Bispecific antigen-binding molecules that bind to HER2 and methods of using the same

Abstract

Provided herein are bispecific antigen-binding molecules that bind to HER2 and methods for using them. The bispecific antigen-binding molecules comprise first and second antigen-binding domains, which bind to two different (preferably non-overlapping) epitopes in the extracellular domain of human HER2. The bispecific antigen-binding molecules cluster on the surface of HER2 IHC2+ cells and IHC3+ cells and are internalized into cellular lysosomes. Also provided are antibody-drug conjugates (ADCs) comprising the antibodies or bispecific antigen-binding molecules provided herein linked to a cytotoxic agent, radionuclide, or other moiety, and methods for treating cancer in a subject by administering the bispecific antigen-binding molecules or ADCs thereof to the subject.

Description

Technical Field

[0001] The present invention relates to bispecific antibodies that specifically bind to human epidermal growth factor receptor 2 (HER2) and modulate HER2 signaling, antigen-binding fragments thereof, antibody-drug conjugates of said antibodies, and methods of using the same.

[0002] Sequence Listing A public copy of the sequence listing has been electronically filed simultaneously with this specification via EFS-Web as a sequence listing in ASCII format with a file name of "10719WO01_Sequence_Listing_ST25.TXT", a creation date of February 26, 2021, and a size of approximately 64 kilobytes. The sequence listing contained in this ASCII format written document is part of this specification and is hereby incorporated by reference in its entirety.

Background Art

[0003] Background Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor encoded by the ERBB2 gene located on the long arm of human chromosome 17 (17q12). The protein is involved in signal transduction pathways leading to cell growth and differentiation. HER2 is a 1255 amino acid, 185 kD transmembrane glycoprotein and has no known ligand for HER2 but consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain. HER2 is thought to be the preferred dimerization partner of other members of the ErbB family, which includes erbB-2, erbB-3, and erbB-4. HER2 can also be activated by complexing with other membrane receptors such as insulin-like growth factor receptor 1. Dimerization, including homodimer formation, results in autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptor, initiating various signal transduction pathways leading to cell proliferation and tumor formation.

[0004] Overexpression of HER2 leads to induction of angiogenesis. This protein is overexpressed in approximately 30% of breast cancers and approximately 30% of gastric / esophageal cancers. Overexpression of HER2 is also seen in other cancers including ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, and head and neck cancer. Breast cancers can have up to 25 - 50 copies of the HER2 gene and up to a 40 - 100-fold increase in HER2 protein. HER2 amplification is an early event in breast and gastric tumorigenesis and is associated with a significantly shorter disease-free survival period.

[0005] Both preclinical and clinical results have shown that tumors overexpressing HER2 respond to anti-HER2 therapy, demonstrating HER2 as a cancer driver. Treatment of HER2-positive tumors with anti-HER2 therapy has led to dramatic improvements in early survival and advanced disease. Various monovalent HER2-blocking antibodies are in clinical development for the treatment of various cancers (U.S. Patent Application Publication Nos. 2019 / 0076438 (Patent Document 1) and 2015 / 0343058 (Patent Document 2)). These antibodies include trastuzumab, pertuzumab, and margetuximab. (Pernas and Tolaney, Therapeutic Advances in Medical Oncology, 11: doi 10.1177 / 1758835919833519, 2019 (Non-Patent Document 1)). Other HER2 antibodies are bispecific or multispecific, such as PRS (Pieris Pharmaceuticals, a monoclonal antibody-bispecific protein targeting HER2 and CD137), GBR1302 (Glenmark Pharmaceuticals, a HER2×CD3 bispecific antibody), ZW25 (Zymeworks, a bispecific antibody that binds to two different epitopes on the extracellular domains of HER2-ECD2 and ECD4), and MCLA-128 (Merus, a bispecific antibody against HER2 and HER3). Some HER2 antibodies are conjugated to a cytotoxic payload, and examples of such are MEDI4276 (Medimmune, a bispecific ADC that binds to two HER2 domains conjugated to tubulysin; see U.S. Patent No. 10,160,812 (Patent Document 3)), SYD985 (Synthon Biopharmaceuticals, an ADC based on trastuzumab and a cleavable linker), and trastuzumab deruxtecan (Daiichi Sankyo, including trastuzumab, a cleavable drug linker, and a topoisomerase I payload).

[0006] Nearly all patients with metastatic HER2-positive cancer will ultimately progress on anti-HER2 therapy due to de novo or acquired resistance. Tumors expressing intermediate levels of HER2 (IHC2+) remain resistant to certain therapies, including trastuzumab-DM1 ado-trastuzumab emtansine (T-DM1), a microtubule-disrupting agent, which is clearly due to inefficient internalization and processing of T-DM1 in lysosomes. Trastuzumab-DMI is only effective in approximately 25% of patients with HER2 breast cancer. There remains a significant unmet medical need for improved anticancer agents with potent efficacy against HER2-expressing cancers, particularly those expressing intermediate levels of HER2.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

[0009] Brief Summary Bispecific antibodies that bind to the human HER2 receptor protein (HER2×HER2) are provided herein. The antibodies are particularly useful for targeting tumor cells that express HER2. Anti-HER2 antibodies and antigen-binding portions thereof may be used alone in unmodified form or may be included as part of an antibody-drug conjugate (ADC).

[0010] [The present invention 1001] a first antigen-binding domain (D1), a second antigen-binding domain (D2), and wherein D1 specifically binds to a first epitope of human HER2, and D2 specifically binds to a second epitope of human HER2, a bispecific antigen-binding molecule. [The present invention 1002] The bispecific antigen-binding molecule of the present invention 1001, wherein D1 and D2 do not compete with each other for binding to human HER2. [The present invention 1003] The following characteristics: (a) binding to ErbB2 with a dissociation half-life of less than about 1 nM as measured by surface plasmon resonance assay; (b) binding to ErbB2 with a t of at least about 30 minutes as measured by surface plasmon resonance assay 1 / 2 ; (c) binding to cell surface HER2 with greater affinity and / or avidity as compared to trastuzumab; (d) binding to HER2 IHC2+ and IHC3+ expressing cells with higher efficiency than trastuzumab; (e) binding to HER2 IHC1+ expressing cells with a greater IC than trastuzumab 50 ; (f) binding to cells expressing moderate or high HER2 levels but not to cells expressing low HER2 levels; (g) forming antibody clusters on the surface of HER2-expressing cells; and (h) being internalized by HER2-expressing cells with higher efficiency than trastuzumab The bispecific antigen-binding molecule of the present invention 1001 or 1002 having one or more of the above. [The present invention 1004] The bispecific antigen-binding molecule according to any one of the present inventions 1001 to 1003, wherein D1 comprises three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. [The present invention 1005] The bispecific antigen-binding molecule of the present invention 1004, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical thereto, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that is at least 95% identical thereto, HCDR3 comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is at least 95% identical thereto, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that is at least 95% identical thereto, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical thereto, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1006] The bispecific antigen-binding molecule of the present invention 1005, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24. [The present invention 1007] The bispecific antigen-binding molecule of the present invention 1005, comprising a heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical thereto, and a light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1008] The bispecific antigen-binding molecule of the present invention 1007, comprising a HCVR that comprises the amino acid sequence of SEQ ID NO: 2 and a LCVR that comprises the amino acid sequence of SEQ ID NO: 18. [The present invention 1009] The bispecific antigen-binding molecule of any one of the present inventions 1001 to 1008, wherein D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 10, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 18. [The present invention 1010] The bispecific antigen-binding molecule of the present invention 1009, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is at least 95% identical thereto, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 95% identical thereto, HCDR3 comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that is at least 95% identical thereto, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that is at least 95% identical thereto, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical thereto, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1011] The bispecific antigen-binding molecule of the present invention 1010, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 12, HCDR2 comprises the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises the amino acid sequence of SEQ ID NO: 16, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24. [The present invention 1012] The bispecific antigen-binding molecule of the present invention 1010, comprising a heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical thereto, and a light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1013] The bispecific antigen-binding molecule of the present invention 1012, comprising a HCVR that comprises the amino acid sequence of SEQ ID NO: 10 and a LCVR that comprises the amino acid sequence of SEQ ID NO: 18. [The present invention 1014] The bispecific antigen-binding molecule according to any one of the present inventions 1001 to 1003, wherein D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within the heavy chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 32, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within the light chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 18. [The present invention 1015] The bispecific antigen-binding molecule of the present invention 1014, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence that is at least 95% identical thereto, HCDR2 comprises the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence that is at least 95% identical thereto, HCDR3 comprises the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence that is at least 95% identical thereto, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that is at least 95% identical thereto, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical thereto, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1016] The bispecific antigen-binding molecule of the present invention 1015, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 34, HCDR2 comprises the amino acid sequence of SEQ ID NO: 36, HCDR3 comprises the amino acid sequence of SEQ ID NO: 38, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24. [The present invention 1017] The bispecific antigen-binding molecule of the present invention 1015, comprising an HCVR that comprises the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence that is at least 95% identical thereto, and an LCVR that comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1018] The bispecific antigen-binding molecule of the present invention 1017, comprising an HCVR that comprises the amino acid sequence of SEQ ID NO: 32 and an LCVR that comprises the amino acid sequence of SEQ ID NO: 18. [The present invention 1019] The bispecific antigen-binding molecule according to any one of the present inventions 1014 to 1018, wherein D2 comprises three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) within the heavy-chain variable region (HCVR) that comprises the amino acid sequence of SEQ ID NO: 40, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) within the light-chain variable region (LCVR) that comprises the amino acid sequence of SEQ ID NO: 18. [The present invention 1020] The bispecific antigen-binding molecule of the present invention 1019, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 42 or an amino acid sequence that is at least 95% identical thereto, HCDR2 comprises the amino acid sequence of SEQ ID NO: 44 or an amino acid sequence that is at least 95% identical thereto, HCDR3 comprises the amino acid sequence of SEQ ID NO: 46 or an amino acid sequence that is at least 95% identical thereto, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that is at least 95% identical thereto, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical thereto, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1021] The bispecific antigen-binding molecule of the present invention 1020, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 42, HCDR2 comprises the amino acid sequence of SEQ ID NO: 44, HCDR3 comprises the amino acid sequence of SEQ ID NO: 46, LCDR1 comprises the amino acid sequence of SEQ ID NO: 20, LCDR2 comprises the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 24. [The present invention 1022] The bispecific antigen-binding molecule of the present invention 1020, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence that is at least 95% identical thereto and an LCVR comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto. [The present invention 1023] The bispecific antigen-binding molecule of the present invention 1022, comprising an HCVR comprising the amino acid sequence of SEQ ID NO: 40 and an LCVR comprising the amino acid sequence of SEQ ID NO: 18. [The present invention 1024] The bispecific antigen-binding molecule of the present invention 1001, conjugated to a cytotoxin. [The present invention 1025] The following characteristics: (a) Exhibiting greater antitumor killing at a lower dosage as compared to trastuzumab conjugated to a cytotoxin; (b) Exhibiting greater antitumor killing at a lower dosage as compared to trastuzumab conjugated to DM1; (c) Exhibiting greater antitumor killing at a lower dosage as compared to Medi-x conjugated to a cytotoxin; (d) Exhibiting greater antitumor killing at a lower dosage as compared to Medi-x conjugated to tubulysin-x. (e) inhibiting the growth of cancers with moderate and high HER2 expression, but not inhibiting the growth of tissues with low HER2 expression; and (f) promoting tumor regression of cancers with moderate and high HER2 expression, but not promoting tumor regression of tissues with low HER2 expression The bispecific antigen-binding molecule of the present invention 1024 having one or more of the above. [The present invention 1026] The bispecific antigen-binding molecule of the present invention 1025, wherein the cytotoxin is selected from the group consisting of biological toxins, chemotherapeutic agents, and radioisotopes. [The present invention 1027] The bispecific antigen-binding molecule of the present invention 1025, wherein the cytotoxin is tubulysin or maytansinoid. [The present invention 1028] The bispecific antigen-binding molecule of the present invention 1001 conjugated to a cytotoxin via a linker. [The present invention 1029] The bispecific antigen-binding molecule of the present invention 1028, wherein the cytotoxin is tubulysin. [The present invention 1030] The tubulysin is TIFF0007695260000001.tif34128 The bispecific antigen-binding molecule of the present invention 1029. [The present invention 1031] The bispecific antigen-binding molecule is TIFF0007695260000002.tif81165 Conjugated to or its positional isomer, wherein TIFF0007695260000003.tif7128 Is a bond with heavy chain glutamine, the bispecific antigen-binding molecule of the present invention 1028. [The present invention 1032] The bispecific antigen-binding molecule of the present invention 1029 conjugated to tubulysin via Q295. [The present invention 1033] The bispecific antigen-binding molecule of the present invention 1029 conjugated to tubulysin via Q297. [The present invention 1034] The linker is azido-PEG 3 -amine, the bispecific antigen-binding molecule of the present invention 1029. [The present invention 1035] The bispecific antigen-binding molecule of the present invention 1030 comprising the CDR within the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the CDR within the D2-HCVR amino acid sequence of SEQ ID NO: 10. [The present invention 1036] The bispecific antigen-binding molecule of the present invention 1030 comprising the CDR within the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the CDR within the D2-HCVR amino acid sequence of SEQ ID NO: 40. [The present invention 1037] The bispecific antigen-binding molecule of the present invention 1028, wherein the cytotoxic agent is maytansinoid. [The present invention 1038] The bispecific antigen-binding molecule of the present invention 1037 comprising the CDR within the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the CDR within the D2-HCVR amino acid sequence of SEQ ID NO: 10. [The present invention 1039] The bispecific antigen-binding molecule of the present invention 1038, comprising the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 40. [The present invention 1040] wherein the maytansinoid is TIFF0007695260000004.tif46128 and in the formula, TIFF0007695260000005.tif6128 is the bond with the linker, the bispecific antigen-binding molecule of the present invention 1037. [The present invention 1041] wherein the linker is TIFF0007695260000006.tif35128 and in the formula, TIFF0007695260000007.tif7128 the bond represented by is the bond with the bispecific antigen-binding molecule, TIFF0007695260000008.tif7128 the bond represented by is the bond with the maytansinoid, the bispecific antigen-binding molecule of the present invention 1040. [The present invention 1042] wherein the maytansinoid is TIFF0007695260000009.tif46128 and in the formula, TIFF0007695260000010.tif6128 is the bond with the linker, the bispecific antigen-binding molecule of the present invention 1037. [The present invention 1043] wherein the linker is TIFF0007695260000011.tif39128 and in the formula, TIFF0007695260000012.tif7128 the bond represented by is the bond with the bispecific antigen-binding molecule, TIFF0007695260000013.tif7128 the bond represented by is the bond with the maytansinoid, the bispecific antigen-binding molecule of the present invention 1042. [The present invention 1044] A pharmaceutical composition comprising the bispecific antigen-binding molecule of the present invention 1001 and a pharmaceutically acceptable carrier. [The present invention 1045] A method of treating cancer in a subject suffering from a tumor overexpressing HER2, comprising administering to the subject a bispecific antigen-binding molecule of any one of the present inventions 1024 to 1043. [The present invention 1046] The method of the present invention 1036, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, gastric cancer, cervical cancer, gastric cancer, endometrial cancer, and ovarian cancer. [The present invention 1047] The method of the present invention 1045, further comprising administering a second anti-cancer therapeutic agent to the subject. [The present invention 1048] A method of treating cancer in a subject, reducing tumor growth, and / or causing tumor regression, comprising administering to a subject in need thereof an antibody-drug conjugate (ADC) comprising a bispecific antigen-binding molecule and a cytotoxic agent, wherein the bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) and a second antigen-binding domain (D2) and D1 specifically binds to a first epitope of human HER2 and D2 specifically binds to a second epitope of human HER2. The method. [The present invention 1049] The method of the present invention 1048, wherein D1 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 or 32, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. [The present invention 1050] The method of the present invention 1048, wherein D2 comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10 or 40, and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. [The present invention 1051] The method of the present invention 1048, wherein the bispecific antigen-binding molecule comprises the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 10. [The present invention 1052] The method of the present invention 1048, wherein the bispecific antigen-binding molecule comprises the CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 40. [The present invention 1053] The method according to any one of the present inventions 1048 to 1052, wherein the cytotoxin is selected from the group consisting of a biological toxin, a chemotherapeutic agent, and a radioisotope. [The present invention 1054] The method according to any one of the present inventions 1048 to 1052, wherein the cytotoxin is tubulysin or maytansinoid. [The present invention 1055] The cytotoxin is tubulysin, and the tubulysin is TIFF0007695260000014.tif34128 The method according to any one of the present inventions 1048 to 1052. [The present invention 1056] The bispecific antigen-binding molecule is TIFF0007695260000015.tif81165 conjugated to or its positional isomer, wherein TIFF0007695260000016.tif7128 is a bond with heavy chain glutamine. The method of the present invention 1055. [The present invention 1057] The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, and the linker is azido-PEG 3 -amine. The method according to any one of the present inventions 1054 to 1056. [The present invention 1058] The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, and the cytotoxin is TIFF0007695260000017.tif46128 wherein TIFF0007695260000018.tif6128is a bond with the linker. The method of the present invention 1048. [The present invention 1059] The linker is TIFF0007695260000019.tif35128 wherein TIFF0007695260000020.tif7128 The bond denoted by represents the bond with the bispecific antigen-binding molecule, TIFF0007695260000021.tif7128 The bond denoted by represents the bond with the cytotoxin, the method of the present invention 1058. [The present invention 1060] The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, and the cytotoxin is TIFF0007695260000022.tif46128 wherein, TIFF0007695260000023.tif6128 is the bond with the linker, the method of the present invention 1048. [The present invention 1061] The linker is TIFF0007695260000024.tif39128 wherein, TIFF0007695260000025.tif7128 The bond denoted by represents the bond with the bispecific antigen-binding molecule, TIFF0007695260000026.tif7128 The bond denoted by represents the bond with the cytotoxin, the method of the present invention 1060. [The present invention 1062] A method for preparing an antibody-drug conjugate, comprising contacting an anti-HER2 antibody or a HER2×HER2 bispecific antigen-binding protein with a compound having the following formula A 1 : TIFF0007695260000027.tif64128 and an aqueous diluent. [The present invention 1063] The method of the present invention 1062, wherein the HER2×HER2 bispecific antigen-binding protein comprises CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 2 and CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 10. [The present invention 1064] The method of the present invention 1062, wherein the HER2×HER2 bispecific antigen-binding protein comprises CDRs within the D1-HCVR amino acid sequence of SEQ ID NO: 32 and CDRs within the D2-HCVR amino acid sequence of SEQ ID NO: 40. [The present invention 1065] The method of the present invention 1063 or 1064, wherein the HER2×HER2 bispecific antigen-binding protein comprises CDRs within the LCVR amino acid sequence of SEQ ID NO: 18. [The present invention 1066] The compound of formula A 1 is present in stoichiometric excess, the method of the present invention 1062. [The present invention 1067] The compound of formula A 1 is a compound of formula A 2 or A 3 : TIFF0007695260000028.tif132128 or a mixture thereof, the method of the present invention 1062. [The present invention 1068] The compound of formula A 2 is stereoisomerically pure, the method of the present invention 1062. [The present invention 1069] The compound of formula A 3 is stereoisomerically pure, the method of the present invention 1062. [The present invention 1070] The compound of A 1 or A 2 is present with a diastereomeric excess of more than 50%, more than 70%, more than 90%, or more than 95%, the method of the present invention 1062. [The present invention 1071] The compound of formula A 1 is a compound of formula (a): TIFF0007695260000029.tif58128 which is prepared by contacting with a compound of formula (b): TIFF0007695260000030.tif50128 in the presence of silica gel and a diluent, the method of the present invention 1062. [The present invention 1072] The following process: (i) A compound of formula (a): TIFF0007695260000031.tif58128 with a compound of formula (b): TIFF0007695260000032.tif50128 contacting in the presence of silica gel and a diluent to synthesize an intermediate, and (ii) contacting a HER2×HER2 bispecific antigen-binding protein with the intermediate and an aqueous diluent An antibody-drug conjugate prepared by. [Inventive item 1073] An anti-HER2 antibody or a HER2×HER2 bispecific antigen-binding protein having the following structure: TIFF0007695260000033.tif76166 A process for preparing an antibody-drug conjugate, comprising contacting with a compound having, wherein the HER2×HER2 bispecific antigen-binding protein is functionalized with an azide group. [Inventive item 1074] The process of Inventive item 1073, wherein the HER2×HER2 bispecific antigen-binding protein functionalized with an azide group is prepared by contacting a HER2×HER2 bispecific antigen-binding protein with transglutaminase and a compound containing a primary amine, a PEG group, and an azide group. [Inventive item 1075] The compound containing a primary amine, a PEG group, and an azide group is TIFF0007695260000034.tif11128 The process of Inventive item 1074. [Inventive item 1076] The product of the process of any one of Inventive items 1073 to 1075. [Inventive item 1077] The bispecific antigen-binding molecule of Inventive item 1028, wherein the cytotoxic agent is camptothecin. [Inventive item 1078] The camptothecin is TIFF0007695260000035.tif46128 The bispecific antigen-binding molecule of Inventive item 1077. [Inventive item 1079] The bispecific antigen-binding molecule is TIFF0007695260000036.tif51170 conjugated with its positional isomer, TIFF0007695260000037.tif7128 is a bond with heavy chain glutamine, the bispecific antigen-binding molecule of Inventive item 1028. [Inventive item 1080] The bispecific antigen-binding molecule is TIFF0007695260000038.tif48128 conjugated to, wherein TIFF0007695260000039.tif6128 is a bond with a linker, the bispecific antigen-binding molecule of Inventive item 1028. [Inventive item 1081] An anti-HER2 antibody or a HER2×HER2 bispecific antigen-binding protein having the following structure: TIFF0007695260000040.tif28159 A process for preparing an antibody-drug conjugate, comprising contacting with a compound having, wherein the HER2×HER2 bispecific antigen-binding protein is functionalized with an azide group. [Inventive item 1082] The process of the present invention 1081, wherein the HER2×HER2 bispecific antigen-binding protein functionalized with an azide group is prepared by contacting the HER2×HER2 bispecific antigen-binding protein with transglutaminase and a compound containing a primary amine, a PEG group, and an azide group. [The present invention 1083] The compound containing a primary amine, a PEG group, and an azide group TIFF0007695260000041.tif21128 is the process of the present invention 1082. [The present invention 1084] The product of the process according to any one of the present inventions 1081 to 1083. [The present invention 1085] The bispecific antigen-binding molecule of the present invention 1001, wherein the first epitope of human HER2 comprises amino acids 141 to 145 and / or 166 to 182 of SEQ ID NO: 54. [The present invention 1086] The bispecific antigen-binding molecule of the present invention 1085, wherein the second epitope of human HER2 comprises amino acids 9 to 23, 41 to 51, 64 to 67, and / or 353 to 359 of SEQ ID NO: 54. [The present invention 1087] The bispecific antigen-binding molecule of the present invention 1085, wherein the first epitope of human HER2 comprises amino acids 141 to 145 and / or 166 to 182 of SEQ ID NO: 54, and the second epitope of human HER2 comprises amino acids 9 to 23, 41 to 51, 64 to 67, and / or 353 to 359 of SEQ ID NO: 54. [The present invention 1088] The bispecific antigen-binding molecule of the present invention 1001, wherein the first epitope of human HER2 comprises amino acids 133 to 148, 174 to 182, and / or 194 to 200 of SEQ ID NO: 54. [The present invention 1089] The bispecific antigen-binding molecule of the present invention 1088, wherein the second epitope of human HER2 comprises amino acids 152 to 161, 258 to 273, and / or 194 to 200 of SEQ ID NO: 54. [The present invention 1090] The bispecific antigen-binding molecule of the present invention 1088, wherein the first epitope of human HER2 comprises amino acids 133 to 148, 174 to 182, and / or 194 to 200 of SEQ ID NO: 54, and the second epitope of human HER2 comprises amino acids 152 to 161, 258 to 273, and / or 194 to 200 of SEQ ID NO: 54. Other embodiments will become apparent from a consideration of the following detailed description.

