Cytotoxic bispecific antibodies that bind DR5 and MUC16 and uses thereof

Abstract

Bispecific binding molecules are disclosed that can bind to human MUC16 and human death receptor 5 and kill cancer cells that express MUC16 and death receptor 5. Also disclosed are pharmaceutical compositions comprising the antibodies and therapeutic methods for using the antibodies.

Description

[Technical Field]

[0001] Related Applications This patent application claims the benefit of U.S. provisional patent applications having serial numbers 63 / 424,323, filed November 10, 2022, and 63 / 443,606, filed February 6, 2023, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] The human MUC16 gene (mucin 16, NCBI Entrez Gene:94025) encodes the type I transmembrane protein MUC16 or mucin 16, a high molecular weight, highly glycosylated protein. The human MUC16 gene is not expressed in most normal tissues except the apical surface of the epithelium of the upper respiratory tract, the ocular surface, the mesothelium lining body cavities (thoracic, abdominal, and pelvic cavities), organs, and the male and female reproductive tract (Lee et al., Pharmaceuticals, 2021, 14, 1053). MUC16 is expressed in most ovarian and endometrial tumors (Kabawat et al. Am. J. Clin. Pathol., 79:98-104; Suh et al., Chemo Open Access 2017, 6:2), pancreatic adenocarcinoma (Haridas et al. PLoS One 2011, 6, e26839; Jiang et al. Appl. Immunohistochem. Mol. Morphol. 2017, 25:620-623; Streppel et al. 2012, Hum. Pathol. 43, 1755-63), some esophageal, gastric, and colorectal adenocarcinomas (Streppel et al. Hum. Pathol. 2012, 43(10):1755-1763), and breast cancer (Moritani et al. al. Hum. Pathol. 2008,39,666-671), as well as non-small cell lung cancer (Chen et al. BMC Cancer 2019,19:171; Kanwal et al. Oncotarget, 2018,9:12226-12239; Lee et al. Pharmaceuticals 2021,14,1053; Liu et al. Dig Dis Sci. 2022 Jun;67:2195-2208). Furthermore, MUC16 is highly expressed in idiopathic alveolar fibrosis (IPF) (Ballester et al. Int. J. Mol. Sci. 2021;22:6502) and possibly other systemic fibrotic diseases (Zhang et al. Prog. Mol. Biol. Transl. Sci. 2019;162:241-252).In IPF, MUC16 is expressed in pathologically hyperplastic alveolar type II epithelial cells and lung fibroblasts in fibroblastic nests, but not in normal lung (Ballester et al. Int. J. Mol. Sci. 2021;22:6502). Antibody-drug conjugates targeting MUC16 have been developed, and bispecific moieties targeting MUC16 and either CD28 or CD3 are currently in early human clinical trials for the treatment of various cancers (NCT01335958, NCT04590326, NCT03564340).

[0003] The extracellular domain of MUC16 consists of an unstructured N-terminal domain and a tandem repeat (TR) region. The TR region contains approximately 60 repeats of 156 amino acids. Approximately 16 homologous SEA (sea urchin sperm protein, enterokinase, and agrin) domains are interspersed within the TR region (White et al., Proteins, 2022;90:1210-1218). A portion of the MUC16 protein is cleaved extracellularly by an unknown mechanism and subsequently shed into the blood. This extracellular portion of MUC16 is a known serum biomarker for ovarian cancer (Bast et al., Int. J. Biol. Markers 1998,13,179-187).

[0004] Death receptor 5 (DR5) is a plasma membrane protein that can induce cell death via apoptosis upon binding to its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Apoptosis can also be mediated by DR5 when bound to an agonist monoclonal antibody (Dubuisson and Micheau, Antibodies 2017, 6, 16). Preclinical studies have reported that stimulation of DR5 with either TRAIL or anti-DR5 antibodies induces cell death in various tumor cells. However, to date, clinical trials have failed to demonstrate significant therapeutic efficacy for either TRAIL or its derivatives, or for anti-DR5 antibodies (Lemke et al. Cell Death Differ., 2014, 21:1350-1364; von Karstedt et al. Nat Rev Cancer. 2017, 17:352-366).

