mice was evaluated. Tumors treated with the control antibody REGN1945-maytansinoid B grew to reach the size limit of the protocol within 24 days (IACUC protocol requires tumors to grow to approximately 1500 mm, exceeding 2 cm in diameter). 3 (requiring sacrifice of animals bearing tumors of 100-200 mg / kg). Treatment of tumors with 2.5, 5, 10, or 15 mg / kg H4H14639D-maytansinoid B induced regression of tumor size compared to the start of treatment. The results are shown in Table 30. [Table 30]

[0320] (Example 26) In vivo efficacy against patient-derived NSCLC tumors CTG-0165 patient-derived tumors, Met-expressing NSCLC, were implanted subcutaneously into the flanks of nu / nu nude mice. Tumor volumes were approximately 150 mm 3 Upon reaching tumor mass, mice were randomized into groups of six and treated with 10 mg / kg of control antibody REGN1945-maytansinoid B or REGN1945-maytansinoid A, or 3 or 10 mg / kg of H4H14639D-maytansinoid A, or H4H14639D-maytansinoid B. All antibodies were administered once a week for three doses. Tumor growth was monitored for 61 days after implantation.

[0321] The effect of H4H14639D-maytansinoid A or H4H14639D-maytansinoid B on the growth of human tumor xenografts in immunodeficient mice was evaluated. Tumors treated with control antibodies REGN1945-maytansinoid A or REGN1945-maytansinoid B grew to reach the size limit of the protocol within 27 days. Growth of tumors treated with 3 mg / kg H4H14639D-maytansinoid A or H4H14639D-maytansinoid B was inhibited for 27 days. Treatment of tumors with 10 mg / kg H4H14639D-maytansinoid A or H4H14639D-maytansinoid B induced a regression of tumor size compared to the start of treatment. Data are provided in Table 31. [Table 31]

[0322] Example 27 Hydrogen / deuterium (H / D) exchange-based epitope mapping of the binding of anti-Met antibodies H4H13312P2, H4H13306P2, and H4H14639D to human MET Experiments were performed to determine the specific region of the human hepatocyte growth factor receptor ectodomain (SEQ ID NO: 155: human Met isoform 1 expressed with myc-myc-hexahistidine (.mmh) tag (Uniprot ID: P08581), hereafter referred to as hMet) that interacts with anti-Met antibodies H4H13312P2, H4H13306P2, and H4H14639D. H4H13312P2 and H4H13306P2 are bivalent monospecific anti-Met antibodies; H4H14639D is a bispecific antibody that contains two heavy chains and a universal light chain that bind distinct epitopes on Met, each derived from H4H13312P2 and H4H13306P2, respectively. (See Example 5).

[0323] The binding epitope of the above antibody is determined by using hydrogen / deuterium (H / D) exchange epitope mapping (HDX-MS) using mass spectrometry.The general description of HDX method is described, for example, in Ehring (1999) Analytical Biochemistry, vol. 267 (no. 2): pp. 252-259; and Engen and Smith (2001) Anal. Chem. vol. 73: pp. 256A-265A.

[0324] Experimental procedure To map the binding epitope(s) of anti-Met antibodies H4H13312P2, H4H13306P2 and H4H14639D on hMET by HDX, each antibody was separately covalently coupled to NHS-activated Sepharose 4 Fast Flow beads (GE Healthcare, Pittsburgh, PA). To confirm the binding epitopes of anti-Met antibodies, two methods were utilized: "On-Antigen" and "On-Complex", as described below.

[0325] In the "on antigen" experimental condition, hMET 2 Deuterated antigens were bound to H4H13312P2 or H4H13306P2 antibody beads by short incubation and then eluted from the beads by ice-cold low pH quench buffer. Eluted samples were manually loaded onto a Waters H / DX-MS system consisting of integrated online peptide digestion, trapping, 9.0 min liquid chromatography (LC) separation, and Synapt G2-Si MS data acquisition.

[0326] In the "on-complex" experimental conditions, hMET was first bound to H4H13312P2 or H4H13306P2 beads and then 2The mixture was deuterated for 5.0 or 10.0 min by incubation in PBS buffer prepared with 0. Deuterated hMET was eluted and analyzed by a Waters H / DX-MS system as described above.

[0327] LC-MS from undeuterated samples for identification of peptic peptides from hMET E Data were processed and searched against human MET using Waters ProteinLynx Global Server (PLGS) software. Identified peptides were imported into DynamX 3.0 software and filtered by two criteria: 1) minimum product per amino acid of 0.3; 2) duplicate file threshold of 3.0. DynamX 3.0 software then automatically calculated the deuterium uptake difference between "on antigen" and "on complex" for each identified peptide across both 5- and 10-min deuteration time points. Individual isotopic peaks of each peptide picked up by DynamX software for centroid calculation were also manually inspected to ensure accuracy of deuterium uptake calculation.

[0328] Generally, a delta value for deuteration above 0.2 was used as the cutoff point for determining specific binding epitopes.

[0329] result When H4H13312P2 antibody beads were used, the LC-MS E A Waters Enzymate™ BEH pepsin column (2.1 x Using online pepsin digestion with a 30 mm, 5 μm microscope, a total of 162 peptic peptides from human MET were reproducibly identified with traceable deuterium uptake in both "on antigen" and "on complex" experiments. These peptides represent 55.7% sequence coverage. Of all these peptides, only five were found to have significantly reduced deuterium uptake upon H4H13312P2 binding ("on complex") compared to deuteration of the antigen alone ("on antigen"). The centroid values ​​of these five peptides under both experimental conditions are listed in Table 32. The region corresponding to residues 192-204 covered by these five peptides was defined as the binding epitope for antibody H4H13312P2 based on HDX data. [Table 32]

[0330] For the HDX experiments performed with H4H13306P2 antibody beads, a total of 98 peptic peptides derived from hMET were reproducibly identified with traceable deuterium incorporation in both "on antigen" and "on complex" experiments. These 98 peptides represent a sequence coverage of 52.1%. Of all these peptides, 12 were observed to have significantly reduced deuterium incorporation upon H4H13306P2 binding ("on complex") compared to deuteration of the antigen alone ("on antigen"). The centroid values ​​of these 12 peptides under both experimental conditions are listed in Table 33. The regions corresponding to residues 305-315 and residues 421-455 covered by these peptides were defined as the binding epitopes for antibody H4H13306P2 based on the HDX data. [Table 33]

[0331] The binding epitope for the bispecific anti-Met antibody H4H14639D was determined using the same methodology outlined above. The binding epitope of H4H14639D on hMET determined by this methodology corresponds to the epitope determined for the parent antibody.

[0332] Binding epitope of anti-Met antibody H4H13312P2: amino acids 192 to 204 of SEQ ID NO: 155: VRRLKETKDGFMF (SEQ ID NO: 156).

[0333] Binding epitope of anti-Met antibody H4H13306P2: amino acids 305 to 315 of SEQ ID NO: 155: LARQIGASLND (SEQ ID NO: 157) and amino acids 421 to 455 of SEQ ID NO: 155: FIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNF (SEQ ID NO: 158).

[0334] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

[Claim 1] The invention as depicted in the drawings.

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