Brief Description of the Drawings

[0011]

Figure 1A

Figure 1B

Figure 1C

[0012]

Figure 2

[0013]

Figure 3

[0014]

Figure 4

[0015]

Figure 5

[0016]

Figure 6

[0017]

Figure 7A

Figure 7B

[0018]

Figure 8

[0019]

Figure 9

[0020]

Figure 10

[0021]

Figure 11

[0022]

Figure 12

[0023]

Figure 13

[0024]

Figure 14

[0025]

Figure 15

[0026]

Figure 16

[0027]

Figure 17

Best Mode for Carrying Out the Invention

[0028] Detailed Description Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. Since the scope of the present invention will be limited only by the appended claims, it should also be understood that the terms used in this specification are used for the purpose of describing only specific embodiments and are not intended to be limiting.

[0029] Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification, the term "about," when used in relation to a particular recited numerical value, means that the value can vary by up to 1% from the recited value. For example, as used in this specification, the expression "about 100" includes 99 and 101, as well as all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0030] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred methods and materials will be described hereinafter. All patents, patent applications, and non-patent publications mentioned in this specification are hereby incorporated by reference in their entirety.

[0031] HER2 Protein As used in this specification, expressions such as "HER2" refer to members of the epidermal growth factor (EGF) receptor family of receptor tyrosine kinases. This protein is also known as NEU, NGL, HER2, TKR1, CD340, HER-2, MLN 19, HER-2 / neu. HER2 can refer to an amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 51 and / or the amino acid sequence set forth in NCBI accession number NP_004439.2.

[0032] All references in this specification to proteins, polypeptides, and protein fragments are intended to refer to the human version of each respective protein, polypeptide, or protein fragment, unless explicitly specified as being from non-human species. Thus, the expression "HER2" means human HER2, for example, unless specified as being from non-human species such as "mouse HER2", "monkey HER2", etc.

[0033] As used herein, the expression "cell surface-expressed HER2" means one or more HER2 proteins or their extracellular domains that are expressed on the surface of a cell, in vitro or in vivo, such that at least a portion of the HER2 protein is exposed on the extracellular side of the cell membrane and accessible to the antigen-binding portion of an antibody. "Cell surface-expressed HER2" can include or consist of HER2 proteins expressed on the surface of cells that normally express the HER2 protein. Alternatively, "cell surface-expressed HER2" can include or consist of HER2 proteins expressed on the surface of cells that do not normally express human HER2 on their surface but have been artificially engineered to express HER2 on their surface.

[0034] HER2×HER2 bispecific antigen-binding molecule Provided herein is a bispecific antigen-binding molecule that includes 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 of two separate HER2 epitopes by the bispecific antigen-binding molecule results in antibody clustering on the HER2-expressing cell surface and internalization of the HER2 protein along with the bound antibody.

[0035] A bispecific antigen-binding molecule comprising a first antigen-binding domain (D1) that specifically binds to a first epitope of human HER2 and a second antigen-binding domain (D2) that specifically binds to a second epitope of human HER2 may be referred to herein as a "HER2×HER2 bispecific antibody", "HER2×HER2", or other related terms.

[0036] In certain embodiments, the D1 and D2 domains of the HER2×HER2 bispecific antibody are non-competing with each other. Non-competing between D1 and D2 with respect to binding to HER2 means that the respective monospecific antigen-binding proteins derived from D1 and D2 do not compete with each other for binding to human HER2. Examples of competitive assays for antigen-binding proteins are known in the art, and non-limiting examples thereof are described elsewhere in this specification.

[0037] In certain embodiments, D1 and D2 bind to different epitopes (e.g., non-overlapping epitopes or partially overlapping epitopes) on HER2, as described elsewhere herein.

[0038] The HER2×HER2 bispecific antigen-binding molecule may be constructed using the antigen-binding domains of two separate monospecific anti-HER2 antibodies. For example, a collection of monoclonal monospecific anti-HER2 antibodies can be generated using standard methods known in the art. The individual antibodies so generated can be tested pairwise against each other for cross-competition for binding to the HER2 protein. If two different anti-HER2 antibodies can bind to HER2 simultaneously (i.e., do not compete with each other), the antigen-binding domain derived from the first anti-HER2 antibody and the antigen-binding domain derived from the second non-competing anti-HER2 antibody can be engineered into a single HER2×HER2 bispecific antibody according to the present disclosure.

[0039] According to the present disclosure, the bispecific antigen-binding molecule can be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides covalently or non-covalently linked to each other. As will be apparent from the present disclosure, any antigen-binding construct having the ability to simultaneously bind to two separate and non-identical epitopes of HER2 is considered a bispecific antigen-binding molecule. Any of the bispecific antigen-binding molecules or variants thereof described herein may be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) as known to those skilled in the art.

[0040] Anti-HER2 bispecific antibody sequence As used herein, the term "antibody" means any antigen-binding molecule or molecular complex that includes at least one complementarity-determining region (CDR) that specifically binds or interacts with a particular antigen (e.g., HER2). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds, i.e., two heavy (H) chains and two light (L) chains, and multimers thereof (e.g., IgM). The term "antibody" also includes immunoglobulin molecules consisting of four polypeptide chains interconnected by disulfide bonds, i.e., two heavy (H) chains and two light (L) chains. Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or V H and is omitted) and a heavy chain constant region. The heavy chain constant region includes three domains C H1 , C H2 , and C H3 . Each light chain includes a light chain variable region (abbreviated herein as LCVR or V L and is omitted) and a light chain constant region. The light chain constant region includes one domain (C L1 ). The V H region and the V L region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) with relatively conserved regions called framework regions (FRs) disposed therebetween. Each V H and V LIt is composed of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of the anti-HER2 antibody (or its antigen-binding portion) may be identical to the human germline sequence or may be naturally or artificially modified. The amino acid consensus sequence can be defined based on the parallel analysis of two or more CDRs.

[0041] Similarly, the term "antibody" includes immunoglobulin molecules containing eight polypeptide chains interconnected by disulfide bonds, namely four heavy (H) chains and four light (L) chains, that is, bispecific antibodies. The term "antibody" also includes immunoglobulin molecules consisting of eight polypeptide chains interconnected by disulfide bonds, namely four heavy (H) chains and four light (L) chains.

[0042] Furthermore, the term "antibody" includes a functionalized immunoglobulin molecule containing at least one HC, and the HC contains azide-PEG3-amine. In some embodiments, azide-PEG3-amine is located at the Q295 site on the antibody HC. In some embodiments, azide-PEG3-amine is located at the Q297 site on the antibody. In some embodiments, the bispecific antibody has two HCs functionalized with azide-PEG3-amine located at both Q295 sites of the HC. In some embodiments, the bispecific antibody has two HCs functionalized with azide-PEG3-amine located at both Q297 sites of the HC. In some embodiments, the bispecific antibody has two HCs functionalized with azide-PEG3-amine located at both Q295 sites and both Q297 sites of the HC. The Q297 site in the HC is obtained by modifying N297 to Q297, which is also referred to as the N297Q modification herein.

[0043] According to one aspect, a HER2 bispecific antibody is provided. Examples of HER2 bispecific antibodies according to this aspect are listed in Tables 1 and 2 herein. Table 1 lists 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 the bispecific antigen-binding molecules (used interchangeably with bispecific antigen-binding proteins herein) disclosed herein. Table 2 lists the nucleic acid sequence identifiers of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of exemplary antibodies.

[0044] As used herein, a bispecific antibody that specifically binds to HER2 is provided, comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0045] As used herein, a bispecific antibody that specifically binds to HER2 is provided, 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0046] Provided herein are bispecific antibodies that specifically bind to HER2 and comprise an amino acid sequence pair of a heavy chain variable region (HCVR) and a light chain variable region (LCVR) (HCVR / LCVR), which pair with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the bispecific antibody comprises two HCVR / LCVR amino acid sequence pairs contained in any of the exemplary HER2 bispecific antibodies listed in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of: SEQ ID NO: 2 / 18, 10 / 18, 32 / 18, and 40 / 18.

[0047] Further provided are bispecific antibodies that specifically bind to HER2 and comprise a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0048] Further provided are bispecific antibodies that specifically bind to HER2 and comprise a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0049] Further provided are bispecific antibodies that specifically bind to HER2 and comprise a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0050] Furthermore, provided is a bispecific antibody that specifically binds to HER2 and comprises a light chain complementarity-determining region 1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0051] Furthermore, provided is a bispecific antibody that specifically binds to HER2 and comprises a light chain complementarity-determining region 2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0052] Furthermore, provided is a bispecific antibody that specifically binds to HER2 and comprises a light chain complementarity-determining region 3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0053] Furthermore, provided is a bispecific antibody that specifically binds to HER2 and comprises an amino acid sequence pair of heavy chain complementarity-determining region 1 and light chain complementarity-determining region 1 (HCDR1 / LCDR1) comprising any of the HCDR1 amino acid sequences paired with any of the LCDR1 amino acid sequences listed in Table 1. According to certain embodiments, the bispecific antibody comprises at least one HCDR1 / LCDR1 amino acid sequence pair contained in any of the exemplary anti-HER2 antibodies listed in Table 1. In certain embodiments, the HCDR1 / LCDR1 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 4 / 20, 12 / 20, 34 / 20, and 42 / 20.

[0054] Furthermore, provided is a bispecific antibody that specifically binds to HER2 and comprises an amino acid sequence pair of HCDR2 and LCDR2 (HCDR2 / LCDR2) that forms a pair with any of the LCDR2 amino acid sequences listed in Table 1 and any of the HCDR2 amino acid sequences listed in Table 1. According to certain embodiments, the bispecific antibody comprises at least one HCDR2 / LCDR2 amino acid sequence pair contained in any of the exemplary anti-HER2 antibodies listed in Table 1. In certain embodiments, the HCDR2 / LCDR2 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 6 / 22, 14 / 22, 36 / 22, and 44 / 22.

[0055] Furthermore, provided herein is a bispecific antibody that specifically binds to HER2 and comprises an amino acid sequence pair of HCDR3 and LCDR3 (HCDR3 / LCDR3) that forms a pair with any of the LCDR3 amino acid sequences listed in Table 1 and any of the HCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the bispecific antibody comprises at least one HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary bispecific antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8 / 24, 16 / 24, 38 / 24, and 46 / 24.

[0056] Furthermore, provided herein is a bispecific antibody that specifically binds to HER2 and comprises a set of six CDRs (i.e., HCDR1 - HCDR2 - HCDR3 - LCDR1 - LCDR2 - LCDR3) contained in any of the exemplary anti-HER2 antibodies listed in Table 1. In certain embodiments, the set of amino acid sequences of HCDR1 - HCDR2 - HCDR3 - LCDR1 - LCDR2 - LCDR3 is selected from the group consisting of SEQ ID NO: 4 - 6 - 8 - 20 - 22 - 24, 12 - 14 - 16 - 20 - 22 - 24, 34 - 36 - 38 - 20 - 22 - 24, and 42 - 44 - 46 - 20 - 22 - 24.

[0057] In related embodiments, a bispecific antibody that specifically binds to HER2 comprises six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) in two sets of two HCVR / LCVR amino acid sequence pairs defined by any of the exemplary HER2 bispecific antibodies listed in Table 1. For example, the HER2 bispecific antibody comprises an HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set containing an HCVR / LCVR amino acid sequence pair selected from the group consisting of: SEQ ID NO: 2 / 18, 10 / 18, 32 / 18, and 40 / 18.

[0058] Methods and techniques for identifying CDRs within HCVR amino acid sequences and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specific HCVR amino acid sequences and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Under common conditions, the Kabat definition is based on sequence variability, the Chothia definition is based on the position of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, 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 for identifying CDR sequences within antibodies.

[0059] A bispecific antibody that specifically binds to HER2 and comprises a heavy chain (HC) comprising an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto is provided herein. For example, a bispecific antibody that specifically binds to HER2 and comprises an HC comprising an amino acid sequence selected from any of the HC amino acid sequences listed in Table 3 but containing an N297Q modification or an equivalent modification is provided herein. Exemplarily, a bispecific antibody having a heavy chain containing an N297Q modification within an HC amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 28, 48, and 50 is provided herein. In some embodiments, the bispecific antibody comprises an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody comprises an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 28. In some embodiments, the bispecific antibody comprises an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 48. In some embodiments, the bispecific antibody comprises an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 50. In some embodiments, the bispecific antibody comprises an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 26 and an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 28. In some embodiments, the bispecific antibody comprises an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 48 and an HC containing an N297Q modification within the HC amino acid sequence of SEQ ID NO: 50.

[0060] A bispecific antibody that specifically binds to HER2 and comprises a light chain (LC) comprising an amino acid sequence selected from any of the LC amino acid sequences listed in Table 4, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto is provided herein.

[0061] It should be noted that in the original text, there is a reference to "Table 3" in the heavy chain part and "Table 4" in the light chain part which seems inconsistent. I translated according to the original content. You may want to check and correct this if necessary.Provided herein are bispecific antibodies that specifically bind to HER2, comprising an amino acid sequence pair (HC / LC) of HC and LC that form a pair with any of the LC amino acid sequences listed in Table 3 and any of the HC amino acid sequences listed in Table 3. According to certain embodiments, the bispecific antibody comprises two HC / LC amino acid sequence pairs contained in any of the exemplary HER2 bispecific antibodies listed in Table 3. In certain embodiments, the HC / LC amino acid sequence pair is selected from the group consisting of: SEQ ID NO: 26 / 30, 28 / 30, 48 / 30, and 50 / 30. In some aspects, the HC comprises the N297Q modification provided above.

[0062] Also provided herein are nucleic acid molecules encoding an anti-HER2 antibody or a portion thereof. For example, the present invention provides a nucleic acid molecule encoding any of the HCVR amino acid sequences listed in Table 1, and in certain embodiments, the nucleic acid molecule is a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0063] Also provided are nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1, and in certain specific embodiments, the nucleic acid molecule is a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0064] Also provided are nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1, and in certain specific embodiments, the nucleic acid molecule is a polynucleotide sequence selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0065] Also provided is a nucleic acid molecule encoding any of the HCDR2 amino acid sequences listed in Table 1. 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0066] Also provided is a nucleic acid molecule encoding any of the HCDR3 amino acid sequences listed in Table 1. 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0067] Also provided is a nucleic acid molecule encoding any of the LCDR1 amino acid sequences listed in Table 1. 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0068] Also provided is a nucleic acid molecule encoding any of the LCDR2 amino acid sequences listed in Table 1. 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0069] Also provided is a nucleic acid molecule encoding any of the LCDR3 amino acid sequences listed in Table 1. 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0070] Also provided is a nucleic acid molecule encoding HCVR, which comprises a set of three CDRs (i.e., HCDR1 - HCDR2 - HCDR3), and the set of HCDR1 - HCDR2 - HCDR3 amino acid sequences is as defined by any of the exemplary HER2 bispecific antibodies listed in Table 1.

[0071] Also provided is a nucleic acid molecule encoding LCVR, which comprises a set of three CDRs (i.e., LCDR1 - LCDR2 - LCDR3), and the set of LCDR1 - LCDR2 - LCDR3 amino acid sequences is as defined by any of the exemplary HER2 bispecific antibodies listed in Table 1.

[0072] Also provided are nucleic acid molecules encoding both an HCVR and an LCVR, where the HCVR comprises the amino acid sequence of any one of the HCVR amino acid sequences listed in Table 1, and the LCVR comprises the amino acid sequence of any one of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any one of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any one of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same HER2 bispecific antibody listed in Table 1.

[0073] Further provided are nucleic acid molecules encoding any one of the HC amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. For example, the nucleic acid molecule can encode any one of the HC amino acid sequences listed in Table 3, where the HC has an N297Q modification.

[0074] Also provided are nucleic acid molecules encoding any one of the LC amino acid sequences listed in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0075] Also provided herein are recombinant expression vectors capable of expressing a polypeptide comprising a variable region of the heavy or light chain of a HER2 bispecific antibody. For example, a recombinant vector comprises any of the nucleic acid molecules described above, i.e., a nucleic acid molecule encoding any of the HCVR sequences, LCVR sequences, and / or CDR sequences as set forth in Table 1. Also within the scope of the disclosure are host cells into which such vectors have been introduced, methods of producing an antibody or a portion thereof by culturing the host cells under conditions that allow production of the antibody or antibody fragment, and methods of recovering the antibody and antibody fragments so produced.

[0076] HER2 bispecific antibodies having an altered glycosylation pattern are provided herein. In some embodiments, for example, modifications that remove undesirable glycosylation sites, or antibodies lacking fucose moieties present on the oligosaccharide chain, may be useful to increase antibody-dependent cell-mediated 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 activity (CDC).

[0077] Antigen-binding domain The bispecific antigen-binding molecules of the disclosure comprise two separate antigen-binding domains (D1 and D2). As used herein, the expression "antigen-binding domain" means any peptide, polypeptide, nucleic acid molecule, scaffold-type molecule, peptide display molecule, or polypeptide-containing construct that can specifically bind to a particular target antigen (e.g., human HER2). Terms such as "specifically binds" as used herein mean that the antigen-binding domain forms a complex with a particular antigen characterized by a dissociation constant (K D ) of 500 pM or less and does not bind other irrelevant antigens under common test conditions. "Irrelevant antigens" are proteins, peptides, or polypeptides having less than 95% amino acid identity to each other.

[0078] Exemplary classifications of antigen-binding domains that can be used in the context of 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 that include ligand-binding portions of receptors that specifically bind to a particular antigen, antigen-binding scaffolds (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, and other scaffolds based on natural repeat proteins [see, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and aptamers or portions thereof.

[0079] Methods for determining whether two molecules specifically bind to each other are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. For example, when 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 when measured in a surface plasmon resonance assay. D Also included are polypeptides that bind to a particular antigen (e.g., a target molecule [T] or an internal translocation effector protein [E]) or a portion thereof.

[0080] The term "surface plasmon resonance" as used herein refers to an optical phenomenon that enables the analysis of real-time interactions by detecting changes in protein concentration within a biosensor matrix, for example, within a BIAcore® system (Biacore Life Sciences Division, GE Healthcare, Piscataway, NJ).

[0081] The term "K D " when used in this specification, means the equilibrium dissociation constant of a particular protein-protein interaction (e.g., antibody-antigen interaction). The K D values disclosed herein refer to the K D values measured at 25 °C by surface plasmon resonance assay.

[0082] As indicated above, the "antigen-binding domain" (D1 and / or D2) may comprise or consist of an antibody or an antigen-binding fragment of an antibody. The term "antibody" as used in this specification means any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that specifically binds or interacts with a particular antigen (e.g., human HER2). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains interconnected by disulfide bonds, namely two heavy (H) chains and two light (L) chains, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy-chain variable region (abbreviated herein as HCVR or V H for brevity) and a heavy-chain constant region. The heavy-chain constant region comprises three domains C H1 , C H2 , and C H3 . Each light chain comprises a light-chain variable region (abbreviated herein as LCVR or V L for brevity) and a light-chain constant region. The light-chain constant region comprises one domain (C L 1). The V H region and the V L region can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) with relatively conserved regions called framework regions (FRs) in between. Each V H and V LIt is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments, the FRs (or antigen-binding portions thereof) of the antibodies provided herein may be identical to human germline sequences or may be modified naturally or artificially. Amino acid consensus sequences can be defined based on the parallel analysis of two or more CDRs.

[0083] The D1 component and / or D2 component of the bispecific antigen-binding molecules provided herein may comprise or consist of antigen-binding fragments of full antibody molecules. Terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., as used herein, include any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from full antibody molecules using any suitable standard methods, such as proteolytic digestion or recombinant genetic engineering techniques, involving the manipulation and expression of DNA encoding the antibody variable region and optionally the constant domain. Such DNA is known and / or can be readily obtained, 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 using molecular biological techniques to, for example, place one or more variable domains and / or constant domains in an appropriate configuration, introduce codons, create cysteine residues, modify, add, or delete amino acids.

[0084] 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) amino acid residues mimicking the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or minimal recognition units consisting of 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, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, etc., are also encompassed within the expression “antigen-binding fragment” when used herein.

[0085] An antigen-binding fragment of an antibody typically contains at least one variable domain. The variable domain can be of any size or amino acid composition and generally contains at least one CDR that is adjacent to or in-frame with one or more framework sequences. The V L domain that associates with the V H domain in an antigen-binding fragment having a V H domain and a V L domain can be arranged relative to each other in any suitable arrangement. For example, the variable regions can be dimers and contain V H -V H 、V H -V L or V L -V L dimers. Alternatively, an antigen-binding fragment of an antibody can contain a monomeric V H or V L domain.

[0086] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragments of the antibodies 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 Linclude. In any of the variable domain and constant domain configurations, including any of the exemplary configurations listed above, the variable domain and the constant domain 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 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible linkage between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment may be a homodimer or heterodimer (or other multimer) of any of the variable region and constant region configurations listed above, non-covalently to each other and / or non-covalently associated with one or more monomeric V H domains or V L domains (e.g., by disulfide bonds).

[0087] The bispecific antigen-binding molecules provided herein may include, or consist of, human antibodies and / or recombinant human antibodies, or fragments thereof. The term "human antibody" as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Nevertheless, a human antibody may include amino acid residues not encoded by the human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, or somatic mutations in vivo), for example, in the CDRs and in certain CDR3s. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0088] The bispecific antigen-binding molecules of the present disclosure may comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. As used herein, the term "recombinant human antibody" encompasses all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (detailed below), antibodies isolated from a recombinant combinatorial human antibody library (detailed below), antibodies isolated from transgenic animals (e.g., mice) 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 including splicing of human immunoglobulin gene sequences to 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, in the case of using transgenic animals for human Ig sequences, somatic mutagenesis in vivo), such that the amino acid sequences of the V H region and the V L region are related to and derived from human germline V H sequences and V L sequences, but may not naturally occur in the human antibody germline repertoire in vivo.

[0089] Methods for making bispecific antibodies are known in the art, and these methods can be used to construct the bispecific antigen-binding molecules disclosed herein. Exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, 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 having knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 Bispecific formats (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein for an overview of the formats described above) are included.

[0090] Examples of antigen-binding domains (D1 and D2) that can be included in the HER2×HER2 bispecific antigen-binding molecules provided herein include antigen-binding domains derived from any of the anti-HER2 sequences disclosed herein. For example, the present disclosure includes an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1, or a D1 or D2 antigen-binding domain comprising an HCVR comprising a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, including HER2×HER2 bispecific antigen-binding molecules.

[0091] Also provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or an LCVR comprising a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0092] Provided herein are HER2×HER2 bispecific antigen-binding molecules comprising an amino acid sequence pair (HCVR / LCVR) of an HCVR and an LCVR that pair with any of the LCVR amino acid sequences listed in Table 1 and any of the HCVR amino acid sequences listed in Table 1. According to certain embodiments, the invention provides HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain comprising an HCVR / LCVR amino acid sequence pair contained in any of the exemplary anti-HER2 antibodies listed in Table 1.

[0093] Also provided herein are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0094] Also provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0095] Also provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0096] Also provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a light chain complementarity-determining region 1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0097] Also provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a light chain complementarity-determining region 2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0098] Also provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a light chain complementarity-determining region 3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0099] Also provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising an amino acid sequence pair of a heavy chain complementarity-determining region 3 (HCDR3) and an LCDR3 (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences paired with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-HER2 antibodies listed in Table 1.

[0100] Further provided are HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-HER2 antibodies listed in Table 1.

[0101] In related embodiments, the present disclosure provides HER2×HER2 bispecific antigen-binding molecules comprising a D1 or D2 antigen-binding domain and comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in an HCVR / LCVR amino acid sequence pair selected from the anti-HER2 sequences listed in Table 1.

[0102] The HER2×HER2 bispecific antigen-binding molecules provided herein may comprise a D1 antigen-binding domain derived from any of the anti-HER2 antibodies in Table 1 and a D2 antigen-binding domain derived from any other anti-HER2 antibody in Table 1. Non-limiting examples of the HER2×HER2 bispecific antibodies of the present disclosure are illustrated in the examples provided herein.

[0103] As a non-limiting illustration, the present disclosure includes HER2×HER2 bispecific antigen-binding molecules 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: 2 / 18, or a set of CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) of the heavy and light chains comprising SEQ ID NOs: 4-6-8-20-22-24, and the D2 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 10 / 18, or a set of CDRs (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) of the heavy and light chains comprising SEQ ID NOs: 12-14-16-20-22-24. An exemplary HER2×HER2 bispecific antibody having the characteristics of these sequences is a bispecific antibody designated H4H17325D, also referred to as bispecific antibody 1 (bsAb1).

[0104] As yet another non-limiting example, the present disclosure includes a HER2×HER2 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: 32 / 18, or a set of CDRs of the heavy and light chains (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 34-36-38-20-22-24, and the D2 antigen-binding domain comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 40 / 18, or a set of CDRs of the heavy and light chains (HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) comprising SEQ ID NOs: 42-44-46-20-22-24. An exemplary HER2×HER2 bispecific antibody having the characteristics of these sequences is a bispecific antibody designated H4H17087D, also referred to as bispecific antibody 2 (bsAb2).

[0105] Constituents of multimer formation In certain embodiments, the bispecific antigen-binding molecules provided herein may include one or more constituents of multimer formation. The constituents of multimer formation can function to maintain the association between the antigen-binding domains (D1 and D2). As used herein, a "constituent of multimer formation" is any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to associate with a second constituent of multimer formation of the same or a similar structure or composition. For example, a constituent of multimer formation can be immunoglobulin C HIt may be a polypeptide containing 3 domains. Non-limiting examples of components for multimer formation are the Fc portions of immunoglobulins, such as isotypes IgG1, IgG2, IgG3, and IgG4, and the Fc domains of IgG selected from any allotype within each isotype group. In certain embodiments, the component for multimer formation is an Fc fragment or an amino acid sequence 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the component for multimer formation is a cysteine residue or a short cysteine-containing peptide. Other multimer-forming domains include peptides or polypeptides containing or consisting of leucine zippers, helix-loop motifs, or coiled-coil motifs.

[0106] In certain embodiments, the bispecific antigen-binding molecules provided herein include two multimer-forming domains M1 and M2, D1 is attached to M1, D2 is attached to M2, and the association of M1 and M2 promotes the physical linkage of D1 and D2 to each other in 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 having a specific amino acid sequence, and M2 can be an Fc domain having the same amino acid sequence as M1. Alternatively, M1 and M2 may differ from each other at one or more amino acid positions. For example, M1 is the first immunoglobulin (Ig) C H It may contain 3 domains, and M2 is the second Ig C H It may contain 3 domains, and the first and second Ig C H The 3 domains have at least one amino acid different from each other, and the at least one amino acid difference reduces the binding of the target-directed construct to protein A compared to a reference construct having the same M1 and M2 sequences. In one embodiment, the Ig C of M1 H The 3 domains bind to protein A, and the Ig C of M2 HThe 3 domain contains mutations that reduce or abolish protein A binding, such as the H95R modification (according to the IMGT exon numbering convention; according to the EU numbering convention, H435R). C of M2 H 3 may further include the Y96F modification (according to IMGT; according to EU, Y436F). C of M2 H As yet another modification that can be found within 3, the following can be mentioned: for the IgG1 Fc domain, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; according to EU, D356E, L358M, N384S, K392N, V397M, and V422I); for the IgG2 Fc domain, N44S, K52N, and V82I (IMGT; according to EU, N384S, K392N, and V422I); and for the IgG4 Fc domain, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; according to EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I).

[0107] The bispecific antigen-binding molecules of the present disclosure may be "isolated". As used herein, an "isolated bispecific antigen-binding molecule" means 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 the tissue or cell in which the antibody was produced is an "isolated bispecific antibody" for the purposes of the present disclosure. An isolated bispecific antigen-binding molecule also includes the molecule in situ within 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 and / or chemical substances.

[0108] The bispecific antigen-binding molecules disclosed herein, or antigen-binding domains thereof (D1 and / or D2), may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the variable domains of the heavy and / or light chains as compared to the corresponding germline sequences from which the antigen-binding protein or antigen-binding domain is derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes bispecific antigen-binding molecules disclosed herein or antigen-binding domains thereof (D1 and / or D2) that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more frameworks and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations").

[0109] One of ordinary skill in the art can readily generate many bispecific antigen-binding molecules or antigen-binding domains thereof (D1 and / or D2) that contain one or more individual germline mutations or combinations thereof, starting from the heavy chain variable region sequences and light chain variable region sequences disclosed herein. In certain embodiments, all of the framework residues and / or CDR residues within the V H domain and / or the V L domain are mutated back to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues, e.g., only the mutated residues found within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2, or CDR3, are mutated back to the original germline sequence. In other embodiments, one or more framework residues and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived).

[0110] Furthermore, the bispecific antigen-binding molecule of the present disclosure, or its antigen-binding domains (D1 and / or D2), may contain any combination of two or more germline mutations within the framework region and / or CDR region. For example, certain individual residues are mutated to the corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence are either maintained or mutated to the corresponding residues of a different germline sequence. A bispecific antigen-binding molecule containing one or more germline mutations, or its antigen-binding domains (D1 and / or D2), once obtained, can be tested for one or more desired properties, such as improvement of binding specificity, increase in binding affinity, improvement or enhancement (where applicable) of antagonist or agonist biological properties, reduction of immunogenicity, etc. The bispecific antigen-binding molecule or its antigen-binding domains (D1 and / or D2) obtained in this general manner is encompassed by the present disclosure.

[0111] Variant The present invention also includes anti-HER2 antibodies and bispecific antigen-binding molecules comprising a variant of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. Exemplary variants included in this aspect include variants of the amino acid sequences of any of the HCVR, LCVR, and / or CDR disclosed herein having one or more substitutions, such as conservative substitutions. In some aspects, the present disclosure provides anti-HER2 antibodies and HER2×HER2 bispecific antigen-binding molecules having an amino acid sequence of HCVR, LCVR, and / or CDR that contains, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, 3 or fewer, 2, or 1 amino acid substitutions relative to any of the amino acid sequences of HCVR, LCVR, and / or CDR described in Table 1 herein, and the modified antibodies and bispecific antigen-binding molecules maintain the binding activity to HER2.

[0112] Exemplary variants included in this aspect of the disclosure also include variants having substantial sequence identity with the amino acid sequence of any of the HCVR, LCVR, and / or CDR disclosed herein. As used herein in the context of amino acid sequences, the terms "substantial identity" or "substantially identical" mean that two amino acid sequences share at least 95%, 98% or 99% sequence identity when optimally aligned by, for example, programs such as GAP or BESTFIT that use default gap weighting. In certain embodiments, the 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). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. If two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward 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, for example, Pearson (1994) Methods Mol. Biol. 24:307-331, which is 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: cysteine and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.Alternatively, a conservative substitution is any change having 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" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0113] Sequence identity between two different amino acid sequences is typically measured using sequence analysis software. The sequence analysis software aligns similar sequences using similarity measures assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For example, the GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides such as homologous polypeptides from different species or homologous polypeptides between a wild-type protein and its variants. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides the alignment of the best overlapping regions between the query sequence and the search sequence and the percent sequence identity (Pearson (2000), supra). Another preferred algorithm for comparing the sequences provided herein with a database containing a large number of sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN using default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0114] HER2×HER2 bispecific antigen-binding molecule comprising an Fc variant According to certain embodiments provided herein, there is provided a HER2×HER2 bispecific antigen-binding protein comprising an Fc domain comprising one or more mutations that enhance or attenuate antibody binding to the FcRn receptor, for example, at an acidic pH as compared to a neutral pH. For example, the present disclosure relates to a HER2×HER2 bispecific antigen-binding protein comprising mutations in the C H 2 or C H 3 region, the mutations increasing the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., L / R / S / P / Q or K) and / or position 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F) and position 434. In one embodiment, the modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), and modifications of 307 and / or 308 (e.g., 308F and / or 308P).

[0115] For example, the present disclosure includes a HER2×HER2 bispecific antigen-binding protein comprising an Fc domain comprising 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). Any possible combination of the aforementioned Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated to be within the scope of the present disclosure.

[0116] Biological characteristics of the antigen-binding molecules provided herein HER2×HER2 bispecific antigen-binding proteins that bind to human HER2 with high affinity (e.g., hErbB2 ecto-mFc) are provided herein. For example, the present disclosure provides an anti-HER2×HER2 bispecific antigen-binding protein that binds to human HER2 with a K of less than about 3 nM as measured by surface plasmon resonance at 25°C using, for example, the assay format defined in Example 8 herein or an assay substantially similar thereto. D According to certain embodiments, an anti-HER2 antibody is provided that binds to human HER2 at 25°C with a K of 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, less than about 0.25 nM, less than about 200 pM, less than about 150 pM, less than about 100 pM, or less than about 50 pM as measured by surface plasmon resonance at 25°C using, for example, the assay format defined in Example 8 herein or an assay substantially similar thereto. D For example, the present disclosure provides an anti-HER2 antibody that binds to human HER2 at 25°C with a dissociation half-life (t

[0117] Also, for example, using the assay format defined in Example 8 herein or an assay substantially similar thereto, a dissociation half-life (t of greater than about 20 minutes as measured by surface plasmon resonance at 25°C. 1 / 2Also provided herein are HER2×HER2 bispecific antigen-binding proteins that bind to human HER2 (e.g., hErbB2 ecto-mFc) in (e.g., at 25° C.). In certain embodiments, using, for example, the assay format defined in Example 8 herein or an assay substantially similar thereto, when measured by surface plasmon resonance, t is greater than about 20 minutes, greater than about 25 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 than those. 1 / 2 Also provided are HER2×HER2 bispecific antigen-binding proteins that bind to human HER2 at 25° C.

[0118] Also provided herein are HER2×HER2 bispecific antigen-binding proteins that exhibit internalization in early endosomes and transport from early endosomes to lysosomes. Also provided herein are HER2×HER2 bispecific antigen-binding proteins that form surface clusters of HER2 and induce internalization.

[0119] The antigen-binding proteins of the present disclosure may possess one or more of the aforementioned biological characteristics, or any combination thereof. The aforementioned list of biological characteristics of the antibody is not intended to be exhaustive. Other biological characteristics of the antibodies provided herein will be apparent to those skilled in the art from a review of the present disclosure, including the examples herein.

[0120] Antibody-drug conjugate (ADC) Also provided herein are antibody-drug conjugates (ADCs) comprising HER2×HER2 bispecific antigen-binding proteins conjugated to a therapeutic moiety such as a cytotoxic agent, a chemotherapeutic agent, or a radioisotope.

[0121] Cytotoxic agents include, but are not limited to, any agent that is detrimental to cell growth, viability, or proliferation, such as tubulin-interacting agents and DNA-damaging agents. In some embodiments, the cytotoxic agent is a tubulin inhibitor. In certain embodiments, the tubulin inhibitor inhibits tubulin polymerization. In some embodiments, the cytotoxic payload is a topoisomerase I inhibitor. In some embodiments, the cytotoxic agent is maytansinoid, auristatin, hemiasterlin, vinblastine, vincristine, pyrrolobenzodiazepine, paclitaxel, docetaxel, cryptophycin, tubulysin, or camptothecin.Examples of suitable cytotoxic agents and chemotherapeutic agents that can be conjugated to an anti-HER2 antibody according to this aspect of the present disclosure include, for example, 1-(2-chloroethyl)-1,2-dimethanesulfonylhydrazide, 1,8-dihydroxy-bicyclo[7.3.1]trideca-4,9-diene-2,6-diyne-13-one, 1-dehydrotestosterone, 5-fluorouracil, 6-mercaptopurine, 6-thioguanine, 9-aminocamptothecin, actinomycin D, amanitin, aminopterin, anguidine, anthracycline, anthramycin (AMC), auristatin, bleomycin, busulfan, butyric acid, calicheamicin (e.g., calicheamicin γ1), camptothecin, carminomycin, carmustine, cemadotin, cisplatin, colchicine, combretastatin, cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin, dacarbazine, diacetoxypentyl doxorubicin, dibromomannitol, dihydroxyanthracenedione, disorazole, dolastatin (e.g., dolastatin 10), doxorubicin, duocarmycin, echinomycin, erythrobicin, emetine, epothilone, esperamicin, estramustine, ethidium bromide, etoposide, fluorouracil, geldanamycin, gramicidin D, glucocorticoid, irinotecan, kinesin spindle protein (KSP) inhibitor, leptomycin, leurosine, lidocaine, lomustine (CCNU), maytansinoid, mechlorethamine, melphalan, mercaptopurine, methopterin, methotrexate, mitramycin, mitomycin, mitoxantrone, N8-acetylspermidine, podophyllotoxin, procaine, propranolol, pteridine, puromycin, pyrrolobenzodiazepine (PBD), rhizoxin, streptozotocin, talisomycin, taxol, tenoposide, tetracaine, thioepacrambucil, tomamycin, topotecan, tubulysin, vinblastine, vincristine, vindesine, vinorelbine, and derivatives of any of them.According to certain embodiments, the cytotoxic agent conjugated to the anti-HER2 antibody is a maytansinoid such as DM1 or DM4, a tubulysin derivative, or a dolastatin derivative. According to certain embodiments, the cytotoxic agent conjugated to the anti-HER2 antibody is an auristatin such as MMAE, MMAF, or a derivative thereof. In some embodiments, the cytotoxic agent is Dxd or a derivative thereof. In some embodiments, the cytotoxic agent is AZ13599185 (see, e.g., Li et al., 2016 Cancer Cell 29, 117-129). Other cytotoxic agents known in the art are also contemplated 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, saponin, pokeweed toxin (i.e., phytolaccatoxin and phytolaccigenin), and other toxins such as those described in Sapra et al., Pharmacol. & Therapeutics, 2013, 138:452-469. In some embodiments, the cytotoxic agent is tubulysin, a maytansinoid, or camptothecin.

[0122] In certain embodiments, the cytotoxin is tubulysin. Suitable tubulysins include those described in U.S. Patent Application No. 16 / 724,164, filed December 20, 2019. In some embodiments, the tubulysin is compound IVa, IVa’, IVb, IVc, IVd, IVe, IVf, IVg, IVh, IVj, IVk, IV-l, IVm, IVn, IVo, IVp, IVq, IVr, IVs, IVt, IVu, IVvA, IVvB, IVw, IVx, IVy, Va, Va’, Vb, Vc, Vd, Ve, Vf, Vg, Vh, Vi, Vj, Vk, Via, VIb, VIc, VId, VIe, VIf, VIg, VIh, VI, VIi, VII, VIII, IX, X, D-5a, or D-5c as cited from U.S. Patent Application No. 16 / 724,164, filed December 20, 2019. In certain embodiments, the tubulysin is compound Ve of U.S. Patent Application No. 16 / 724,164, filed December 20, 2019. In some embodiments, the tubulysin has the following structure. TIFF0007695260000042.tif46128

[0123] Tubulysin 1A can be prepared using the methods disclosed in U.S. Patent Application No. 16 / 724,164, filed December 20, 2019.

[0124] In certain embodiments, the cytotoxin is a maytansinoid, such as a derivative of maytansine. Suitable maytansinoids include DM1, DM4, or derivatives, stereoisomers, or isotopologs thereof. Also, suitable maytansinoids include, but are not limited to, those disclosed in WO2014 / 145090A1, WO2015 / 031396A1, U.S. Patent Application Publication No. 2016 / 0375147 A1, and U.S. Patent Application Publication No. 2017 / 0209591 A1, which are hereby incorporated by reference in their entirety. In some embodiments, the maytansinoid is DM1.

[0125] In some embodiments, the maytansinoid has the following structure: TIFF0007695260000043.tif45128 wherein A is an optionally substituted arylene or heteroarylene.

[0126] In some embodiments, the maitansinoid has the following structure: TIFF0007695260000044.tif41128 wherein A is an optionally substituted arylene or heteroarylene.

[0127] In some embodiments, the maitansinoid has the following structure: TIFF0007695260000045.tif45128 wherein n is an integer from 1 to 12, and R 1 is alkyl.

[0128] In some embodiments, the maitansinoid is as follows. TIFF0007695260000046.tif212141 TIFF0007695260000047.tif213132 TIFF0007695260000048.tif177133 TIFF0007695260000049.tif120170

[0129] In some embodiments, the maitansinoid is as follows. TIFF0007695260000050.tif37128

[0130] In some embodiments, the maitansinoid is as follows. TIFF0007695260000051.tif37128

[0131] In some embodiments, the cytotoxic agent is camptothecin, such as an analogue or derivative of camptothecin. In some embodiments, the HER2×HER2 bispecific antigen-binding protein, such as an antibody, is conjugated to exatecan, deruxtecan, DX-8951, DXd, camptothecin, or a derivative or analogue thereof. In certain embodiments, the cytotoxic agent is DXd. In certain embodiments, the cytotoxic agent is as follows. TIFF0007695260000052.tif46128

[0132] Also provided herein is an antibody-radionuclide conjugate (ARC) comprising an anti-HER2 antibody conjugated to one or more radionuclides. Examples of radionuclides that can be used in the context of the aspects of the present disclosure include, but are not limited to, for example, 225 Ac, 212 Bi, 213 Bi, 131 I, 186 Re, 227 Th, 222 Rn, 223 Ra, 224 Ra, and 90 Y.

[0133] In certain embodiments, an ADC is provided that includes a HER2×HER2 bispecific antigen-binding protein conjugated to a cytotoxic agent (e.g., any of the cytotoxic agents disclosed above) via a linker molecule. The 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; Phillips, G.L., Ed.; Springer Verlag: New York, 2013; Antibody-Drug Conjugates; Ducry, L., Ed.; Humana Press, 2013; Antibody-Drug Conjugates; Wang, J., Shen, W.-C., and Zaro, J.L., Eds.; Springer International Publishing, 2015, the entire contents of each of which are incorporated herein by reference. Generally, a conjugation-mediated linker suitable for the antibody conjugates described herein is one that has sufficient stability to utilize the antibody's circulation half-life while also being able to release the payload after internalization of the conjugate mediated by the antigen. The linker may or may not be cleavable. Cleavable linkers include those that are cleaved by intracellular metabolism such as cleavage via hydrolysis, reduction, or enzymatic reaction after internalization. Non-cleavable linkers include those that release the attached payload via lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzyme-cleavable linkers, reduction-labile linkers, self-immolative linkers, and non-cleavable linkers.Also, suitable linkers include, but are not limited to, peptides, glucuronides, succinimide-thioethers, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, dipeptide units, valine-citrulline units, and para-aminobenzyl (PAB) units, or linkers that contain or consist of them.

[0134] Any linker molecule or linker technology known in the art can be used to make 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, MC (6-maleimidocaproyl), MP (maleimidopropanoyl), val-cit (valine-citrulline), val-ala (valine-alanine), dipeptide moieties in protease-cleavable linkers, ala-phe (alanine-phenylalanine), dipeptide moieties in protease-cleavable linkers, 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 linkers that contain or consist of variants and combinations thereof. Additional exemplary linkers that can be used in the context of the present disclosure are provided, for example, in U.S. Patent No. 7,754,681, the contents of which are incorporated herein by reference in their entirety, and Ducry, Bioconjugate Chem., 2010, 21:5-13, and the references cited therein.

[0135] In certain embodiments, the linker is stable under physiological conditions. In certain embodiments, the linker is cleavable and can release at least the payload portion, for example, in the presence of an enzyme or within a specific pH range or pH value. In some embodiments, the linker includes an enzymatically cleavable moiety. Exemplary enzymatically cleavable moieties include, but are not limited to, peptide bonds, ester bonds, hydrazones, and disulfide bonds. In some embodiments, the linker includes a cathepsin-cleavable linker.

[0136] In some embodiments, the linker includes a non-cleavable moiety.

[0137] Suitable linkers also include, but are not limited to, for example, linkers that are chemically bound to two cysteine residues of a single binder such as an antibody. Such linkers can serve to mimic the disulfide bonds of the antibody that are broken as a result of the conjugation process.

[0138] In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker comprises 2 amino acids. In some embodiments, the linker comprises 3 amino acids. In some embodiments, the linker comprises 4 amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinaceous, non-proteinaceous, 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 amino acid side chains 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. In some embodiments, the linker comprises a dipeptide, tripeptide, or tetrapeptide. In some embodiments, the linker comprises a peptide, and the peptide is valine-citrulline (val-cit or VC), glutamic acid-valine-citrulline (EVC), or glycine-glycine-phenylalanine-glycine (GGFC).

[0139] In some embodiments, the linker comprises a self-immolative group. The self-immolative group can be any such group known to those of skill in the art. In certain embodiments, the self-immolative group is p-aminobenzyl (PAB), or a derivative thereof. Useful derivatives include p-aminobenzyloxycarbonyl (PABC). In some embodiments, the linker comprises a moiety having the following structure. TIFF0007695260000053.tif16128

[0140] One of ordinary skill in the art will recognize that the self-immolative group is capable of undergoing a chemical reaction that releases the linker's remaining atoms from the payload.

[0141] In some embodiments, the linker is as follows: TIFF0007695260000054.tif34128 wherein, TIFF0007695260000055.tif7128 is a bond (e.g., via a lysine residue) to an antibody or antigen-binding protein, TIFF0007695260000056.tif6128 is a bond to a cytotoxic agent (e.g., DM1). In some embodiments, the linker is as follows: TIFF0007695260000057.tif34128 wherein, TIFF0007695260000058.tif7128 is a bond (e.g., via a lysine residue) to an antibody or antigen-binding protein, TIFF0007695260000059.tif6128 is a bond to a cytotoxic agent (e.g., DM1). In certain embodiments, the linker is as follows. TIFF0007695260000060.tif34128

[0142] In certain embodiments, the linker is as follows. TIFF0007695260000061.tif34128

[0143] In some embodiments, the linker is derived from maleimidylmethyl-4-trans-cyclohexanecarboxylate. TIFF0007695260000062.tif39128

[0144] In some embodiments, the linker is as follows: TIFF0007695260000063.tif39128 wherein, TIFF0007695260000064.tif7128 is a binding to an antibody or antigen-binding protein (e.g., via a lysine residue), TIFF0007695260000065.tif6128 is a binding to a cytotoxic agent (e.g., a compound having the following formula). TIFF0007695260000066.tif46128

[0145] Suitable linkers include, but are not limited to, linkers containing one or more cyclic moieties. In some embodiments, the cyclic moiety is derived from a cycloaddition reaction. In certain embodiments, the cyclic moiety is derived from a 1-3-cycloaddition reaction between an azide and an alkyne, such as a cycloalkyne. In some embodiments, the cyclic moiety is as follows. TIFF0007695260000067.tif25128

[0146] In some embodiments, the linker includes one or more spacers. Suitable spacers include, for example, moieties that covalently or via ionic interactions link two linker moieties, a linker moiety having a payload, or a linker moiety having an antibody. In certain embodiments, the spacer is a PEG group.

[0147] The present disclosure includes an ADC in which a linker connects a HER2×HER2 bispecific antigen-binding protein to a drug or cytotoxin via an addition at a specific amino acid within the antibody or antigen-binding molecule. Exemplary amino acid additions that can be used in the context of this aspect include, for example, lysine (see, e.g., U.S. Patent No. 5,208,020; U.S. Patent Application Publication No. 2010 / 0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO2005 / 089808; U.S. Patent No. 5,714,586; U.S. Patent Application Publication No. 2013 / 0101546; and U.S. Patent Application Publication No. 2012 / 0585592), cysteine (see, e.g., U.S. Patent Application Publication No. 2007 / 0258987; WO2013 / 055993; WO2013 / 055990; WO 2013 / 053873; WO2013 / 053872; WO2011 / 130598; U.S. Patent Application Publication No. 2013 / 0101546; and U.S. Patent No. 7,750,116), 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).The linker may also be conjugated to the antigen-binding protein via addition of carbohydrates (see, e.g., U.S. Patent Application Publication No. 2008 / 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). Also, site-specific conjugation techniques may be employed to conjugate directly 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, glutamine conjugation via transglutaminase (see, e.g., Schibli, Angew Chemie Inter Ed. 2010, 49, 9995).

[0148] According to certain embodiments, the present disclosure provides an ADC in which the HER2×HER2 bispecific antigen-binding protein described herein is conjugated to a linker-drug composition (e.g., the compound “7” described below and also referred to herein as “M0026”) described in International Patent Application Publication WO2014 / 145090, which is incorporated herein by reference in its entirety. TIFF0007695260000068.tif46128

[0149] Also provided herein are antibody-drug conjugates comprising a monospecific anti-HER2 antibody and a HER2×HER2 bispecific antibody, wherein the HER2×HER2 bispecific antibody is conjugated to a cytotoxic agent. In certain embodiments, the cytotoxic agent is a maytansinoid. In certain embodiments, the maytansinoid is a compound having the following formula: In TIFF0007695260000069.tif45128, n is an integer from 1 to 12, and R 1 is alkyl. In certain embodiments, the maytansinoid is as follows. In TIFF0007695260000070.tif45133, in certain embodiments, the cytotoxic agent is a maytansinoid, and this maytansinoid is covalently attached to the antibody via a non-cleavable linker. In certain embodiments, the cytotoxic agent is a maytansinoid, and this maytansinoid is covalently attached to the antibody via a cleavable linker.

[0150] In one embodiment, the bispecific antibody is conjugated to the following: In TIFF0007695260000071.tif54128, where In TIFF0007695260000072.tif7128, is the binding to the antibody.

[0151] In one embodiment, the bispecific antibody is conjugated to the following: In TIFF0007695260000073.tif54128, where In TIFF0007695260000074.tif7128, is the binding to the antibody.

[0152] In one embodiment, the bispecific antibody is conjugated to the following: In TIFF0007695260000075.tif54128, where In TIFF0007695260000076.tif7128, is the binding to the antibody.

[0153] In one embodiment, the bispecific antibody is conjugated to the following: In TIFF0007695260000077.tif46150, where In TIFF0007695260000078.tif7128, is the binding to the antibody.

[0154] In some embodiments, the bispecific antibody is conjugated to the following: TIFF0007695260000079.tif43128 wherein L is a linker, TIFF0007695260000080.tif7128 is a binding to an antibody.

[0155] In some embodiments, the bispecific antibody is the following: TIFF0007695260000081.tif63165 or its positional isomer, conjugated thereto, wherein SP is a spacer, TIFF0007695260000082.tif7128 is a binding to an antibody.

[0156] In some embodiments, the bispecific antibody is the following: TIFF0007695260000083.tif81165 or its positional isomer, conjugated thereto, wherein TIFF0007695260000084.tif7128 is a binding to the glutamine residue of the antibody. In certain embodiments, the glutamine is the heavy chain Q295 glutamine. In certain embodiments, the bispecific antibody is conjugated to heavy chain Q295 and Q297, said Q297 being derived from the N297Q mutation.

[0157] In some embodiments, the bispecific antibody is bound to the following: TIFF0007695260000085.tif50165 wherein TIFF0007695260000086.tif7128 is a binding to the glutamine residue of the antibody. In certain embodiments, the glutamine is the heavy chain Q295 glutamine. In certain embodiments, the bispecific antibody is conjugated to heavy chain Q295 and Q297, said Q297 being derived from the N297Q mutation.

[0158] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Wherein: Ab is a HER2×HER2 bispecific antigen-binding protein described herein, L is a linker, Pay is a cytotoxic agent, n is an integer from 1 to 12.

[0159] In some embodiments, n is 2. In some embodiments, n is 4. In some embodiments, Pay is as follows: TIFF0007695260000087.tif67128.

[0160] In some embodiments, L is a cleavable linker. In some embodiments, L contains a peptide. In some embodiments, L contains val-cit. In some embodiments, L contains the following. TIFF0007695260000088.tif16128 In some embodiments, L contains a PEG group. In some embodiments, L contains a cyclic moiety. In certain embodiments, the cyclic moiety is the product of a 1,3-cycloaddition of an azide and a cycloalkyne. In some embodiments, -L-Pay is TIFF0007695260000089.tif14128 and SP 1 and SP 2 are each independently a spacer, TIFF0007695260000090.tif10128 is a cyclic moiety, TIFF0007695260000091.tif6128 is a bond with heavy chain glutamine. In some embodiments, the glutamine is Q295 glutamine. In some embodiments, n is 2, and in the formula, two L-Pays are conjugated to Q295. In some embodiments, n is 4, and in the formula, two L-Pays are conjugated to Q295 and two L-Pays are conjugated to Q297. In some embodiments, the cyclic moiety is the product of a cycloaddition reaction. In certain embodiments, the cyclic moiety is the product of a cycloaddition reaction of an azide and a cycloalkyne. In certain embodiments, the cyclic moiety is as follows: TIFF0007695260000092.tif is 25128. In certain embodiments, SP 1 contains a PEG moiety. In certain embodiments, SP 2 contains a dipeptide. In certain embodiments, SP2 contains val-cit. In certain embodiments, SP 2 is as follows: includes TIFF0007695260000093.tif 16128. In certain embodiments, SP 2 contains a PEG moiety. In some embodiments, SP2-Pay is as follows: TIFF0007695260000094.tif is 47165. In certain embodiments, TIFF0007695260000095.tif 10128 is TIFF0007695260000096.tif 59165 or a positional isomer thereof.

[0161] In certain embodiments, TIFF0007695260000097.tif 14128 is TIFF0007695260000098.tif 81165 (tubulysin 1b) or a positional isomer thereof, and TIFF0007695260000099.tif 7128 is the binding to said antibody.

[0162] In some embodiments, Ab is conjugated to a linker payload or payload disclosed in WO2015 / 157592, for example, compound T32 disclosed therein. In some embodiments, Ab is conjugated to deruxtecan (DXd) via a linker optionally containing GGFG.

[0163] In some embodiments, Ab is a HER2×HER2 bispecific antigen-binding protein comprising a CDR within the D1-HCVR amino acid sequence of SEQ ID NO: 2 and a CDR within the D2-HCVR amino acid sequence of SEQ ID NO: 10. In some aspects, the HER2×HER2 bispecific antigen-binding protein further comprises a CDR within the LCVR amino acid sequence of SEQ ID NO: 18. In some embodiments, Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10. In some aspects, the HER2×HER2 bispecific antigen-binding protein further comprises the LCVR amino acid sequence of SEQ ID NO: 18.

[0164] In some embodiments, Ab is a HER2×HER2 bispecific antigen-binding protein comprising a CDR within the D1-HCVR amino acid sequence of SEQ ID NO: 32 and a CDR within the D2-HCVR amino acid sequence of SEQ ID NO: 40. In some aspects, the HER2×HER2 bispecific antigen-binding protein further comprises a CDR within the LCVR amino acid sequence of SEQ ID NO: 18. In some embodiments, Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40. In some aspects, the HER2×HER2 bispecific antigen-binding protein further comprises the LCVR amino acid sequence of SEQ ID NO: 18.

[0165] 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.

[0166] In some embodiments, L comprises a moiety having the following structure. TIFF0007695260000100.tif34128

[0167] In some embodiments, L comprises a moiety having the following structure. TIFF0007695260000101.tif34128

[0168] In some embodiments, L includes a portion having the following structure. TIFF0007695260000102.tif34128

[0169] In some embodiments, L includes a portion having the following structure. TIFF0007695260000103.tif39128

[0170] In some embodiments, Pay is tubulysin.

[0171] In some embodiments, Pay is maytansinoid.

[0172] In some embodiments, Pay is as follows: TIFF0007695260000104.tif43128 wherein R 1 is alkyl.

[0173] In some embodiments, Pay is as follows. TIFF0007695260000105.tif46128

[0174] In some embodiments, Pay is as follows. TIFF0007695260000106.tif46128

[0175] In some embodiments, n is an integer from 2 to 5.

[0176] In some embodiments, -L-Pay is as follows: TIFF0007695260000107.tif54128 wherein TIFF0007695260000108.tif7128 is a binding to an antibody.

[0177] In some embodiments, -L-Pay is as follows: TIFF0007695260000109.tif54128 wherein TIFF0007695260000110.tif7128 is a binding with an antibody.

[0178] In some embodiments, -L-Pay is as follows: TIFF0007695260000111.tif54128 wherein, TIFF0007695260000112.tif7128 is a binding with an antibody.

[0179] In some embodiments, -L-Pay is as follows: TIFF0007695260000113.tif46150 wherein, TIFF0007695260000114.tif7128 is a binding with an antibody.

[0180] In some embodiments, -Pay is as follows. TIFF0007695260000115.tif48128

[0181] In some embodiments, -L-Pay is as follows. TIFF0007695260000116.tif50165 In a specific embodiment, L-Pay is as follows, TIFF0007695260000117.tif50165 n is 2.

[0182] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Wherein: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10, L-Pay is as follows: TIFF0007695260000118.tif54128 wherein, TIFF0007695260000119.tif7128 is a binding with an antigen-binding protein, and n is an integer from 2 to 5.

[0183] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10, L-Pay is as follows: TIFF0007695260000120.tif54128 Wherein, TIFF0007695260000121.tif7128 is the binding to the antigen-binding protein, and n is an integer from 2 to 5.

[0184] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10, L-Pay is as follows: TIFF0007695260000122.tif54128 Wherein, TIFF0007695260000123.tif7128 is the binding to the antigen-binding protein, and n is an integer from 2 to 5.

[0185] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10, L-Pay is as follows: TIFF0007695260000124.tif46150 Wherein, TIFF0007695260000125.tif7128 is a binding to an antigen-binding protein, and n is an integer from 2 to 5.

[0186] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40, L-Pay is as follows: TIFF0007695260000126.tif54128 Wherein, TIFF0007695260000127.tif7128 is a binding to an antigen-binding protein, and n is an integer from 2 to 5.

[0187] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40, L-Pay is as follows: TIFF0007695260000128.tif54128 Wherein, TIFF0007695260000129.tif7128 is a binding to an antigen-binding protein, and n is an integer from 2 to 5.

[0188] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40, L-Pay is as follows: TIFF0007695260000130.tif54128 wherein TIFF0007695260000131.tif7128 is a bond with an antigen-binding protein, and n is an integer from 2 to 5.

[0189] In some embodiments, the conjugate has the following structure: Ab - [L - Pay] n Wherein: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1 - HCVR amino acid sequence of SEQ ID NO: 32 and the D2 - HCVR amino acid sequence of SEQ ID NO: 40, L - Pay is as follows: TIFF0007695260000132.tif46150 wherein TIFF0007695260000133.tif7128 is a bond with an antigen-binding protein, and n is an integer from 2 to 5.

[0190] In some embodiments, the conjugate has the following structure: Ab - [L - Pay] n Wherein: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1 - HCVR amino acid sequence of SEQ ID NO: 2 and the D2 - HCVR amino acid sequence of SEQ ID NO: 10, and Pay is as follows: TIFF0007695260000134.tif55128 wherein TIFF0007695260000135.tif7128 is a bond with an antigen-binding protein, and n is an integer from 2 to 5.

[0191] In some embodiments, the conjugate has the following structure: Ab - [L - Pay] n Wherein: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40, and Pay is as follows: TIFF0007695260000136.tif55128wherein, TIFF0007695260000137.tif7128is the binding to the antigen-binding protein, and n is an integer from 2 to 5.

[0192] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10, and L-Pay is as follows: TIFF0007695260000138.tif81165wherein, TIFF0007695260000139.tif7128is the binding to heavy chain glutamine. In some embodiments, n is 2. In one embodiment, n is 2 and L-Pay is bound to glutamine Q295. In some embodiments, n is 4. In one embodiment, n is 4 and L-Pay is bound to glutamines Q295 and Q297.

[0193] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n wherein: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40, and L-Pay is as follows: TIFF0007695260000140.tif81165wherein, TIFF0007695260000141.tif7128 is a binding to heavy chain glutamine. In some embodiments, n is 2. In one embodiment, n is 2 and L-Pay is bound to Q295 glutamine. In some embodiments, n is 4. In one embodiment, n is 4 and L-Pay is bound to Q295 glutamine and Q297 glutamine.

[0194] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10, and Pay is as follows. TIFF0007695260000142.tif48128

[0195] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40, and Pay is as follows. TIFF0007695260000143.tif48128

[0196] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10, L-Pay is as follows: TIFF0007695260000144.tif50165 Where, TIFF0007695260000145.tif7128 is the binding of the heavy chain glutamine of the binding protein. In some embodiments, n is 2. In one embodiment, n is 2 and L-Pay is bound to Q295 glutamine. In some embodiments, n is 4. In one embodiment, n is 4 and L-Pay is bound to Q295 glutamine and Q297 glutamine.

[0197] In some embodiments, the conjugate has the following structure: Ab-[L-Pay] n Where: Ab is a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40, L-Pay is as follows: TIFF0007695260000146.tif50165 Wherein, TIFF0007695260000147.tif7128 is the binding of the heavy chain glutamine of the binding protein. In some embodiments, n is 2. In one embodiment, n is 2 and L-Pay is bound to Q295 glutamine. In some embodiments, n is 4. In one embodiment, n is 4 and L-Pay is bound to Q295 glutamine and Q297 glutamine.

[0198] The antibody-drug conjugates described herein can be prepared using conjugation conditions known to those skilled in the art (see, for example, Doronina et al. Nature Biotechnology 2003, 21, 7, 778, which is incorporated herein by reference in its entirety). In some embodiments, an antibody-drug conjugate of a HER2×HER2 bispecific antigen-binding protein is prepared by contacting the HER2×HER2 bispecific antigen-binding protein described herein with a compound comprising a desired linker and a cytotoxic agent, wherein the linker bears a moiety reactive with the antibody or antigen-binding protein at a desired residue of the antibody or antigen-binding protein, for example.

[0199] In some embodiments, the antibody-drug conjugates provided herein are conjugated at glutamine Q295 or Q297 of the heavy chain. Antibodies containing heavy chain Q297 glutamine can be prepared, for example, via the N297Q mutation using techniques known in the art. See, for example, Bioconjugate Chem. 2014, 25, 3, 569 (2014). Such conjugation methods can provide antibody-drug conjugates having a DAR of 2 or 4. In some embodiments, such methods involve introducing a first reactive moiety at heavy chain glutamine by reacting an antibody with a primary amine compound containing the first reactive moiety in the presence of transglutaminase to generate an antibody containing the first reactive moiety at Q295 and optionally Q297. This product can then react with a payload having a linker and a complementary second reactive moiety to provide the antibody-drug conjugates provided herein. In some embodiments, the first reactive moiety is an azide. In some embodiments, the second reactive moiety is a cycloalkyne. In some embodiments, the payload having a linker and a complementary second reactive moiety is as follows. TIFF0007695260000148.tif76166 This can be prepared using the method described in U.S. Patent Application No. 16 / 724,164, filed on December 20, 2019 (for example, the compound LP4 described therein). In certain embodiments, the primary amine compound is as follows. TIFF0007695260000149.tif11128

[0200] In some embodiments, the payload having a linker and a complementary second reactive moiety is as follows. TIFF0007695260000150.tif29163 In certain embodiments, the primary amine compound is as follows. TIFF0007695260000151.tif25128

[0201] In some embodiments, the HER2×HER2 bispecific antigen-binding protein described herein is of Formula A below 1 Compound having: TIFF0007695260000152.tif64128 A process for preparing an antibody-drug conjugate is provided herein, which comprises contacting with a compound having and an aqueous diluent.

[0202] In some embodiments, the compound of Formula A 1 is present in stoichiometric excess. In some embodiments, the compound of Formula A 1 is present in a 5- to 6-fold stoichiometric excess. In some embodiments, the aqueous diluent comprises HEPES. In some embodiments, the aqueous diluent comprises DMA.

[0203] In some embodiments, the compound of Formula A 1 is a compound of Formula A below 2 or A 3 of the compound. TIFF0007695260000153.tif61128 TIFF0007695260000154.tif64128

[0204] In some embodiments, the compound of Formula A 2The compound is stereoisomerically pure A 3 In some embodiments, the compound of formula A 1 The compound of formula A 1 Or A 2 The compound of formula A 1 Or A 2 The compound of formula A or A exists with 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%.

[0205] The term "diastereomeric excess" refers to the difference in the mole fraction of the desired single diastereomer compared to the remaining diastereomers in the composition. The diastereomeric excess is calculated as follows: (amount of single diastereomer) - (amount of other diastereomers) / 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]. The diastereomeric excess can be calculated similarly for any one of 1, 2, 3, or 4.

[0206] In some embodiments, the compound of formula A 1 The compound of formula A is prepared by contacting the compound of the following formula (a): TIFF0007695260000155.tif58128 with the compound of the following formula (b): TIFF0007695260000156.tif49128 in the presence of silica gel and a diluent. In some embodiments, the diluent contains an organic solvent and water.

[0207] Also provided herein are products prepared by the following process: (i) The compound of formula (a): Contact TIFF0007695260000157.tif62128 with the compound of the following formula (b): TIFF0007695260000158.tif49128 in the presence of silica gel and a diluent to synthesize an intermediate, and (ii) Contact the HER2×HER2 bispecific antigen-binding protein described herein with the above intermediate and an aqueous diluent.

[0208] In some embodiments, provided herein is a process for preparing an antibody-drug conjugate comprising contacting the HER2×HER2 bispecific antigen-binding protein described herein with a compound having the following formula B and an aqueous diluent: TIFF0007695260000159.tif57146 wherein LG is a leaving group.

[0209] In some embodiments, the compound of formula B is present in stoichiometric excess. In some embodiments, the compound of formula B is present in a 5- to 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, for example, an NHS or a trifluorophenyl ester.

[0210] In some embodiments, the compound of formula B is the following formula B 1 compound: TIFF0007695260000160.tif58156.

[0211] In some embodiments, the compound of formula B 1 is the compound of the following formula C: TIFF0007695260000161.tif57148 is prepared by contacting it with N-hydroxysuccinimide (NHS), a peptide coupling reagent, and an organic diluent. Suitable peptide coupling reagents include those that activate, i.e., render reactive, the 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.

[0212] In some embodiments, the compound of formula C is the compound of formula D below: TIFF0007695260000162.tif59128 is prepared by contacting it 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.

[0213] Epitope mapping and related techniques The epitope to which the antibody or antigen-binding domain binds may 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 HER2 protein. Alternatively, the related epitope may consist of multiple non-contiguous amino acids (or amino acid sequences) of HER2.

[0214] As described elsewhere in this specification, for example in Examples 21 or 22, the individual antigen-binding domains (D1 and D2) of a HER2×HER2 bispecific antigen-binding molecule can bind to epitopes that are distinct from each other, or non-overlapping, or partially overlapping. As used herein, a "partially overlapping epitope" means that when determined by any epitope mapping method known in the art (e.g., X-ray crystallography, alanine scanning mutagenesis, hydrogen / deuterium exchange [HDX], domain swapping, etc.), the first epitope and the second epitope share less than 5, less than 4, less than 3, or only 1 common amino acid. The D1 domain and the D2 domain may be non-competing with each other. For example, in certain embodiments, binding of the D1 domain of a particular HER2×HER2 bispecific antigen-binding molecule to its epitope on ErbB2 does not inhibit (or only minimally inhibits) binding of the D2 domain of the HER2×HER2 bispecific antigen-binding molecule to its epitope on HER2. By the non-overlapping (or at most partially overlapping) nature of the respective epitopes of the D1 and D2 components, the HER2×HER2 bispecific antigen-binding molecule can bind to a single HER2 molecule on the cell surface. Furthermore, the HER2×HER2 bispecific binding molecule can cluster at ErbB2 on the cell surface and induce HER2 internalization.

[0215] A variety of techniques known to those skilled in the art can be used to determine the epitopes on HER2 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, site-directed mutagenesis (e.g., alanine scanning mutagenesis, arginine scanning mutagenesis, etc.), peptide blot analysis (Reineke, 2004, Methods Mol Biol 248:443-463), protease protection assays, and peptide cleavage analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be employed (Tomer, 2000, Protein Science 9:487-496). Another method that can be used to identify the amino acids within a polypeptide that interact with an antibody is hydrogen / deuterium exchange detected by mass spectrometry. As a general term, hydrogen / deuterium exchange involves labeling the target protein with deuterium and then binding the antibody to this deuterium-labeled protein. Next, the protein / antibody complex is transferred to water, and hydrogen-deuterium exchange is allowed to occur for 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 mass spectrometry analysis, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267(2):252-259; Engen and Smith (2001) Anal.Chem.73:256A-265A. The amino acids within a polypeptide with which an antibody interacts can also be identified using X-ray crystallographic analysis.

[0216] HER2 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 described in Table 1 herein) are further provided herein. Similarly, HER2 bispecific antibodies that compete for binding to HER2 with any of the specific exemplary antibodies described herein (e.g., antibodies comprising any of the amino acid sequences described in Table 1 herein) are also provided herein.

[0217] By using standard methods known in the art and exemplified herein, it is possible to readily determine whether an antibody binds to the same epitope as a reference anti-HER2 antibody or competes with the reference anti-HER2 antibody for binding. For example, to determine whether a test antibody binds to the same epitope as the reference anti-HER2 antibody provided herein, the reference antibody is bound to the HER2 protein. Next, the ability of the test antibody to bind to the HER2 molecule is evaluated. If the test antibody can bind to HER2 after saturation binding with the reference anti-HER2 antibody, it can be concluded that this test antibody binds to an epitope different from the reference anti-HER2 antibody. On the other hand, if the test antibody cannot bind to the HER2 molecule after saturation binding with the reference anti-HER2 antibody, this test antibody may bind to the same epitope as the epitope bound by the reference anti-HER2 antibody. Additional standard experiments (e.g., peptide mutagenesis and binding analysis) are then performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody or whether steric hindrance (or another phenomenon) is the cause of 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 one embodiment, when measured in a competitive binding assay, if one antibody at, for example, 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess inhibits the binding of the other antibody by at least 50%, but preferably 75%, 90%, or even 99%, the two antibodies bind to the same (or overlapping) epitope (see, e.g., Junghans et al., Cancer Res. 1990:50:1495-1502). Alternatively, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies are considered to bind to the same epitope. If only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, the two antibodies are considered to have "overlapping epitopes".

[0218] To determine whether the antibody competes (or cross-competes) with the reference anti-HER2 antibody for binding, the above-described binding procedure is carried out in the following two ways: In the first way, the reference antibody is bound to the HER2 protein under saturation conditions, and then the binding of the test antibody to the HER2 molecule is evaluated. In the second way, the test antibody is bound to the HER2 molecule under saturation conditions, and then the binding of the reference antibody to the HER2 molecule is evaluated. In both ways, if only the first (saturating) antibody was able to bind to the HER2 molecule, the test antibody and the reference antibody are concluded to compete for binding to HER2. As will be understood by those skilled in the art, antibodies that compete for binding to the reference antibody may not necessarily bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0219] Preparation of human antibodies The HER2×HER2 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 method can be used in the context of the present disclosure to generate human antibodies that specifically bind to human HER2.

[0220] For example, using the VELOCIMMUNE™ technology, or any other similar known method for generating fully human monoclonal antibodies, a high affinity chimeric antibody against HER2 having a human variable region and a murine constant region is first isolated. As described in the following experimental sections, antibodies are characterized and selected for desirable features including affinity, dimerization blocking activity, selectivity, epitope, etc. If desired, the murine constant region is replaced with a desired human constant region, such as wild-type or modified IgG1 or IgG4, to generate a fully human anti-HER2 antibody. While the constant region selected may vary depending on the particular application, the features of high affinity antigen binding and target specificity reside in the variable region. In one example, the fully human anti-HER2 antibody is isolated directly from antigen positive B cells.

[0221] Biological equivalents The HER2 bispecific antibodies and antibody fragments provided herein include proteins having an amino acid sequence different from the amino acid sequences of the antibodies described but retaining the ability to bind to human HER2. Such variant antibodies and antibody fragments include one or more amino acid additions, deletions, or substitutions as compared to the parental sequence, but exhibit a biological activity that is essentially equivalent to the biological activity of the described antibodies. Similarly, DNA sequences encoding the anti-HER2 antibodies of the disclosure include sequences that include one or more nucleotide additions, deletions, or substitutions as compared to the disclosed sequences but encode anti-HER2 antibodies or antibody fragments that are essentially biologically equivalent to the anti-HER2 antibodies or antibody fragments of the disclosure. Examples of such variant amino acid and DNA sequences are discussed above.

[0222] Two antigen-binding proteins or antibodies are considered to be biologically equivalent if, for example, under similar experimental conditions, when administered at the same molar dose either as a single dose or multiple doses, they do not show a significant difference in the rate and extent of absorption. Some antibodies have the same extent of absorption but different rates of absorption, yet the difference in absorption rate is intentional and reflected in the labeling and is not essential for achieving the effective in vivo drug concentration, for example, in chronic use, and is considered not medically important for the specific pharmaceutical being tested, so they can be considered equivalent or a pharmaceutical alternative when they can be considered biologically equivalent.

[0223] In one embodiment, two antigen-binding proteins are biologically equivalent if there are no clinically significant differences in their safety, purity, and potency.

[0224] In one embodiment, two antigen-binding proteins are biologically equivalent if, compared to a continuous therapy without switching between the reference product and the biological product one or more times, there is no expected increase in the risk of adverse effects, including a clinically significant change in immunogenicity, or the patient can make such a switch without a decrease in efficacy.

[0225] In one embodiment, two antigen-binding proteins are biologically equivalent if they both act by one or more common mechanisms to the known extent of such mechanisms for one or more conditions of use.

[0226] Biological equivalence can be demonstrated by in vivo and in vitro methods. Examples of methods for measuring biological equivalence include, for example, (a) in vivo tests in humans or other mammals in which the concentration of an antibody or its metabolite is measured 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 in which the appropriate acute pharmacological effects of the antibody (or its target) are measured as a function of time, and (d) appropriately controlled clinical trials that demonstrate the safety, efficacy, or bioavailability or biological equivalence of the antibody.

[0227] Biologically equivalent variants of the HER2 bispecific 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 substituted with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges upon refolding. In other situations, biologically equivalent antibodies can include HER2 bispecific antibody variants that contain amino acid changes that modify the glycosylation properties of the antibody, such as mutations that eliminate or remove glycosylation.

[0228] Species selectivity and species cross-reactivity According to certain embodiments, the present disclosure provides anti-HER2 antibodies (and antigen-binding molecules comprising anti-HER2 antigen-binding domains) that bind to human anti-HER2 but not to HER2 from other species. The present disclosure also includes anti-HER2 antibodies (and antigen-binding molecules comprising anti-HER2 antigen-binding domains) that bind to human anti-HER2 and HER2 derived from one or more non-human species. For example, the anti-HER2 antibodies and antigen-binding molecules can bind to human HER2, but in some cases, 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, cynomolgus monkey, marmoset, rhesus monkey, or chimpanzee HER2.

[0229] Bispecific antibody As described elsewhere herein, the present disclosure provides a bispecific antigen-binding molecule comprising two different antigen-binding domains, wherein a first antigen-binding domain (D1) binds to a first epitope on HER2 and a second antigen-binding domain (D2) binds to a second epitope on HER2. In certain embodiments, the first and / or second epitope is within the extracellular domain of HER2, for example within SEQ ID NO: 54. In certain embodiments, the first and second epitopes on HER2 to which the D1 domain and the D2 domain bind are distinct, or non-overlapping, or partially overlapping. According to this aspect, the D1 domain can comprise the amino acid sequence of any of the HCVR / LCVR or CDR described in Table 1 herein, and the D2 domain can comprise the amino acid sequence of any other HCVR / LCVR or CDR described 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 obtained antigen-binding protein of D1 does not compete with the obtained antigen-binding protein of D2 for binding to HER2).

[0230] In some embodiments, the human HER2 epitopes to which the anti-HER2×anti-HER2 bispecific antibody binds include amino acids 9-23 (SEQ ID NO: 57), amino acids 41-51 (SEQ ID NO: 58), amino acids 64-77 (SEQ ID NO: 59), amino acids 133-148 (SEQ ID NO: 61), amino acids 141-145 (SEQ ID NO: 155), amino acids 152-161 (SEQ ID NO: 64), amino acids 166-182 (SEQ ID NO: 56), amino acids 174-182 (SEQ ID NO: 162), amino acids 194-200 (SEQ ID NO: 163), amino acids 258-273 (SEQ ID NO: 65), and / or amino acids 353-359 (SEQ ID NO: 60) of SEQ ID NO: 54. In some embodiments, the first epitope of human HER2 includes amino acids 141-145 and / or 166-182 of SEQ ID NO: 54, and the second epitope of human HER2 includes amino acids 9-23, 41-51, 64-67, and / or 353-359 of SEQ ID NO: 54. In some embodiments, the first epitope of human HER2 includes amino acids 133-148, 174-182, and / or 194-200 of SEQ ID NO: 54, and the second epitope of human HER2 includes 152-161, 258-273, and / or 194-200 of SEQ ID NO: 54.

[0231] Exemplary bispecific antibody formats that can be used in the context of the present disclosure include the use of a first immunoglobulin (Ig) C H 3 domain and a second Ig C H 3 domain, wherein the first and second Ig C H 3 domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A, and the second Ig C H 3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (according to the IMGT exon numbering convention; H435R according to the EU numbering convention). The second C H 3 may further include the Y96F modification (according to IMGT, Y436F according to EU). The second CH Yet another modification that can be found within 3 is as follows: In the case of IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; in EU, D356E, L358M, N384S, K392N, V397M, and V422I); in the case of IgG2 antibodies, N44S, K52N, and V82I (IMGT; in EU, N384S, K392N, and V422I); and in the case of IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; in EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). It is envisioned that modifications of the bispecific antibody formats described above are also within the scope of the present disclosure.

[0232] Other exemplary bispecific formats that can be used in the context of the present disclosure include, but are not limited to, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chains (such as common light chains having knobs-into-holes), CrossMab, CrossFab, (SEED) bodies, leucine zippers, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2 Bispecific formats are included (see, for example, Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein for an overview of the formats described above). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates, which are then self-assembled into multimeric complexes having defined composition, valency, and geometric arrangement. (See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: Dec. 4, 2012].)

[0233] Therapeutic Agents and Administration Pharmaceutical compositions comprising an anti-HER2 antibody or HER2×HER2 bispecific antigen-binding molecule of the invention are provided herein, including HER2×HER2 ADCs. The pharmaceutical compositions may be formulated with suitable carriers, excipients, and other agents that provide improvements in transport, delivery, tolerability, and the like.

[0234] Therapeutic Use of Antibodies Methods are provided herein that include administering to a subject in need thereof a therapeutic composition comprising a HER2×HER2 bispecific antigen-binding molecule, including HER2×HER2 ADCs, comprising any D1 and D2 components described herein. The therapeutic composition can comprise any HER2×HER2 bispecific antigen-binding molecule disclosed herein, including HER2×HER2 ADCs, and a pharmaceutically acceptable carrier or diluent.

[0235] HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADCs, are useful for the treatment, prevention, and / or amelioration of any disease or disorder that can be treated by, inter alia, being related to or mediated by HER2 expression, signaling, or activity; or by blocking HER2 dimerization; or by otherwise inhibiting HER2 activity and / or signaling; and / or by promoting receptor internalization; and / or by decreasing the number of cell surface receptors.

[0236] For example, the HER2×HER2 bispecific antigen-binding molecules of the present disclosure, including HER2×HER2 ADC, are useful for the treatment of tumors that express (or overexpress) HER2. In some embodiments, the HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADC, bind to cells that express high levels of HER2, such as IHC3+. In some embodiments, the HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADC, bind to cells that express moderate levels of HER2, such as IHC2+. In some embodiments, the HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADC, poorly bind to cells that express low levels of HER2, such as IHC1+.

[0237] In some embodiments, the HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADC, exhibit cytotoxicity against cells with moderate or high HER2 expression but not against those with low HER2 expression. In some aspects, the cytotoxicity is less than about 5%, less than about 4%, less than about 3%, or less than about 2% of cells that express low levels of HER2.

[0238] In certain embodiments, the HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADC, are used to treat one or more of the following cancers: breast cancer, cervical cancer, gastric cancer (e.g., gastric cancer with HER2 amplification), esophageal cancer, colorectal cancer, endometrial cancer, glioblastoma, head and neck cancer (e.g., head and neck squamous cell carcinoma [HNSCC]), ovarian cancer, lung cancer (e.g., non-small cell lung cancer [NSCLC]), small cell lung cancer, acute myeloid leukemia, adult T-cell leukemia, astrocytoma, bladder cancer, cholangiocarcinoma, chronic myeloid leukemia, Kaposi sarcoma, kidney cancer, leiomyosarcoma, liver cancer, lymphoma, malignant glioma, malignant mesothelioma, melanoma, mesothelioma, MFH / fibrosarcoma, multiple myeloma, nasopharyngeal cancer, osteosarcoma, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, synovial sarcoma, thyroid cancer, and Wilms tumor.

[0239] In some embodiments, HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADCs, can be used to treat primary and / or metastatic tumors that express high or intermediate levels of HER2, i.e., tumors that occur in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tracts, muscle, bone, skin and appendages, connective tissue, spleen, immune system, hematopoietic cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye.

[0240] In the context of the treatment methods described herein, HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADCs, may be administered as a monotherapy (i.e., as the sole therapeutic agent), in combination with one or more additional therapeutic agents (examples are described elsewhere herein), or as an ADC (examples are described elsewhere herein).

[0241] Combination Therapies and Formulations Provided herein are compositions and therapeutic formulations comprising any anti-HER2 antibody and HER2×HER2 bispecific antigen-binding molecule, including HER2×HER2 ADCs, described herein, in combination with one or more additional therapeutic active ingredients, and methods of treatment comprising administering such compositions to a subject in need thereof.

[0242] Anti-HER2 antibodies and HER2×HER2 bispecific antigen-binding molecules, including HER2×HER2 ADC, may be formulated and / or administered together in combination with one or more additional therapeutic active ingredients selected from the group consisting of: another antagonist of Her2 / ErbB2 (e.g., an anti-ErbB2 [e.g., trastuzumab or T-DM1 (KADCYLA®), or trastuzumab deruxtecan (T-SXD; DNA topoisomerase 1 inhibitor)], or a small molecule inhibitor of ErbB2 activity), an antagonist of another EGFR family member such as ErbB3 or ErbB4 (e.g., an anti-ErbB3 or anti-ErbB4 antibody, or a small molecule inhibitor of ErbB3 activity or ErbB4 activity), a MET antagonist (e.g., an anti-MET antibody [e.g., onartuzumab, emibetuzumab, and H4H14639D], or a small molecule inhibitor of MET), an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of EGFRvIII (e.g., an anti-EGFRvIII antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), a PDGFR-α inhibitor (e.g., an anti-PDGFR-α antibody), a PDGFR-β inhibitor (e.g., an anti-PDGFR-β antibody, or a small molecule kinase inhibitor such as imatinib mesylate or sunitinib malate), a PDGF ligand inhibitor (e.g., an anti-PDGF-A, -B, -C, or -D antibody, aptamer, siRNA, etc.), a VEGF antagonist (e.g., a VEGF-Trap such as aflibercept, e.g., U.S. Patent No. 7,087,See No. 411 (also referred to herein as "VEGF-inhibitory fusion protein"), anti-VEGF antibody (e.g., bevacizumab), small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonist (e.g., REGN421, etc., anti-DLL4 antibody disclosed in U.S. Patent Application Publication No. 2009 / 0142354), Ang2 antagonist (e.g., H1H685P, etc., anti-Ang2 antibody disclosed in U.S. Patent Application Publication No. 2011 / 0027286), FOLH1 antagonist (e.g., anti-FOLH1 antibody), antagonist of STEAP1 or STEAP2 (e.g., anti-STEAP1 antibody or anti-STEAP2 antibody), TMPRSS2 antagonist (e.g., anti-TMPRSS2 antibody), MSLN antagonist (e.g., anti-MSLN antibody), CA9 antagonist (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), CD20×CD3 bispecific antibody, PD-1 blocker (e.g., anti-PD-1 antibody such as pembrolizumab or nivolumab), etc. Other agents that can be beneficially administered in combination with the antibodies provided herein include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, such as 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, etc., or their respective receptors.,

[0243] As an example, a PD-1 inhibitor such as an anti-PD-1 antibody can be combined with the HER2×HER2 antibody-drug conjugate described herein. The target patient population includes, in particular, patients having tumors that overexpress HER2 mutations (e.g., IHC2+ or IHC3+), such as patients having breast cancer that expresses HER2.

[0244] Compositions and therapeutic agents are provided herein that comprise any of the anti-HER2 antibodies and HER2×HER2 bispecific antigen-binding molecules described herein, including HER2×HER2 ADC, in combination with one or more chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, etc.; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, etc.; antibiotics such as actinomycin, aclacinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, calminomycin, cardinophilin, chromomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozotocin, tubercidin, ubenimex, dinostatin, zorubicin, etc.; antimetabolites such as methotrexate and 5-fluorouracil (5-FU), etc.;Folic acid analogs, such as denopterin, methotrexate, pteropterin, trimethoprim, etc.; purine analogs, such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine, etc.; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyruridine, doxifluridine, enocitabine, floxuridine, etc.; androgens, such as calusterone, drostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane, etc.; folic acid replenishers, such as folinic acid, etc.; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (trademark); razoxane; schizophyllan; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2’’-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (「Ara-C」); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (Taxol (trademark), Bristol-Myers Squibb Oncology, Princeton, New Jersey) and docetaxel (Taxotere (trademark), Aventis, Antony, France), etc.; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin, etc.; vinblastine; platinum;Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase inhibitor RFS 2000; Difluoromethylornithine (DMFO); Retinoic acid; Esperamicin; Capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included. This definition also includes antihormonal agents that act to control or inhibit the action of hormones on tumors, for example, antiestrogen agents including tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogen agents such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included.;

[0245] The HER2×HER2 bispecific antigen-binding molecule may also be administered in combination with and / or co-formulated with an antiviral agent, an antibiotic, an analgesic, a corticosteroid, a steroid, oxygen, an antioxidant, a COX inhibitor, a cardioprotective agent, a metal chelator, IFN-gamma, and / or an NSAID.;

[0246] For example, additional therapeutic active ingredients such as any of the agents or derivatives thereof listed above may be administered immediately prior to, simultaneously with, or immediately after administration of the HER2×HER2 bispecific antigen-binding molecule (for the purposes of this disclosure, such dosing regimens are considered administration of the antibody "combined" with the additional therapeutic active ingredient). The present disclosure includes pharmaceutical compositions in which the HER2×HER2 bispecific antigen-binding molecule is co-formulated with one or more additional therapeutic active ingredients described elsewhere in this specification.;

[0247] Dosing regimen According to certain embodiments, a HER2×HER2 bispecific antigen-binding molecule (or a pharmaceutical composition comprising an anti-HER2 antibody, a HER2×HER2 bispecific antigen-binding molecule, or a HER2×HER2 ADC and any additional therapeutic active agent mentioned herein) including multiple doses of a HER2×HER2 ADC may be administered to a subject over a defined period of time. A method according to this aspect includes sequentially administering multiple doses of the HER2×HER2 bispecific antigen-binding molecules provided herein to a subject. As used herein, "sequentially administering" means that each dose of the antibody is administered to the subject at different times, such as on different days separated by a predetermined interval (e.g., time, day, week, or month). The present invention includes methods that include sequentially administering a single initial dose of a HER2×HER2 bispecific antigen-binding molecule, then one or more secondary doses of a HER2×HER2 bispecific antigen-binding molecule, and then optionally one or more tertiary doses of a HER2×HER2 bispecific antigen-binding molecule to a patient.

[0248] The terms "initial dose", "secondary dose", and "tertiary dose" refer to the temporal order of administration of the HER2×HER2 bispecific antigen-binding molecule. Thus, an "initial dose" is a dose administered at the start 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 dose, secondary dose, and tertiary dose all contain the same amount of the HER2×HER2 bispecific antigen-binding molecule, but may generally differ from each other with respect to the dosing frequency. However, in certain embodiments, the amount of antibody contained in the initial dose, secondary dose, and / or tertiary dose may differ from each other during the course of treatment (e.g., increased or decreased by appropriate adjustment). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of a treatment regimen, and subsequent doses are administered at a less frequent basis (e.g., a "maintenance dose").

[0249] Diagnostic use of the antibody In addition, the HER2×HER2 bispecific antigen-binding molecule of the present disclosure can be used to detect and / or measure HER2 or HER2-expressing cells in a sample, for example, for diagnostic purposes. For example, an anti-HER2 antibody or a fragment thereof can be used to diagnose a condition or disease characterized by abnormal expression of HER2 (such as overexpression, underexpression, lack of expression, etc.). Exemplary diagnostic assays for HER2 include, for example, contacting a sample obtained from a patient with a HER2×HER2 bispecific antigen-binding molecule, which antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled HER2×HER2 bispecific antigen-binding molecule can be combined with a secondary antibody that is itself detectably labeled for use in diagnostic applications. The detectable label or reporter molecule can be 3 H, 14 C, 32 P, 35 S, or 125 a radioisotope such as I; a fluorescent moiety or chemiluminescent moiety such as fluorescein or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. Specific assay examples that can be used to detect or measure HER2 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). In some embodiments, the HER2×HER2 bispecific antigen-binding molecule is labeled as described in WO2018 / 044540, which is hereby incorporated by reference in its entirety. In some embodiments, the HER2×HER2 bispecific antigen is labeled as follows. TIFF0007695260000163.tif57133

[0250] In some embodiments, a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO: 10 is labeled on biosensor 1.

[0251] In some embodiments, a HER2×HER2 bispecific antigen-binding protein comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO: 40 is labeled on the biosensor 1.

[0252] Samples that can be used in the HER2 diagnostic assay according to the present disclosure include any tissue or body fluid sample obtained from a subject. Generally, the HER2 level in a specific sample obtained from a healthy patient (e.g., a patient not suffering from a disease or condition associated with abnormal HER2 levels or activity) is measured to first establish a baseline or standard HER2 level. The reference level of HER2 can then be compared to the HER2 level measured in a sample obtained from an individual suspected of having a HER2-related disease or condition.

Example

[0253] The following examples are presented to provide those skilled 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 regard as their invention. Efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. 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.

[0254] The comparative antibodies used in the following examples include the following: · Trastuzumab-MCC-DM1, an anti-HER2 ADC containing the DM1 payload. See, for example, WO2015 / 031396A1. This ADC is referred to herein as CompAb1-MCC-Maytansinoid A. The parent anti-HER2 antibody, Herceptin, is referred to herein as CompAb1. · DS8201A, an anti-HER2 ADC containing a DxD payload. See, for example, Ogitani et al. (Clinical Cancer Research, October 2016, 22(20): doi:10.1158 / 1078-0432.ccr-15-2822) and Tamura (Lancet 2019). This ADC is referred to herein as CompAb1-camptothecin-LP. The parental antibody is Herceptin, also referred to as CompAb1. · MEDI4276, a HER2×HER2 bispecific antibody ADC containing an AZ13599185 payload. The antibody contains SEQ ID NO: 52 (DomainIV_VL.(G4S)4 Linker_v3.DomainIV_VH.(G4S)3) and SEQ ID NO: 53 (Bs2AB_VK.hKappa). The linker payload is compound T32 shown below, which is in WO2015 / 157592 and Faria et al. (Antibodies, 2019, 8(11) doi:10.3390 / antib8010011). The parental bispecific antibody is referred to herein as CompAb2, and the ADC is referred to herein as CompAb2-tubulysin 2A-LP. · The isotype control antibody is referred to herein as IC1. The IC1 antibody can be conjugated to tubulysin 1A-LP, MCC-maytansinoid A, camptothecin, or tubulysin 2A-LP. TIFF0007695260000164.tif58128

[0255] Example 1. Generation of anti-HER2 antibodies The anti-HER2 antibody was obtained by immunizing a genetically engineered mouse containing DNA encoding the human immunoglobulin heavy chain variable region and kappa light chain variable region with hErbB2 ecto-mFc (company, catalog number, location), an immunogen containing a recombinant human HER2 extracellular domain fused to mouse Fc. The mouse used for immunization expresses a "universal light chain". That is, the antibodies produced by this mouse have different heavy chain variable regions but essentially the same light chain variable domain.

[0256] The immune response of the antibody was monitored by a HER2-specific immunoassay. When the desired immune response was obtained, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce HER2-specific antibodies. Using this technique, several anti-HER2 chimeric antibodies (i.e., antibodies possessing human variable domains and mouse constant domains) were obtained. Further, as described in U.S. Patent Application Publication No. 2007 / 0280945 A1, several fully human anti-HER2 antibodies were isolated directly from antigen-positive B cells without fusion with myeloma cells.

[0257] The specific biological properties of exemplary anti-HER2 antibodies generated according to the method of this example, and bispecific antibodies constructed from this antibody, are described in detail in the examples set forth below.

[0258] Example 2. Construction of Bispecific Antibodies Having Two Different Antigen-Binding Domains Specific for Different Epitopes of HER2 This example describes the construction of bispecific antibodies containing two different antigen-binding domains (D1 and D2). D1 and D2 are derived from different anti-HER2 antibodies and, as a result, bind to separate epitopes on the HER2 extracellular domain.

[0259] The individual anti-HER2 antigen-binding domains used to construct the bispecific antibodies of this example were derived from various bivalent monospecific anti-HER2 antibodies generated according to Example 1 herein. All HER2×HER2 bispecific antibodies described herein contain the same ("common") light chain (including the light chain variable region [LCVR] amino acid sequence of SEQ ID NO: 18, as well as the light chain CDR [LCDR1, LCDR2, and LCDR3] amino acid sequences of SEQ ID NO: 20, 22, and 24). Therefore, the antigen-binding domains (D1 and D2) of all bispecific antibodies described in this example both contain this common light chain variable region, but these bispecific antibodies differ from each other with respect to their D1 heavy chain variable region (HCVR) and heavy chain CDR (HCDR), and their D2 heavy chain variable region (HCVR) and heavy chain CDR (HCDR). The components of the bispecific antibodies of this example are summarized in Table 1.

[0260] In some embodiments, the bispecific antibody contains an N297Q modification in one or both heavy chains. Residue 297 within the heavy chain is identified according to the Kabat numbering convention. Therefore, the HC chain sequences shown in Table 3 may include the N297Q modification.

[0261] (Table 1) Amino acid sequences of HER2×HER2 bispecific antibodies TIFF0007695260000165.tif63167

[0262] (Table 2) HER2×HER2 bispecific nucleic acid sequences TIFF0007695260000166.tif62167

[0263] (Table 3) HER2×HER2 bispecific full-length heavy and light chain amino acid sequences TIFF0007695260000167.tif29128

[0264] Example 3. Surface binding of HER2×HER2 bispecific antibodies In this example, the binding ability of CompAb1 and the HER2×HER2 bispecific antibody to the cell surface of ZR751 cells (ATCC catalog number CRL-1500), JIMT1 cells (DSMZ catalog number ACC589), and MDAMB361 cells (ATCC catalog number HTB-27) was tested.

[0265] For the assay, cells were grown in a 5% CO2, 37°C, pre-treated 75 cm 2 flask (Corning, number 430641U) in the medium specified above. On the day of the assay, the cells were trypsinized and incubated with the Violet viability marker (Biolegend, number 77477) in PBS (10 minutes, 4°C, 1 mL). Subsequently, the cells were centrifuged (1500 RPM, 5 minutes, 4°C), resuspended in DMEM + 10% FBS, seeded into a U-bottom 96-well plate at 200,000 cells / well, and incubated with the indicated dilutions of the Alexa 674-labeled antibody (Thermo, catalog number A37573) (20 minutes, DMEM + 10% FBS, 4°C, 50 μL / well). Then, the cells were centrifuged and washed twice (5 minutes, DMEM + 10% FBS, 4°C, 200 μL / well). Thereafter, the cells were fixed with 1% paraformaldehyde (Electron Microcopy Sciences, number 15710) in PBS (10 minutes, 4°C, 50 μL / well), the fixative was diluted with an additional 150 μL of PBS, and the cells were subjected to flow cytometry analysis using a Fortessa X20 instrument (BD Biosciences).

[0266] As shown in Tables 4 - 6, in the three cell lines tested, both HER2×HER2 bispecific antibodies bound with greater affinity and avidity than CompAb1, while the isotype control antibody (IC1) showed little or no binding. See also Figures 1A, 1B, and 1C.

[0267] (Table 4) Cell surface binding of HER2×HER2 bispecific antibody to MDAMB361 cells TIFF0007695260000168.tif73128

[0268] (Table 5) Cell surface binding of HER2×HER2 bispecific antibody to JIMT1 cells TIFF0007695260000169.tif73128

[0269] (Table 6) Binding of HER2×HER2 bispecific antibody to ZR751 cells TIFF0007695260000170.tif73128

[0270] Example 4. Surface binding of HER2×HER2 in a panel of cell lines To test the binding ability of the HER2×HER bispecific antibody of the present invention to cell lines expressing various levels of HER2, a high-content imaging assay was performed. For the assay, 16 cancer cell lines and 4 normal primary cultures were used (see Table 7). The cell lines were classified for relative HER2 expression by Western blot analysis.

[0271] (Table 7) Cell lines and relative HER2 expression TIFF0007695260000171.tif129152

[0272] Cells were plated at 2.5×10 on a 96-well optical plate (Greiner, catalog number 655936) with a black wall coated with collagen 5Cells were seeded at a density of one cell per well in the appropriate culture medium and incubated overnight at 37 °C in 5% CO2. The next day, the cells were incubated with 10 μg / mL Alexa647-labeled (Thermo, catalog number A37573) antibody in 50 μL of DMEM at 4 °C for 30 minutes. Subsequently, the cells were washed twice with cold DMEM + 10% FBS and incubated with 4% paraformaldehyde (Electron Microscopy Sciences, catalog number 15710) + 0.075% saponin (Sigma, catalog number S4521) + 10 μg / mL Hoechst (Life Technologies, catalog number H3569) in PBS for 10 minutes at room temperature. Then, the solution was replaced with PBS.

[0273] Image Xpress Micro Images were acquired using a high-content microscope (Molecular Devices) and quantified by dividing the average intensity fluorescence of AF647 by the nuclear signal using ImageJ.

[0274] Table 8 shows that the CompAb1 and HER2×HER2 bispecific antibodies (BsAb1 and BsAb2) efficiently bound to cancer cell lines expressing moderate and high levels of HER2, but poorly bound to normal cells expressing low HER2 levels. Consistent with the experiments of Example 3, the HER2×HER2 bispecific antibodies bound more efficiently to cell lines expressing moderate HER2 levels than CompAb1. See also Figure 2.

[0275] (Table 8) Binding of HER2×HER2 bispecific antibodies to cancer cell lines expressing various levels of HER2 TIFF0007695260000172.tif116128

[0276] Example 5: HER2×HER2 Internalization and Cluster Formation This example tested the ability of the HER2×HER2 bispecific antibody to form clusters and translocate internally on T47D cells (ATCC catalog number HTB-133). For the 2D monolayer assay, T47D cells were seeded at 25,000 cells / well in complete medium on a collagen-coated 96-well optical plate (Greiner, catalog number 655936) and incubated overnight at 5% CO2, 37°C. For 3D spheroids, cells were seeded at 10,000 cells / well in a low-adhesion 96-well plate (Corning, number 4515) and incubated for 72 hours. On the day of the assay, the cells were incubated for 30 minutes with cold (4°C) medium containing 10 μg / mL of Alexa647 (Thermo, catalog number A37573) labeled antibody. The cells were then washed twice (5 minutes, 100 μL, 4°C, complete medium) and incubated in complete medium at 37°C for 60 minutes. The cells were incubated for 30 minutes with cold (4°C) medium containing 4 μg / mL of Alexa488 anti-human Fab (Jackson, number 109-547-003). The cells were then washed twice (5 minutes, 100 μL, 4°C, complete medium) and incubated for 10 minutes at room temperature with 4% paraformaldehyde (Electron Microcopy Sciences, catalog number 15710) + 0.075% saponin (Sigma, catalog number S4521) + 10 μg / ml Hoechst (LifeTech, catalog number H3569) in PBS. The solution was then replaced with PBS. Images were acquired with a Zeiss Spinning Disc Confocal Microscope and analyzed using Zeiss Zen Blue software. Quantification of the number of intracellular vesicles and surface clusters was performed on a single confocal section of a representative image.

[0277] As seen in Tables 9 and 10, the HER2×HER2 bispecific antibody forms surface clusters and translocates internally more efficiently than CompAb1.

[0278] (Table 9) 3D spheroid (SD55) TIFF0007695260000173.tif27128

[0279] (Table 10) 2D monolayer (SD53) TIFF0007695260000174.tif27128

[0280] Example 6. DM1 antibody conjugation and characterization of the conjugate Antibodies (BsAb1, BsAb2, and IC1; 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 M1 (SMCC - DM1) for 2 hours at ambient temperature. The conjugate was purified by size - exclusion chromatography or large - scale ultrafiltration and sterile - filtered. Protein concentration was determined by UV spectral analysis. Size - exclusion HPLC was used to confirm that all conjugates used were >90% monomer, and RP - HPLC was used to confirm that there was <1% of unconjugated linker - payload. All conjugate antibodies were analyzed by UV for linker - payload loading values according to Hamblett et al. (American Association for Cancer Research. 2004 Oct 15;10(20):7063 - 70). The ratio of payload to antibody is reported in Table 11.

[0281] (Table 11) Percent yield and ratio of payload to antibody for each antibody - drug conjugate TIFF0007695260000175.tif36128

[0282] Example 6A Conjugation of maytansinoid B with an antibody In this example, antibodies (BsAb1, BsAb2) were conjugated to maytansinoid B: TIFF0007695260000176.tif49128 to Form TIFF0007695260000177.tif46152, where Ab is BsAb1 or BsAB2.

[0283] Antibodies BsAb1 and BsAb2 at 10 - 20 mg / ml were conjugated with excess maytansine - 3 - N - methyl - L - alanine - N - Me - beta - alanine - carbamyl - (p - amino)benzyl - citrulline - valine - adipoyl - succinate (Compound B 1 ). The conjugate was purified by size - exclusion chromatography or large - scale diafiltration and sterile - filtered. Protein concentration was determined by UV spectral analysis. All conjugate antibodies were analyzed for linker - payload loading values by UV and / or by the mass difference between the native and conjugate forms according to Hamblett et al. (American Association for Cancer Research. 2004 Oct 15;10(20):7063 - 70).

[0284] Compound B1 was synthesized according to the method described for "Compound 1" in US Patent Application Publication No. 2018 / 0134794 A1 (US Patent Application No. 15 / 814,095), which is hereby incorporated by reference in its entirety. TIFF0007695260000178.tif58156

[0285] Example 7. Site - specific conjugation of HER2×HER2 bispecific antibodies with tubulysin 1A and Campt - 1 In this example, bispecific antibodies BsAb1 and BsAb2 were site - specifically conjugated to two or more LPs of the following structure: TIFF0007695260000179.tif81165 This structure contains the payload tubulysin 1A. Further, bispecific antibodies BsAb1 and BsAb2 were site - specifically conjugated as follows. TIFF0007695260000180.tif50165

[0286] Site-specific conjugates of cyclooctyne-spacer-payload to wild-type antibodies or their antigen-binding fragments were produced in three steps. The first step was the deglycosylation of the wild-type antibody. The second step was the enzymatic addition of a small molecule, such as azido-PEG3-amine, to the Q295 site of the deglycosylated antibody using a microbial transglutaminase (MTG)-based enzyme (hereinafter, "MTG-based" conjugation). In the third step, [2+3] cycloaddition, for example, the addition of cyclooctyne-spacer-payload to the azido-functionalized antibody via 1,3-dipolar cycloaddition between azide and cyclooctyne (also known as copper-free click chemistry) was employed. Baskin, J.M.; Prescher, J.A.; Laughlin, S.T.; Agard, N.J.; Chang, P.V.; Miller, I.A.; Lo, A.; Codelli, J.A.; Bertozzi, C.R. PNAS 2007, 104 (43), 16793-7. See Figure 3A for an example of a linker-spacer-payload having a DIBAC moiety conjugated to an azido-functionalized antibody via [2+3] cycloaddition. This process provides site-specific and stoichiometric conjugates with an isolated yield of approximately 50-80%. Figure 3B is an example of a three-step site-specific conjugation performed as follows.

[0287] Step 1: Preparation of deglycosylated antibody. Deglycosylation was performed to expose the conjugation site. An anti-HER2 human IgG4 bispecific antibody (40 mg, 27 mg / mL in PBS, pH 5.5-8.0) was mixed with PNGase F enzyme (New England BioLabs, 500,000 U / mL, 2 uL of enzyme per mg of antibody, 80 uL total). The reaction mixture was incubated overnight at 37 °C with gentle stirring. Deglycosylation was monitored by ESI-MS. At the end of the reaction, the reaction mixture was used directly in the next step.

[0288] Step 2: Preparation of azido-functionalized antibody. 1.5 mL of deglycosylated HER2×HER2 bispecific antibody (40 mg) in PBS (pH 7.2) was incubated with 200 molar equivalents of azido-PEG3-amine (MW = 218.26 g / mol) in the presence of MTG (ACTIVA TI, Ajinomoto Co., Japan) (0.06 mg of MTG / mg of antibody). The reaction mixture was incubated at 37 °C for 4 h and then at 25 °C overnight with gentle mixing. The reaction was monitored by ESI-MS. At the end of the reaction, excess azido-PEG3-amine and MTG were removed by SEC (Superdex 200 PG, GE Healthcare) to generate azido-functionalized antibody. Azido-PEG3-amine was added to two Q295 sites on the antibody, resulting in a 404 Da increase in the antibody-PEG3-azide conjugate of 2DAR.

[0289] This process can also be carried out for antibodies having an N297Q modification in one or both heavy chains. In this example, azido-PEG3-amine was added to two Q295 sites and at least one 297Q site on the antibody, resulting in a 3DAR or 4DAR antibody-PEG3-azide conjugate.

[0290] Step 3: Preparation of site-specific conjugate by [2+3] click reaction between azido-functionalized glutaminyl-modified antibody and cyclooctyne containing linker payload (LP).

[0291] Generally, the azido-functionalized glutamyl-modified antibody was dissolved in ≥6 molar equivalents of LP, such as a compound of the following structure, dissolved in a suitable organic solvent (e.g., DMSO, DMF or DMA; the reaction mixture contains 10-20% organic solvent v / v): The azide-functionalized antibody-LP conjugate was prepared by incubating with TIFF0007695260000181.tif76166 at 25 °C to 37 °C for 3 to 24 hours. The progress of the reaction was monitored by ESI-MS. The absence of azide-functionalized antibody (mAb-PEG3-N3) indicated the completion of conjugation. Excess linker-payload (LP) and organic solvents were removed by desalting column or size exclusion chromatography (SEC). The purified conjugate was analyzed by SEC-HPLC and ESI-MS. The monomer purity of the conjugate was >99% by SEC-HPLC analysis.

[0292] As a specific example, 2.4 mL of azide-functionalized HER2×HER2 antibody, such as BsAb1, BsAb2 (31 mg) in PBS, was treated with 6 equivalents of tubulysin 1A-LP (concentration 10 mg / mL in DMA) at 30 °C overnight. Excess linker payload (LP) was removed by SEC (Superdex 200 PG, GE Healthcare). The final product was characterized by UV, SEC-HPLC (see Figure 4), and ESI-MS.

[0293] In a similar manner, BsAb1 and BsAb2, both azide-functionalized at Q295 with the described bisazidoalkyl-substituted amine (bisSP1), were treated with CAMPT-1-LP having the following structure to obtain BsAb1-Campt1 and BsAb2-Campt1 shown below. TIFF0007695260000182.tif154170

[0294] CAMPT-1-LP (i.e., LP1) was synthesized as described in Scheme 1 and Examples i-iii below. The starting material L1-1 (CAS 2226472-26-8) was synthesized according to WO2018089373A2, which is hereby incorporated by reference in its entirety. Scheme 1. Synthesis of vcPAB-carbamate linker-payload CAMPT-1-LP TIFF0007695260000183.tif112165

[0295] Example i: N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl]-1-[2-(cyclooct-2-en-1-yloxy)acetamide]-3,6,9,12-tetraoxapentadecan-15-amide (L1-2) TIFF0007695260000184.tif23128

[0296] To a solution of compound L1-1 (0.17 g, 0.33 mmol) in DMF (10 mL) were successively added DIPEA (0.13 g, 1.0 mmol) and vcPAB (0.13 g, 0.34 mmol), and the reaction mixture was stirred at room temperature for 1 hour. The completion of the reaction was monitored by LCMS. The resulting mixture was directly purified by reverse-phase flash chromatography (0 - 80% acetonitrile in water) to give compound L1-2 (0.18 g, 70% yield) as a colorless oil. ESI m / z: 791.3 (M+H) + 。 1 H NMR (400 MHz, DMSO d6)δ 9.91(s,1H),8.11(d,J=8.4Hz,1H),7.89(d,J=8.8Hz,1H),7.61(t,J=5.6Hz,1H),7.55(d,J=8.4Hz,2H),7.23(d,J=8.4Hz,2H),5.98(t,J=5.6Hz,1H),5.42(s,2H),5.10(br s,1H),),4.43(s,2H),4.39-4.37(m,1H),4.30-4.21(m,2H),3.87(d,J=14.8Hz,1H),3.75(d,J=14.8Hz,1H),3.62-3.58(m,2H),3.50-3.46(m,12H),3.43(t,J=6.0Hz,2H),3.27-3.22(m,2H),3.06-2.92(m,2H),2.41-2.32(m,2H),2.26-2.05(m,3H),1.99-1.66(m,6H),1.62-1.55(m,3H),1.44-1.35(m,3H),0.89(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H)ppm。

[0297] Example ii: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-en-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl 4-nitrophenyl carbonate (L1-3) TIFF0007695260000185.tif28139

[0298] A suspension of compound L1-2 (80 mg, 0.10 mmol), DMAP (12 mg, 0.10 mmol), and DIPEA (26 mg, 0.20 mmol) in dry DMF (5 mL) was stirred at room temperature for 10 minutes, then bis(4-nitrophenyl) carbonate (61 mg, 0.20 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LCMS. The resulting mixture was purified directly by reverse-phase flash chromatography (0 - 80% acetonitrile in water) to give compound L1-3 (53 mg, 55% yield) as a white solid. ESI m / z: 956.3 (M+H)+ .

[0299] Example iii: {4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{1-[2-(cyclooct-2-en-1-yloxy)acetamido]-3,6,9,12-tetraoxapentadecan-15-amido}-3-methylbutanamido]pentanamido]phenyl}methyl N-({[({[(10S,23S)-10-ethyl-18-fluoro-10-hydroxy-19-methyl-5,9-dioxo-8-oxa-4,15-diazahexacyclo[14.7.1.0 2 , 14 .0 4 , 13 .0 6 , 11 .0 20 , 24 tetracosa-1,6(11),12,14,16,18,20(24)-heptaene-23-yl]carbamoyl}methoxy)methyl]carbamoyl}methyl)carbamate (LP1 / CAMPT-1-LP) TIFF0007695260000186.tif36163

[0300] To a yellow solution of compound L1-3 (16 mg, 17 μmol) and exatecan mesylate (12 mg, 17 μmol) in dry DMF (2 mL) was added DIPEA (6.5 mg, 51 μmol), and the clear reaction solution was stirred at room temperature for 2 hours. The completion of the reaction was monitored by LCMS. The resulting mixture was purified directly by reverse-phase flash chromatography (0 - 60% acetonitrile in aqueous TFA (0.01%)) to give the linker payload LP1 (15 mg, 63% yield as the TFA salt) as a white solid. ESI m / z: 698.8 (M / 2 + H) + . 1 1H NMR (400 MHz, DMSO d6)δ 9.99(s,1H),8.80(t,J = 6.8Hz,1H),8.50(d,J = 9.2Hz,1H),8.12(d,J = 7.2Hz,1H),7.87(d,J = 8.8Hz,1H),7.79(d,J = 10.8Hz,1H),7.62 - 7.58(m,3H),7.42(t,J = 6.0Hz,1H),7.31(s,1H),7.28(d,J = 8.4Hz,2H),6.53(br s,1H),5.98(t,J = 5.2Hz,1H),5.63 - 5.57(m,1H),5.46 - 5.37(m,3H),5.21(s,2H),4.93(s,2H),4.63(d,J = 6.4Hz,2H),4.41 - 4.35(m,1H),4.29 - 4.21(m,2H),4.02(s,2H),3.87(d,J = 14.4Hz,1H),3.75(d,J = 14.8Hz,1H),3.63 - 3.58(m,4H),3.50 - 3.48(m,12H),3.46 - 3.41(m,2H),3.27 - 3.24(m,2H),3.23 - 3.12(m,2H),3.07 - 2.91(m,2H),2.47 - 2.45(m,0.5H),2.41 - 2.33(m,4.5H),2.25 - 2.04(m,5H),1.99 - 1.69(m,9H),1.63 - 1.54(m,3H),1.44 - 1.33(m,3H),0.88 - 0.82(m,9H) ppm. (Protons of TFA were not observed). 19 19F NMR (376 MHz, DMSO d6 )δ - 74 (TFA), - 111 (Ar - F) ppm.

[0301] Table 12 is a list of the DAR (ESI - MS) values of the synthesized antibody tubulysin and camptothecin conjugate (ADC).

[0302] Characterization of antibodies and ADCs by SEC - HPLC and LC - ESI - MS The purified conjugate was analyzed by SEC - HPLC and ESI - MS using representative SEC and ESI - MS.

[0303] The SEC experiment was performed using a Waters 1515 instrument at a flow rate of 0.80 mL / min with PBS pH 7.2 on a Superdex™ 200 Increase (1.0×30 cm) column and monitored at λ = 280 nm using a Waters 2998 PDA. The analytical sample was composed of 30 - 80 μL of the test sample. The SEC results in Figure 4 show the typical retention times of monomeric mAb and its conjugate with minimal aggregation or degradation.

[0304] The intact mass of the ADC sample was measured by LC - ESI - MS to determine the drug - payload distribution profile and calculate the average DAR. Each test sample (20 - 50 ng, 5 μL) was reduced with DTT and then loaded onto an Acquity UPLC Protein BEH C4 column (10 Kpsi, 300 Å, 1.7 μm, 75 μm×100 mm, catalog number 186003810). After desalting for 3 minutes, the protein was eluted and the mass spectrum was acquired by a Waters Synapt G2 - Si mass spectrometer. As summarized in Table 12, most site - specific ADCs have an approximate DAR of 2.

[0305] (Table 12) Antibody - Drug Conjugate DAR TIFF0007695260000187.tif66166

[0306] The ADCs generated in this experiment were used in the following examples.

[0307] Example 8. Binding Affinity and Kinetic Constants Obtained from Surface Plasmon Resonance of HER2×HER2 Human Bispecific Monoclonal Antibody and Conjugate HER2×HER2 Human Bispecific Monoclonal Antibody Equilibrium Dissociation Constant (K DThe value) was determined using a real-time surface plasmon resonance biosensor assay with a MASS-2 instrument. The MASS-2 sensor surface was derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (REGN2567), and the anti-HER2×HER2 ADC and the non-modified parental antibody expressed with a human constant region were captured. The Biacore binding assay was performed in 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 hErbB2 (hErbB2-MMH) expressing a C-terminal myc-myc-hexahistidine tag was prepared in-house. Different concentrations (3-fold dilutions) of hErbB2-MMH (in the range of 90 nM to 1.1 nM) prepared in HBS-EP running buffer were injected at a flow rate of 30 μL / min onto the anti-HER2×HER2 ADC or the antibody capture surface. The binding of hErbB2-MMH to each of the captured ADC and the monoclonal antibody was monitored for 3 minutes. Subsequently, the dissociation of hErbB2-MMH was monitored for 10 minutes in HBS-EP running buffer. The anti-human Fc surface was regenerated by a short injection of 20 mM H3PO4. All binding kinetics experiments were performed at 25°C.

[0308] Using Scrubber2.0c curve fitting software, the real-time sensorgram was fitted to a 1:1 binding model to determine the kinetic association rate constant (k a ) and the dissociation rate constant (k d ). All sensorgrams were double-referenced by subtracting the signal of the buffer injection sensorgram from the corresponding analyte sensorgram, thereby removing artifacts caused by the dissociation of the antibody from the capture surface. The binding dissociation equilibrium constant (K D ) and the dissociation half-life (t 1 / 2 ) were calculated from the kinetic rate constants as follows: K D (M) = k d / k a , and t 1 / 2 (min) = ln2 / (60 × k d)

[0309] (Table 13) Binding affinity of bispecific anti-HER2 mAb and ADC at 25 °C measured by Biacore TIFF0007695260000188.tif122161

[0310] As shown in Table 13, the bispecific HER2×HER2 antibodies described herein showed a T 1 / 2 value exceeding 1155 minutes at maximum.

[0311] Example 9: Processing of cathepsin B-cleavable linker The ability of CompAb1 and BsAb2 to induce cleavage of the cathepsin B-cleavable linker on T47D cells (ATCC catalog number HTB-133) was tested. T47D cells were seeded at 25,000 cells / well on collagen-coated 96-well optical plates (Greiner, catalog number 655936) in complete medium (RPMI1640 + 10% FBS + 5 ml penicillin / streptomycin / glutamine + 1 mM NaPyr + 10 mM Hepes + 10 μg / ml insulin) and incubated overnight at 5% CO2, 37 °C. The next day, the cells were incubated for 30 minutes in cold (4 °C) medium containing 10 μg / mL fluorescent antibody (antibody labeled by Biosensor 1, see WO2018 / 044540 incorporated herein by reference). The cells were then washed twice (5 minutes, 100 μl, 4 °C, complete medium), recording medium (DPBS + 2% FBS + 10 mM HEPES) was added, and confocal live imaging was started immediately thereafter. Images were acquired with a Zeiss Spinning Disc Confocal Microscope and analyzed using Zeiss Zen Blue software. Quantification of the integrated fluorescence of Alexa 568 (cleaved biosensor) was performed in a single confocal section of 36 different fields.

[0312] The biosensor emits an AF647 signal when intact. When the biosensor-conjugated antibody translocates internally into the endo / lysosome and the cathepsin B linker is cleaved, signals for both AF647 and AF568 are emitted. Table 14 shows that the HER2×HER2 bispecific antibody induces processing of the cathepsin B-cleavable biosensor more efficiently than CompAb1. See also Figure 5.

[0313] (Table 14) Fluorescence of cleaved biosensor in CompAb1 compared to BsAb2 TIFF0007695260000189.tif148159

[0314] Example 10. In Vitro Cytotoxicity by HER2×HER2 Conjugate in HER2+ Cell Lines To test the ability of CompAb1 and BsAb1 conjugated to either maytansinoid A or tubulysin A1-LP payload to kill bioassay cells, an in vitro cytotoxicity assay was performed. The assay was performed on cells with various levels of HER2 expression treated for 6 days at reduced ADC concentrations. Cell viability was measured after treatment using Cell Titer Glow (Promega, number G7571). For the assay, cells were grown overnight in their respective media at 5% CO2, 37°C. The next day, either CompAb1 conjugated to either MCC-maytansinoid A or tubulysin A1-LP payload, the HER2×HER2 bispecific antibody, or a control antibody was added to the cells at final concentrations ranging from 66.67 nM to 0.01 nM in DMEM and 10% FBS and incubated for 6 days. After incubation, 100 uL of Cell Titer Glo was added to each well, incubated at room temperature for 5 minutes, and shaken at 500 RPM. The luminescence signal proportional to the amount of APT in each well was measured using an Envision 2105 multi-node plate reader (Perkin Elmer). IC 50Values were determined from a two-phase decay equation over a 10-point response curve (GraphPad Prism). All IC 50 values are expressed in nM concentration.

[0315] Table 15 shows that in cells expressing moderate HER2 levels (ZR751 and JIMT1), the effect of CompAb1-MCC-maytansinoid A was not different from that of the control ADC (IC 50 values of 30 - 40 nM), BsAb1-MCC-maytansinoid A showed moderate efficacy (IC 50 values of 6 - 7 nM), CompAb1-tubulysin 1A-LP showed better efficacy (IC 50 values of 0.15 - 0.2 nM), and BsAb1-tubulysin 1A-LP showed the highest cytotoxicity among all tested ADCs (IC 50 values of 0.06 - 0.07 nM). In cells expressing higher HER2 levels (MDAMB361, MDAMB453, and SKBR3), CompAb1-MCC-maytansinoid A induced efficient cytotoxicity (IC 50 values of 0.04 - 0.5 nM), BsAb1-MCC-maytansinoid A showed higher efficacy (IC 50 values of 0.02 - 0.1 nM), CompAb1-tubulysin 1A-LP showed even higher efficacy (IC 50 values of 0.006 - 0.008 nM), and BsAb1-tubulysin 1A-LP showed the highest cytotoxicity among all tested ADCs (IC 50 values of 0.002 - 0.004 nM).

[0316] (Table 15) Efficacy of ADCs against cells expressing different HER2 levels TIFF0007695260000190.tif66162

[0317] Example 11. In vitro cytotoxicity in HER2 low-expressing cell lines To test the killing effect of BsAb1 tubulysin 1A-LP, BsAb2 tubulysin 1A-LP, and the comparator from Medimmune, HER2×HER2-tubulysin (CompAb2-tubulysin 2A-LP), on bioassay cells, an in vitro cytotoxicity assay was performed. The assay was carried out on normal primary cultures and breast cancer cells expressing low HER2 levels. The cells were treated with decreasing concentrations of the ADC for 6 days, and cell viability was measured after treatment using Cell Titer Glow (Promega, number G7571).

[0318] For the assay, the cells were grown overnight in their respective media at 5% CO2, 37°C. The next day, either BsAb1-tubulysin 1A-LP, BsAb2-tubulysin 1A-LP, CompAb2-tubulysin 2A-LP, or their respective unbound control antibodies were added to the cells at final concentrations ranging from 66.67 nM to 0.01 nM in DMEM and 10% FBS and incubated for 6 days. After incubation, 100 uL of Cell Titer Glow was added to each well, incubated at room temperature for 5 minutes, and shaken at 500 RPM. The luminescence signal proportional to the amount of APT in each well was measured using an Envision 2105 multi-node plate reader (Perkin Elmer). IC 50 values were determined from a two-phase decay equation over a 10-point response curve (GraphPad Prism). All IC 50 values are represented in nM concentration.

[0319] Table 16 shows that BsAb1-tubulysin 1A-LP and BsAb2-tubulysin 1A-LP had little or no killing effect in cells expressing low HER2 levels (IC 50 values 8 -> 67 nM). This surprising benefit demonstrates the utility of the ADC in killing tumor cells without targeting normal tissues. In contrast, CompAb2-tubulysin 2A-LP, an ADC reported to have antitumor effects in HER2 IHC 0+ xenografts and toxicity in humans, had a low IC of 0.3 nM50 Induced cytotoxicity.

[0320] (Table 16) Cytotoxicity of ADC against HER2 low-expressing cell lines TIFF0007695260000191.tif48153

[0321] Example 12. Dose-dependence of HER2×HER2 bispecific antibody ADC in JIMT1 xenografts To test the efficacy of BsAb1-trastuzumab 1A-LP in HER2 IHC2+ JIMT1 xenograft models, in vivo tumor assays were performed. For the assay, 6-week-old female SCID mice (C.B-Igh-1b / IcrTac-Prkdcscid, Taconic Biosciences, n = 50) were used. For transplantation, JIMT1 (DSMZ catalog number ACC589) cells were mixed with Matrigel (Corning, catalog number 354234), and 150 μL of a suspension of cells and Matrigel containing 4×10 6 cells were injected subcutaneously into SCID mice. Eleven days later, the indicated ADCs were injected subcutaneously at the indicated doses into SCID mice randomized based on tumor size. The size of the xenograft in each mouse was measured using Caliper (Roboz, catalog number RS6466).

[0322] The average tumor size per treatment group at each measured time point is shown in Table 17. The HER2×HER2 bispecific ADC, BsAb1-trastuzumab 1A-LP (DAR 2.1), showed antitumor cytotoxicity in a dose-dependent manner. In mice receiving a single dose of 3 mg / kg of the HER2×HER2 bispecific ADC BsAb1-trastuzumab 1A-LP, the size of the JIMT1 xenograft significantly and continuously decreased to an average size of 10 mm 3 (day 123). In mice receiving a single dose of 1 mg / kg of the HER2×HER2 bispecific ADC, BsAb1-trastuzumab 1A-LP, the size of the JIMT1 xenograft decreased to an average size of 74 mm 3It decreased on the (28th day). In contrast, in mice that received 3, 1, or 0.3 mg / kg of isotype control ADC (IC1-trastuzumab 1A-LP; DAR2) or saline, there was little or no anti-tumor effect. See also FIGS. 6A and 6B.

[0323] (Table 17) JIMT1 xenograft tumor size (mm 3 ) TIFF0007695260000192.tif168170

[0324] Example 13. In Vivo Efficacy of HER2×HER2 Bispecific ADC Compared with CompAb1-MCC-Maytansinoid A To compare the efficacy of BsAb1-trastuzumab 1A-LP, BsAb2-trastuzumab 1A-LP, and CompAb1-MCC-maytansinoid A in a HER2 IHC2+ JIMT1 xenograft model, an in vivo tumor assay was performed. For the assay, 6-week-old female SCID mice (C.B-Igh-1b / IcrTac-Prkdcscid, Taconic Biosciences, n = 50) were used. For transplantation, JIMT1 (DSMZ, ACC589) cells were mixed with Matrigel (Corning, catalog number 354234), and 150 uL of a suspension of cells and Matrigel containing 4×10 6 cells were injected subcutaneously into SCID mice. At 14 or 28 days later, the indicated ADCs were injected subcutaneously into SCID mice randomized based on tumor size. The size of the xenograft in each mouse was measured using a caliper (Roboz, catalog number RS6466).

[0325] The average tumor size per treatment group at each measured time point is shown in Table 18. In mice that received a single dose of 3 mg / kg of BsAb1-trastuzumab 1A-LP or BsAb2-trastuzumab 1A-LP, the size of the JIMT1 xenograft was 18 and 6 mm in average size, respectively 3(On days 83 and 97, respectively), it decreased significantly and continuously. In contrast, in mice that received two doses of 10 mg / kg of CompAb1-MCC-Maytansinoid A, there was little or no antitumor effect, similar to mice that received a single dose of 3 mg / kg of IC1-Tubulysin 1A-LP or two doses of 10 mg / kg of IC1-MCC-Maytansinoid A. See also Figures 7A and 7B.

[0326] (Table 18) JIMT1 xenograft tumor size (mm 3 ) TIFF0007695260000193.tif195170

[0327] Example 14. Efficacy of BsAb1-Tubulysin 1A-LP Compared to CompAb1-MCC-Maytansinoid A in MDAMB361 Xenografts To compare the efficacy of BsAb1-Tubulysin 1A-LP and CompAb1-MCC-Maytansinoid A in a HER2 IHC2+ MDAMB361 xenograft model, an in vivo tumor assay was performed. For the assay, 6-week-old female SCID mice (C.B-Igh-1b / IcrTac-Prkdcscid, Taconic Biosciences, n = 50) were used. One day before cell transplantation, 17β-estradiol pellets (0.72 mg, 60-day release, Innovative research of America, number SE-121) were subcutaneously implanted into the mice. The next day, MDAMB361 cells (DSMZ, ACC589) were mixed with Matrigel (Corning, catalog number 354234), and 150 μL of a suspension of cells and Matrigel containing 6 × 10 6 cells were injected into the SCID mice. Nineteen days later, the indicated ADCs were subcutaneously injected at the indicated doses into the SCID mice randomized based on tumor size. A second dose was administered to the BsAb1-Tubulysin 1A-LP treatment cohort on day 52 to evaluate the development of drug resistance. The size of the xenograft in each mouse was measured using a Caliper (Roboz, catalog number RS6466).

[0328] For each time point measured, the average tumor size per treatment group is shown in Table 19. In mice receiving a single dose of 3 mg / kg of BsAb1-trastuzumab 1A-LP (DAR 2.1), the size of the MDA-MB 361 xenograft decreased from an average size of 202 mm 3 to an average size of 122 mm 3 partially by day 38. A second dose administered on day 52 caused a further regression of the xenograft tumor size to 70 mm 3 by day 76. In contrast, in mice receiving a single dose of 3 mg / kg of CompAb1-MCC-maytansinoid A (DAR 3.1) or control ADC (IC1-MCC-maytansinoid A, DAR 3.6, or IC1-trastuzumab 1A-LP, DAR 2) or saline, there was little or no anti-tumor effect. See also FIGS. 8A and 8B.

[0329] (Table 19) MDA-MB 361 xenograft tumor size (mm 3 ) TIFF0007695260000194.tif190149

[0330] Example 15. Efficacy of BsAb1-trastuzumab 1A-LP compared to CompAb1-MCC-maytansinoid A in N87 xenografts To compare the efficacy of BsAb1-trastuzumab 1A-LP and CompAb1-MCC-maytansinoid A in a HER2 IHC3+ N87 xenograft model, an in vivo tumor assay was performed. For the assay, 6-week-old female SCID mice (CBySmn.CB17-Prkdcscid / J, Jackson Labs, number 001803, n = 50) were used. For transplantation, N87 (ATCC, HTB-5822) cells were mixed with Matrigel (Corning, catalog number 354234) and 3.6×10 6A 150 μL suspension of cells and Matrigel containing the cells was injected into SCID mice. Twelve days later, the indicated ADCs were subcutaneously injected at the indicated doses into SCID mice randomized based on tumor size. The cohort treated with 1 mg / kg of BsAb1-tubricin 1A-LP was administered two additional doses on days 25 and 39. The size of the xenograft in each mouse was measured using a caliper (Roboz, catalog number RS6466).

[0331] Table 20 shows the average tumor size per treatment group at each measured time point. In mice receiving a single dose of 3 mg / kg of BsAb1-tubricin 1A-LP (DAR 2.1), the N87 xenograft size significantly and continuously decreased to an average size of 25 mm 3 (day 57). In mice receiving a single administration of 10 mg / kg of CompAb1-MCC-maytansinoid A (DAR 3.1), the size of the N87 xenograft significantly decreased to an average size of 15 mm 3 but the tumor escaped (i.e., became resistant to treatment) earlier (day 51 vs. day 88) than in mice treated with BsAb1-tubricin 1A-LP. In mice receiving three administrations of 1 mg / kg of BsAb1-tubricin 1A-LP, the size of the N87 xenograft partially decreased to an average size of 109 mm 3 (day 35). In contrast, in mice receiving a single administration of the control ADCs (3 mg / kg of IC1-tubricin 1A-LP, DAR 2, or 10 mg / kg of IC1-MCC-maytansinoid A, DAR 3.6), there was no anti-tumor effect. See also Figure 9.

[0332] (Table 20) N87 xenograft tumor size (mm 3 ) TIFF0007695260000195.tif193144

[0333] Example 16. In Vivo Comparison of Three ADCs in JIMT1 Xenografts To compare the efficacy of BsAb1-trastuzumab 1A-LP with CompAb1-MCC-maytansinoid A (DAR 3.1) and CompAb1-L-camptothecin (DAR 8) in a HER2 IHC2+ JIMT1 xenograft model, an in vivo tumor assay was performed. For the assay, 6-week-old female SCID mice (CBySmn.CB17-Prkdcscid / J, Jackson Labs, number 001803, n = 50) were used. For transplantation, JIMT1 (DSMZ, ACC589) cells were mixed with Matrigel (Corning, catalog number 354234), and 150 uL of a suspension of cells and Matrigel containing 4 × 10 6 cells were injected into SCID mice. On days 13, 20, and 27, the indicated ADCs were subcutaneously injected at the indicated doses into SCID mice randomized based on tumor size. One cohort received a single dose of 3 mg / kg of BsAb1-trastuzumab 1A-LP on day 13. The size of the xenograft in each mouse was measured using a caliper (Roboz, catalog number RS6466). The mean tumor size per treatment group at each measured time point is shown in Table 21.

[0334] In mice that received a single dose of 3 mg / kg of BsAb1-trastuzumab 1A-LP (DAR 2.1), the size of the JIMT1 xenograft significantly and continuously decreased to an average size of 3 mm 3 (day 79). In mice that received three weekly doses of 1 mg / kg of BsAb1-trastuzumab 1A-LP, the size of the JIMT1 xenograft partially decreased from an average size of 179 mm 3 to 63 mm 3 by day 44. In contrast, in mice that received three weekly doses of 10 mg / kg of CompAb1-L-camptothecin, tumor progression slowed and the average size was 374 mm 3had only reached. In contrast, in mice that received weekly doses of 10 mg / kg of CompAb1-MCC-maytansinoid A or control ADCs (3 mg / kg of IC1-tubulysin 1A-LP, 10 mg / kg of IC1-L-camptothecin, or 10 mg / kg of IC1-MCC-maytansinoid A), there was no anti-tumor effect. See also FIGS. 10A and 10B.

[0335] (Table 21) JIMT1 xenograft tumor size (mm 3 ) TIFF0007695260000196.tif128170

[0336] Example 17. Efficacy of HER2×HER2-M830 bispecific antibody drug conjugate compared to CompAb1-MCC-maytansinoid A in JIMT1 xenografts To compare the efficacy of BsAb1-tubulysin 1A-LP and CompAb1-MCC-maytansinoid A (DAR 3.1) in HER2 IHC2+ CTG0807, an in vivo tumor assay was performed. For the assay, 6- to 8-week-old female athymic nude Fox1nu mice (Envifo, Indianapolis, Indiana) were used.

[0337] Pre-test animals: When a sufficient number of stock animals reached 1.0 - 1.5 cm 3 tumors were harvested for re-implantation into pre-test animals. For pre-test animals, tumor pieces harvested from stock animals were implanted unilaterally in the left flank. Test animals: The pre-test tumor volume was recorded for each experiment starting 7 - 10 days after implantation. When the average tumor volume of the tumors reached 150 - 300 mm 3 the animals were matched by tumor volume into treatment or control groups for use in dosing and dosing was initiated on day 0. Tumor volume was measured twice a week.

[0338] The matched test animals were injected intravenously at the indicated doses. The average tumor size per treatment group at each measured time point is shown in Table 22.

[0339] In mice that received three weekly doses of 3 mg / kg of BsAb1-trastuzumab 1A-LP (DAR 2.1), the CTG0807 PDX tumor size decreased completely (0 mm 3 ) and continuously. In mice that received three weekly doses of 1 mg / kg of BsAb1-trastuzumab 1A-LP, tumor growth was delayed and the tumor size remained below 300 mm 3 for 30 days. In mice that received three weekly doses of 10 mg / kg of CompAb1-MCC-maytansinoid A (DAR 3.1), growth continued during and after treatment, but at a slightly slower rate than tumors treated with three weekly doses of PBS or ADC controls (10 mg / kg of IC1-MCC-maytansinoid A or 3 mg / kg of IC1-trastuzumab 1A-LP). See also FIGS. 11A and 11B.

[0340] (Table 22) CTG0807 PDX tumor size (mm 3 ) TIFF0007695260000197.tif49170TIFF0007695260000198.tif219170TIFF0007695260000199.tif47170

[0341] Example 18. Efficacy of BsAb1-trastuzumab 1A-LP compared to CompAb1-MCC-maytansinoid A in JIMT1 xenografts To compare the efficacy of BsAb1-trastuzumab 1A-LP and CompAb1-MCC-maytansinoid A in HER2 IHC2+ CTG1184, an in vivo tumor assay was performed. For the assay, 6- to 8-week-old female athymic nude Fox1nu mice (Envifo, Indianapolis, Indiana) were used.

[0342] Animals before the test: A sufficient number of stock animals were 1.0 - 1.5 cm 3When the [volume] reached [a certain value], the tumor was harvested for re-implantation into pre-test animals. For pre-test animals, tumor pieces harvested from stock animals were implanted unilaterally in the left flank. Test animals: The pre-test tumor volume was recorded for each experiment starting 7 - 10 days after implantation. When the average tumor volume of the tumor reached 150 - 300 mm 3 When the [volume] reached [a certain value], the animals were matched according to tumor volume into treatment or control groups for dosing and dosing was initiated on day 0. Tumor volume was measured twice a week.

[0343] The matched test animals were injected intravenously at the indicated doses. The average tumor size per treatment group at each measured time point is shown in Table 23.

[0344] In mice receiving three weekly doses of 3 mg / kg and 1 mg / kg of BsAb1-trastuzumab 1A-LP (DAR 2.1), the CTG1184 PDX tumor sizes decreased significantly and continuously to mean values of 6 (day 52) and 13 mm 3 (day 45), respectively. In mice receiving three weekly doses of 3 mg / kg of IC1-trastuzumab 1A-LP (control ADC) or 10 mg / kg of CompAb1-MCC-maytansinoid A (DAR 3.1), growth continued during and after treatment, but at a slower rate than tumors treated with three weekly doses of PBS or 10 mg / kg of IC1-MCC-maytansinoid A (control ADC). See also Figures 12A and 12B.

[0345] (Table 23) CTG1184 PDX tumor size after the indicated ADC treatment. TIFF0007695260000200.tif222170

[0346] Example 19. Immunohistochemical (IHC) staining to evaluate the level of HER2 in tumor cell lines and patient-derived xenografts. The PDX models used in Example 17 and 1 were confirmed to be HER IHC 2+ / 3+ for CTG1184 according to the following method. Tissue samples were fixed in 10% neutral buffered formalin for 12 hours at 4°C in the dark, washed three times with PBS, dehydrated in a series of ethanol gradients (once each at 70%, 80%, 90%, and four times at 100%, each for 30 minutes), processed in xylene and paraffin using Tissue-Tek® (three times, each for 30 minutes), and then embedded to generate 4-μm tissue sections. IHC staining was performed using the protocol attached to the HercepTest™ kit. The HER2 IHC score was determined by visual comparison of the tissue sections with control sections.

[0347] (Table 24) IHC scores of cell lines and patient-derived xenografts TIFF0007695260000201.tif53128

[0348] In summary, the inventors observed that the efficient internalization of the HER2×HER2 antibody dramatically increased the processing of the cathepsin B-cleavable linker in intracellular vesicles. Accordingly, HER2×HER2-tubulysin killed cell lines expressing not only high HER2 levels but also moderate HER2 levels at sub-nanomolar IC 50 values. Furthermore, HER2×HER2-tubulysin induced complete and sustained tumor regression in a number of HER2 IHC 3+ and 2+ tumor xenografts and PDX models.

[0349] Example 20. Efficacy of BsAb1-Campt1 and BsAb2-Campt2 compared to Comp ADC in N87 xenografts. To compare the efficacy of BsAb1-Camp1 ADC and BsAb2-Camp1 ADC with CompAb1-MCC-maytansinoid A, COMPAb1-GGFG-Dxd, and BsAb2-tubulysin 1A-LP in the HER2 IHC3+ N87 xenograft model, an in vivo tumor assay was performed. For the assay, 6-week-old female SCID mice (CBySmn.CB17-Prkdcscid / J, Jackson Labs, number 001803, n = 50) were used. For transplantation, N87 (ATCC, HTB-5822) cells were mixed with Matrigel (Corning, catalog number 354234), and 150 uL of a suspension of cells and Matrigel containing 4×10 6 cells were injected into SCID mice. Seven days later, the indicated ADCs were subcutaneously injected at the indicated doses into SCID mice randomized based on tumor size. A cohort treated with 1 mg / kg of H4H17087D-tubulysin 1A-LP received two additional doses on days 14 and 21. The size of the xenograft in each mouse was measured using a caliper (Roboz, catalog number RS6466).

[0350] In mice that received a single dose of 10 mg / kg of BsAb1-Campt1 or BsAb2-Camp1, the N87 tumors completely regressed by 110 days. The anti-tumor activity was equivalent to that of 10 mg / kg of CompAb1-GGFG-Dxd and 3 mg / kg of BsAb2-tubulysin 1A-LP. In mice that received a single dose of 10 mg / kg of CompAb1-MCC-maytansinoid A, the size of the N87 xenografts initially decreased significantly, but 5 out of 5 tumors evaded treatment by day 50. In mice that received three doses of 1 mg / kg of BsAb2-tubulysin 1A-LP, a similar average anti-tumor response was observed, but only 2 out of 5 tumors evaded treatment. In contrast, mice that received a single dose of the control ADC had no anti-tumor effect. See Figures 13A and 13B.

[0351] Example 21: Epitope mapping data of the binding of BsAb2 to Erbb2.mmH. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residues of human epidermal growth factor receptor 2 that interact with the bivalent parental antibody containing the binding arms of BsAb2 (parental Ab1 and parental Ab2). In this experiment, a recombinant production version of the extracellular domain (ECD) region of HER2 with a 6-histidine tag was used (SEQ ID NO: 54). A description of the general outline of the HDX-MS method is provided, for example, in Ehring (1999) Analytical Biochemistry 267(2):252-259; and Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0352] The HDX-MS experiment was performed on a customized platform consisting of a custom HDX automation system for deuterium labeling and quenching, a Waters Acquity Binary Solvent Manager for sample digestion and capture, another Waters Acquity Binary Solvent Manager for analytical gradients, and a Thermo Q Exactive HF mass spectrometer for peptide identification and mass measurement.

[0353] The D2O-labeled solution was prepared in a D2O-based buffer (50 mM phosphate, 100 mM sodium chloride, pD 7.0). For deuterium labeling, 10 μL of 0.93 mg / mL Her2 protein or Her2 protein pre-mixed with either bivalent parent (molar ratio of antigen to antibody 1:0.7) was incubated in duplicate at 20 °C with 90 μL of the D2O-labeled solution at various time points (non-deuterated control at 0 min, deuterium labeling at 15 s, 60 s, 600 s, 3600 s, and 6000 s). The deuteration reaction was quenched by adding 100 μL of 4 M guanidine hydrochloride, 0.85 M TCEP buffer (acidified to pH 2.3 with HCl) to each sample and incubating at 15 °C for 180 s. The quenched samples were then injected into the LC system and subjected to on-line pepsin digestion at 15 °C. The digested peptides were captured on a C8 column (1 mm × 50 mm, Novabioassays) at -6 °C with a 12.5 min gradient of 2 - 32% solvent B (mobile phase A: 0.2% formic acid in water, mobile phase B: 0.2% formic acid in acetonitrile). The eluted peptides were analyzed by a Thermo Q Exactive HF mass spectrometer in LC-MS / MS or LC-MS mode.

[0354] The LC-MS / MS data of the non-deuterated Her2 samples were subjected to a search against a database containing the amino acid sequence of the Her2 protein and its reverse sequence using the Byonic search engine (Protein Metrics). The search parameters were set as default using non-specific enzyme digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into the HDX WorkBench software (version 3.3) to calculate the deuterium uptake (D-uptake) and percentage of deuterium uptake (%D) for all deuterated samples. The residue numbers of the peptides were derived from the actual protein sequence including tags (N-terminal residue T is the first amino acid). TIFF0007695260000202.tif18157

[0355] A total of 351 peptides derived from Her2 were identified from both Her2 alone and Her2 in the complex containing the parental Ab1 sample, representing 100% sequence coverage of Her2. Any peptide showing a decrease of more than 5% in the percentage of deuterium incorporation was defined as significantly protected (Δ%D < -5%). The Her2 ECD is the YQDTI sequence (SEQ ID NO: 55) of amino acids 141 - 145 and the RSRACHPCSPMCKGSRC sequence (SEQ ID NO: 56) of amino acids 166 - 182, which were assigned as epitopes on the human Her2 ECD targeted by the parental Ab1. When mAb parental Ab1 binds to these sequences, a significant decrease in deuterium incorporation was shown. Deuterium incorporation data for peptides covering epitope regions I (amino acids 141 - 145, YQDTI, SEQ ID NO: 55) and II (amino acids 166 - 182, RSRACHPCSPMCKGSRC, SEQ ID NO: 56) are shown in Figure 14.

[0356] A total of 366 peptides derived from Her2 were identified from both Her2 alone and Her2 in the complex containing the parental Ab2 sample, representing 100% sequence coverage of Her2. Any peptide showing a decrease of more than 5% in the percentage of deuterium incorporation was defined as significantly protected (Δ%D < -5%). The Her2 ECD showed a significant decrease in deuterium incorporation when binding to the parental Ab2 at the MKLRLPASPETHLDM sequence (SEQ ID NO: 57) of amino acids 9 - 23, the TYLPTNASLSF sequence (SEQ ID NO: 58) of amino acids 41 - 51, and the IAHNQVRQVPLQRL sequence (SEQ ID NO: 59) of amino acids 64 - 77. Furthermore, there was a significant decrease in deuterium incorporation at the AFLPESF sequence (SEQ ID NO: 60) of amino acids 353 - 359, which may be due to an allosteric effect. Deuterium incorporation data for peptides covering epitope regions I (amino acids 9 - 23, MKLRLPASPETHLDM, SEQ ID NO: 57), II (amino acids 41 - 51, TYLPTNASLSF, SEQ ID NO: 58), III (amino acids 64 - 77, IAHNQVRQVPLQRL, SEQ ID NO: 59), and the peripheral epitope region IV (amino acids 353 - 359, AFLPESF, SEQ ID NO: 60) are shown in Figure 15.

[0357] Example 22. Epitope mapping data for the binding of BsAb1 to Erbb2.mmH. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residues of HER2 that interact with the bivalent parental antibody containing the binding arms of BsAb1 (parental Ab3 and parental Ab4). The same recombinant HER2 protein as described in Example 21 was used (SEQ ID NO: 54).

[0358] The HDX-MS experiment was performed on a customized platform consisting of a custom HDX automation system for deuterium labeling and quenching, a Waters Acquity Binary Solvent Manager for sample digestion and capture, another Waters Acquity Binary Solvent Manager for analytical gradient, and a Thermo Q Exactive HF mass spectrometer for peptide identification and mass measurement.

[0359] The D2O-labeled solution was prepared in a D2O-based buffer (50 mM phosphate, 100 mM sodium chloride, pD 7.0). For deuterium labeling, 10 μL of 0.93 mg / mL Her2 protein or Her2 protein pre-mixed with either parental Ab3 or parental Ab4 (molar ratio of antigen to antibody 1:0.7) was incubated in duplicate with 90 μL of the D2O-labeled solution at 20 °C at various time points (non-deuterated control at 0 min, deuterium labeling at 15 s, 60 s, 600 s, and 3600 s). The deuteration reaction was quenched by adding 100 μL of 4 M guanidine hydrochloride and 0.85 M TCEP buffer (acidified to pH 2.3 with HCl) to each sample and incubating at 15 °C for 180 s. The quenched samples were then injected into an LC system and subjected to on-line pepsin digestion at 15 °C. The digested peptides were captured on a C8 column (1 mm × 50 mm, Novabioassays) at -6 °C with a 12.5 min gradient of 2 - 32% solvent B (mobile phase A: 0.2% formic acid in water, mobile phase B: 0.2% formic acid in acetonitrile). The eluted peptides were analyzed by a Thermo Q Exactive HF mass spectrometer in LC-MS / MS or LC-MS mode.

[0360] The LC-MS / MS data of the non-deuterated Her2 samples were subjected to a search against a database containing the amino acid sequence of the Her2 protein and its reverse sequence using the Byonic search engine (Protein Metrics). The search parameters were set as default using non-specific enzymatic digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into the HDX WorkBench software (version 3.3) to calculate the deuterium uptake (D-uptake) and percentage of deuterium uptake (%D) for all deuterated samples. The residue numbers of the peptides were derived from the actual protein sequence including the tags (N-terminal residue T is the first amino acid). TIFF0007695260000203.tif18157

[0361] A total of 386 Her2-derived peptides were identified from both Her2 alone and Her2 in the complex containing the parental Ab3 sample, representing 99.8% sequence coverage of Her2. Any peptide showing a decrease of more than 5% in the percentage of deuterium incorporation was defined as significantly protected (Δ%D < -5%). The Her2 ECD is the IQRNPQLCYQDTILWK sequence (SEQ ID NO: 61), which is amino acids 133 - 148, and the SPMCKGSRC sequence (SEQ ID NO: 62), which is amino acids 174 - 182, assigned as the epitopes on human Her2 ECD targeted by the parental Ab3. When binding to the parental Ab3, it showed a significant decrease in deuterium incorporation. Furthermore, the human Her2 ECD showed a moderate decrease in deuterium incorporation when binding to the parental Ab3 with the TRTVCAG sequence (SEQ ID NO: 63), which is amino acids 194 - 200. Deuterium incorporation data for peptides covering epitope regions I (amino acids 133 - 148, IQRNPQLCYQDTILWK, SEQ ID NO: 61) and II (amino acids 174 - 182, SPMCKGSRC, SEQ ID NO: 62), as well as the marginal epitope region III (amino acids 194 - 200, TRTVCAG, SEQ ID NO: 63) are shown in Figure 16.

[0362] A total of 385 Her2-derived peptides were identified from both Her2 alone and Her2 in the complex containing the parental Ab4 sample, representing 99.7% sequence coverage of Her2. Any peptide showing a decrease of more than 5% in the percentage of deuterium incorporation was defined as significantly protected (Δ%D < -5%). The Her2 ECD is the HKNNQLALTL sequence (SEQ ID NO: 64) of amino acids 152 - 161, which was assigned as the major epitope on the human Her2 ECD targeted by the parental Ab4. When binding to the parental Ab4, it showed a significant decrease in deuterium incorporation. Furthermore, the human Her2 ECD showed a moderate decrease in deuterium incorporation when binding to the parental Ab4 with the TRTVCAG sequence (SEQ ID NO: 63) of amino acids 194 - 200 and the ESMPNPEGRYTFGASC sequence (SEQ ID NO: 65) of amino acids 258 - 273. The deuterium incorporation data of the peptides covering the detected epitope region I (amino acids 152 - 161, HKNNQLALTL, SEQ ID NO: 64), the peripheral epitope region II (amino acids 194 - 200, TRTVCAG, SEQ ID NO: 63), and region III (amino acids 258 - 273, ESMPNPEGRYTFGASC, SEQ ID NO: 65) are shown in Figure 17.

[0363] The present invention is not limited to the scope according to the specific embodiments described herein. Indeed, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and the accompanying figures in addition to those described herein. Such changes are intended to be within the scope of the appended claims.

Claims

**Claim 1** A bispecific antigen-binding molecule comprising a first antigen-binding domain (D1) and a second antigen-binding domain (D2), wherein D1 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 2, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, and D2 either comprises a HCVR containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 10, and an LCVR containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, or D1 comprises a HCVR containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 32, and an LCVR containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, and D2 comprises a HCVR containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 40, and an LCVR containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, D1 specifically binds to a first epitope of human HER2, and D2 specifically binds to a second epitope of human HER2, a bispecific antigen-binding molecule. **Claim 2** The bispecific antigen-binding molecule according to claim 1, wherein D1 and D2 do not compete with each other for binding to human HER2. **Claim 3** A heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or D1 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

24. The bispecific antigen-binding molecule according to claim 1 or 2. **Claim 4** The bispecific antigen-binding molecule according to any one of claims 1 to 3, wherein D1 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical thereto, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto.

5. The bispecific antigen-binding molecule according to any one of claims 1 to 4, wherein D1 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 2 and an LCVR comprising the amino acid sequence of SEQ ID NO:

18.

6. The bispecific antigen-binding molecule according to any one of claims 1 to 5, wherein D2 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical thereto, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto.

7. The bispecific antigen-binding molecule according to any one of claims 1 to 6, wherein D2 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 10 and an LCVR comprising the amino acid sequence of SEQ ID NO:

18.

8. The bispecific antigen-binding molecule according to any one of claims 1 to 3, wherein D1 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence that is at least 95% identical thereto, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto.

9. The bispecific antigen-binding molecule according to any one of claims 1 to 3 and 8, wherein D1 comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 32 and an LCVR comprising the amino acid sequence of SEQ ID NO:

18.

10. The bispecific antigen-binding molecule according to any one of claims 1 to 3, 8, and 9, wherein D2 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence that is at least 95% identical thereto, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical thereto. Claim 11 The bispecific antigen-binding molecule according to any one of claims 1 to 3 and 8 to 10, wherein D2 comprises an HCV-R comprising the amino acid sequence of SEQ ID NO: 40 and an LCV-R comprising the amino acid sequence of SEQ ID NO:

18. Claim 12 The bispecific antigen-binding molecule according to any one of claims 1 to 11, which is conjugated to a cytotoxin. Claim 13 The following features: (a) showing greater antitumor activity at a lower dosage as compared to trastuzumab conjugated to a cytotoxin; (b) showing greater antitumor activity at a lower dosage as compared to trastuzumab conjugated to DM1; (c) showing greater antitumor activity at a lower dosage as compared to CompAb2 conjugated to a cytotoxin; (d) showing greater antitumor activity at a lower dosage as compared to CompAb2 conjugated to tubulysin; (e) inhibiting the growth of cancers with moderate and high HER2 expression, but not inhibiting the growth of tissues with low HER2 expression; and (f) promoting tumor regression of cancers with moderate and high HER2 expression, but not promoting tumor regression of tissues with low HER2 expression The bispecific antigen-binding molecule according to claim 12, having one or more of the above. Claim 14 The bispecific antigen-binding molecule according to claim 13, wherein the cytotoxin is selected from the group consisting of a biotoxin, a chemotherapeutic agent, and a radioisotope. Claim 15 The bispecific antigen-binding molecule according to claim 13, wherein the cytotoxin is tubulysin, camptothecin, or a maytansinoid. Claim 16 The bispecific antigen-binding molecule according to any one of claims 1 to 15, which is conjugated to a cytotoxin via a linker.

17. The bispecific antigen-binding molecule according to claim 16, wherein the cytotoxin is tubulysin.

18. The tubulysin is The bispecific antigen-binding molecule according to claim 17.

19. The bispecific antigen-binding molecule is conjugated to or its positional isomer, wherein is a bond with heavy chain glutamine. The bispecific antigen-binding molecule according to claim 16.

20. The bispecific antigen-binding molecule according to claim 17, comprising a heavy chain conjugated to tubulysin via Q295.

21. comprising a heavy chain containing the N297Q mutation (EU index numbering rule), (i) conjugated to tubulysin via the glutamine residue of heavy chain Q295, and (ii) conjugated to tubulysin via the glutamine residue of the N297Q mutation, The bispecific antigen-binding molecule according to claim 17.

22. The linker is azido-PEG ３ -amine. The bispecific antigen-binding molecule according to claim 17.

23. The bispecific antigen-binding molecule according to claim 18, comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO:

10.

24. The bispecific antigen-binding molecule according to claim 18, comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO:

40.

25. The bispecific antigen-binding molecule according to claim 16, wherein the cytotoxin is a maytansinoid.

26. The bispecific antigen-binding molecule according to claim 25, comprising the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO:

10.

27. The bispecific antigen-binding molecule according to claim 25, comprising the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO:

40.

28. The maytansinoid is wherein is the bond with the linker, the bispecific antigen-binding molecule according to claim 25.

29. The linker is wherein the bond represented by is the bond with the bispecific antigen-binding molecule, the bond represented by is the bond with the maytansinoid, the bispecific antigen-binding molecule according to claim 28.

30. The maytansinoid is wherein is the bond with the linker, the bispecific antigen-binding molecule according to claim 25.

31. The linker is wherein the bond represented by is the bond with the bispecific antigen-binding molecule, the bond represented by is the bond with the maytansinoid, the bispecific antigen-binding molecule according to claim 30.

32. A pharmaceutical composition comprising the bispecific antigen-binding molecule according to any one of claims 1 to 31 and a pharmaceutically acceptable carrier.

33. A pharmaceutical composition for use in the treatment of cancer in a subject suffering from a tumor overexpressing HER2, comprising the bispecific antigen-binding molecule according to any one of claims 12 to 31.

34. The pharmaceutical composition according to claim 33, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, gastric cancer, cervical cancer, gastric cancer, endometrial cancer, and ovarian cancer.

35. The pharmaceutical composition according to claim 33, further combined with a second anti-cancer therapeutic agent.

36. A pharmaceutical composition for treating cancer, reducing tumor growth, and / or causing tumor regression, comprising an antibody-drug conjugate (ADC) containing a bispecific antigen-binding molecule and a cytotoxic agent, wherein the bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) and a second antigen-binding domain (D2), and D1 comprises an HCVVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and an LCVR containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, and D2 comprises an HCVVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10, and an LCVR containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, or D1 comprises an HCVVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 32, and an LCVR containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, and D2 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 40, and a light chain variable region (LCVR) containing three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, D1 specifically binds to the first epitope of human HER2, and D2 specifically binds to the second epitope of human HER2, Pharmaceutical composition.

37. D1 comprises a heavy chain variable region (HCVR) containing HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a light chain variable region (LCVR) containing LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) containing HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, a light chain variable region (LCVR) containing LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or D1 comprises a heavy chain variable region (HCVR) containing HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 38, a light chain variable region (LCVR) containing LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 42 for HCDR1, the amino acid sequence of SEQ ID NO: 44 for HCDR2, and the amino acid sequence of SEQ ID NO: 46 for HCDR3, and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 20 for LCDR1, the amino acid sequence of SEQ ID NO: 22 for LCDR2, and the amino acid sequence of SEQ ID NO: 24 for LCDR3. The pharmaceutical composition according to claim 36.

38. The pharmaceutical composition according to claim 36 or 37, wherein D1 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18.

39. The pharmaceutical composition according to claim 36 or 37, wherein D1 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 32 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18.

40. The pharmaceutical composition according to any one of claims 36 to 39, wherein D2 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18.

41. The pharmaceutical composition according to any one of claims 36 to 39, wherein D2 comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 40 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18.

42. The pharmaceutical composition according to claim 36, 37, 38, or 40, wherein the bispecific antigen-binding molecule comprises the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO:

10.

43. The pharmaceutical composition according to claim 36, 37, 39, or 41, wherein the bispecific antigen-binding molecule comprises the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO:

40.

44. The pharmaceutical composition according to any one of claims 36 to 43, wherein the cytotoxin is selected from the group consisting of a biological toxin, a chemotherapeutic agent, and a radioisotope.

45. The pharmaceutical composition according to any one of claims 36 to 43, wherein the cytotoxin is tubulysin or a maytansinoid.

46. The pharmaceutical composition according to any one of claims 36 to 43, wherein the cytotoxin is tubulysin, and the tubulysin is The pharmaceutical composition according to any one of claims 36 to 43, which is

47. The bispecific antigen-binding molecule is conjugated to or a positional isomer thereof, wherein is a bond with heavy chain glutamine. The pharmaceutical composition according to claim 46.

48. The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, and the linker is azido-PEG ３ - amine. The pharmaceutical composition according to any one of claims 45 to 47.

49. The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, and the cytotoxin is wherein is a bond with the linker. The pharmaceutical composition according to any one of claims 36 to 43.

50. The linker is wherein the bond denoted by represents the bond with the bispecific antigen-binding molecule, and the bond denoted by represents the bond with the cytotoxin. The pharmaceutical composition according to claim 49.

51. The cytotoxin is conjugated to the bispecific antigen-binding molecule via a linker, and the cytotoxin is wherein is a bond with the linker. The pharmaceutical composition according to any one of claims 36 to 43.

52. wherein the linker is and in the formula, the bond represented by is the bond with the bispecific antigen-binding molecule, and the bond represented by is the bond with the cytotoxin. The pharmaceutical composition according to claim 51.

53. A method for preparing an antibody-drug conjugate, comprising contacting an HER2×HER2 bispecific antigen-binding protein with a compound having the following formula A １ : and an aqueous diluent, wherein the bispecific antigen-binding protein comprises a first antigen-binding domain (D1) and a second antigen-binding domain (D2) and D1 comprises an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, and D2 comprises an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, or D1 comprises an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 32, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, and D2 comprises an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 10, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, or D1 comprises an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 32, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18, and D2 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 40, and a light chain variable region (LCVR) containing three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO:

18. Method.

54. D1 comprises a heavy chain variable region (HCVR) containing HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region (LCVR) containing LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) containing HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region (LCVR) containing LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or D1 comprises a heavy chain variable region (HCVR) containing HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 38, and a light chain variable region (LCVR) containing LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) containing HCDR1 comprising the amino acid sequence of SEQ ID NO: 42, HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (LCVR) containing LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

24. The method according to claim 53.

55. The method according to claim 53 or 54, wherein the HER2×HER2 bispecific antigen-binding protein comprises the D1-HCVR amino acid sequence of SEQ ID NO: 2 and the D2-HCVR amino acid sequence of SEQ ID NO:

10.

56. The method according to claim 53 or 54, wherein the HER2×HER2 bispecific antigen-binding protein comprises the D1-HCVR amino acid sequence of SEQ ID NO: 32 and the D2-HCVR amino acid sequence of SEQ ID NO:

40.

57. The method according to claim 55 or 56, wherein the HER2×HER2 bispecific antigen-binding protein comprises the LCVR amino acid sequence of SEQ ID NO:

18.

58. The compound of formula A １ is present in stoichiometric excess in the method according to any one of claims 53 to 57.

59. The compound of formula A １ is a compound of formula A ２ or A ３ of the compound: or a mixture thereof in the method according to any one of claims 53 to 58.

60. The compound of formula A ２ is stereoisomerically pure in the method according to claim 59.

61. The compound of formula A ３ is stereoisomerically pure in the method according to claim 59.

62. The compound of A １ or A ２ is present with a diastereomeric excess of more than 50%, more than 70%, more than 90%, or more than 95% in the method according to claim 59.

63. The compound of formula A １ is a compound of formula (a): to a compound of formula (b): The method according to any one of claims 53 to 62, which is prepared by contacting in the presence of silica gel and a diluent.

64. The following process: (i) A compound of formula (a): is contacted with a compound of formula (b): in the presence of silica gel and a diluent to synthesize an intermediate, and (ii) contacting the HER2×HER2 bispecific antigen-binding protein with the intermediate and an aqueous diluent An antibody-drug conjugate prepared by wherein the bispecific antigen-binding protein is a first antigen-binding domain (D1) and a second antigen-binding domain (D2) comprising D1 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 2, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, and D2 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, or D1 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 32, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, and D2 comprises an HCVR containing three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 40, and an LCVR containing three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18. An antibody-drug conjugate.

65. D1 comprises a heavy chain variable region (HCVR) containing an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain variable region (LCVR) containing an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) containing an HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain variable region (LCVR) containing an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or D1 comprises a heavy chain variable region (HCVR) containing an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 38, and a light chain variable region (LCVR) containing an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) containing an HCDR1 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, and a light chain variable region (LCVR) containing an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

24. The antibody-drug conjugate according to claim 64. **Claim 66** A process for preparing an antibody-drug conjugate, comprising contacting a HER2×HER2 bispecific antigen-binding protein with a compound having the following structure: wherein the HER2×HER2 bispecific antigen-binding protein is functionalized with an azide group, and comprises a first antigen-binding domain (D1) and a second antigen-binding domain (D2), wherein D1 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 2, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, and wherein D2 either comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, or wherein D1 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within the amino acid sequence of SEQ ID NO: 32, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO: 18, and wherein D2 comprises a heavy chain variable region (HCVR) containing three heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) within the amino acid sequence of SEQ ID NO: 40, and a light chain variable region (LCVR) containing three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within the amino acid sequence of SEQ ID NO:

18. ​ Process.

67. D1 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or D1 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 38, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, The process according to claim 66.

68. The process according to claim 66 or 67, wherein the HER2×HER2 bispecific antigen-binding protein functionalized with an azide group is prepared by contacting the HER2×HER2 bispecific antigen-binding protein with transglutaminase and a compound comprising a primary amine, a PEG group, and an azide group.

69. The process according to claim 68, wherein the compound comprising a primary amine, a PEG group, and an azide group is .

70. A product of the process according to any one of claims 66 to 69.

71. The bispecific antigen-binding molecule according to claim 15 or the pharmaceutical composition according to claim 44, wherein the cytotoxic agent is camptothecin.

72. The bispecific antigen-binding molecule or pharmaceutical composition according to claim 71, wherein the camptothecin is .

73. The bispecific antigen-binding molecule according to claim 15 or the pharmaceutical composition according to claim 44, wherein the bispecific antigen-binding molecule is conjugated with or a positional isomer thereof, wherein is a bond with heavy chain glutamine.

74. The bispecific antigen-binding molecule according to claim 15 or the pharmaceutical composition according to claim 44, wherein the bispecific antigen-binding molecule is conjugated with wherein is a bond with a linker.

75. A process for preparing an antibody-drug conjugate comprising contacting a HER2×HER2 bispecific antigen-binding protein with a compound having the following structure: wherein the HER2×HER2 bispecific antigen-binding protein is functionalized with an azide group and comprises a first antigen-binding domain (D1) and a second antigen-binding domain (D2) . D1 includes an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) including the amino acid sequence of SEQ ID NO: 2, and an LCVR containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) including the amino acid sequence of SEQ ID NO: 18, and D2 includes an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) including the amino acid sequence of SEQ ID NO: 10, and an LCVR containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) including the amino acid sequence of SEQ ID NO: 18, or D1 includes an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) including the amino acid sequence of SEQ ID NO: 32, and an LCVR containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) including the amino acid sequence of SEQ ID NO: 18, and D2 includes an HCVR containing three heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) including the amino acid sequence of SEQ ID NO: 40, and an LCVR containing three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) including the amino acid sequence of SEQ ID NO: 18, Process.

76. D1 includes a heavy-chain variable region (HCVR) containing HCDR1 including the amino acid sequence of SEQ ID NO: 4, HCDR2 including the amino acid sequence of SEQ ID NO: 6, and HCDR3 including the amino acid sequence of SEQ ID NO: 8, and a light-chain variable region (LCVR) containing LCDR1 including the amino acid sequence of SEQ ID NO: 20, LCDR2 including the amino acid sequence of SEQ ID NO: 22, and LCDR3 including the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 12, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 14, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 16, a light chain variable region (LCVR) comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, or D1 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 36, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 38, a light chain variable region (LCVR) comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24, and D2 comprises a heavy chain variable region (HCVR) comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 42, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 44, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 46, a light chain variable region (LCVR) comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

24. The process according to claim 75.

77. The process according to claim 75 or 76, wherein the HER2×HER2 bispecific antigen-binding protein functionalized with an azide group is prepared by contacting the HER2×HER2 bispecific antigen-binding protein with transglutaminase and a compound comprising a primary amine, a PEG group, and an azide group.

78. The compound comprising a primary amine, a PEG group, and an azide group is The process according to claim 77.

79. The product of the process according to any one of claims 75 to 78.

80. The bispecific antigen-binding molecule according to claim 1, wherein the first epitope of human HER2 comprises amino acids 141-145 and / or 166-182 of SEQ ID NO:

54.

81. The bispecific antigen-binding molecule according to claim 80, wherein the second epitope of human HER2 comprises amino acids 9-23, 41-51, 64-67, and / or 353-359 of SEQ ID NO:

54.

82. The bispecific antigen-binding molecule according to claim 80, wherein the first epitope of human HER2 comprises amino acids 141-145 and / or 166-182 of SEQ ID NO: 54, and the second epitope of human HER2 comprises amino acids 9-23, 41-51, 64-67, and / or 353-359 of SEQ ID NO:

54.

83. The bispecific antigen-binding molecule according to claim 1, wherein the first epitope of human HER2 comprises amino acids 133-148, 174-182, and / or 194-200 of SEQ ID NO:

54.

84. The bispecific antigen-binding molecule according to claim 83, wherein the second epitope of human HER2 comprises amino acids 152-161, 258-273, and / or 194-200 of SEQ ID NO:

54.

85. The bispecific antigen-binding molecule according to claim 83, wherein the first epitope of human HER2 comprises amino acids 133-148, 174-182, and / or 194-200 of SEQ ID NO: 54, and the second epitope of human HER2 comprises amino acids 152-161, 258-273, and / or 194-200 of SEQ ID NO:

54.

86. The following characteristics: (a) Binding to ErbB2 with an equilibrium dissociation constant (K Ｄ ) of less than 1 nM as measured by surface plasmon resonance assay; (b) Binding to ErbB2 with a dissociation half-life (t １／２ ) of at least 30 minutes as measured by surface plasmon resonance assay; (c) binding to cell surface HER2 with greater affinity and / or avidity when compared to trastuzumab; (d) binding to HER2 IHC2+ and IHC3+ expressing cells with higher efficiency than trastuzumab; (e) greater IC than trastuzumab ５０ binding to HER2 IHC1+ expressing cells; (f) binding to cells expressing moderate or high HER2 levels but not to cells expressing low HER2 levels; (g) forming antibody clusters on the surface of HER2 expressing cells; and (h) being internalized by HER2 expressing cells with higher efficiency than trastuzumab A bispecific antigen-binding molecule according to any one of claims 1 to 31, having one or more of the above.

Images