[0005] Bispecific molecules targeting DR5 and various second antigens are known in the art, some of which are in early human clinical trials, for example, an anti-CDH17 / anti-DR5 bispecific antibody (Garcia-Martinez et al., Mol. Cancer Ther. 2021, 20:96-108; U.S. Patent No. 10,858,438) is currently in clinical development. Other second antigens targeted by such DR5 bispecifics include folate receptor alpha (U.S. Patent Application Publication No. 20200283537), fibroblast activation protein (FAP) (U.S. Patent Application No. 9,926,379), melanoma-associated chondroitin sulfate proteoglycan and Roundabout Homolog 4 (PCT Patent Application Publication No. WO 2011039126 A1), and lymphotoxin beta receptor (Michaelson et al., mAbs (2009), 1:128-141). The anti-FAP / anti-DR5 bispecific antibody RG7386 was tested in a Phase 1 clinical trial (NCT02558140), but the trial was discontinued and development of RG7386 was abandoned. Anti-CD44v6 / anti-DR5 bispecific antibodies did not enhance the agonistic effect of DR5 on CD44v6-expressing cells (U.S. Pat. No. 10,858,438 B2), indicating that the ability of such anti-DR5 bispecific antibodies to kill antigen-expressing cells is not universal to all cell surface antigens.

[0006] The mixed results of the prior art indicate the need for further development of anti-DR5 bispecific molecules that exhibit the ability to kill abnormal cells such as cancer cells.Furthermore, the results of the prior art indicate that it is not possible to reasonably predict whether a bispecific antibody that targets DR5 together with a tumor-associated antigen will be effective in inducing apoptosis of cancer cells.Therefore, there remains an unmet need for cancer therapeutic agents. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides bispecific binding molecules comprising a first antigen-binding domain that specifically binds to the extracellular domain of human MUC16 and a second antigen-binding domain that specifically binds to human DR5. These bispecific molecules are efficient at killing cells that co-express MUC16 and DR5 and are more potent at killing such cells than the monospecific DR5 antibody from which the second antigen-binding domain is derived (e.g., an anti-DR5 antibody having the same set of three heavy chain CDRs (CDR-H1, CDR-H2, and CDR-H3) and light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) present in the bispecific binding molecule).

[0008] Thus, in a first aspect of the invention, the first antigen-binding domain (which binds to the extracellular portion of human MUC16) binds to an epitope present in two or more tandem repeat / SEA segments within the extracellular domain of MUC16.

[0009] In a second aspect of the invention, the first antigen-binding domain has MUC16 binding competed for by one or more of the following antibodies: OC125, H185 (Invitrogen catalogue no. MA5-11579), M11 (American Tissue Culture Collection accession no. PTA-6206), OV197 (Fujirebio Diagnostic), 5E11 (Millipore Sigma catalogue no. MABC1608-25UG), AR9.6, H1H8794, VK-8, B43.13 (also known as oregovomab), or 3A5 (sofituzumab).

[0010] In a third aspect of the present invention, the first antigen-binding domain binds to a polypeptide or peptide consisting essentially of at least one amino acid sequence of SEQ ID NOs: 176-181, or an extracellular fragment of MUC16 isolated from cell culture medium of OVCAR-3 cells.

[0011] In a fourth aspect of the present invention, the first antigen-binding domain comprises six specific heavy and light chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In one embodiment of the fourth aspect, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 2 to 5; CDR2-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 6 to 61 (CDR3-H3); CDR-L1 comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 65. In more specific embodiments of the fourth aspect, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2; CDR-H3 comprises the amino acid sequence of SEQ ID NO: 6; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 63; and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 65. Each of the above CDR sequences conforms to the Kabat rules for determining CDRs.

[0012] In an alternative embodiment of the fourth aspect, the set of six CDR sequences (and their corresponding CDR rules) characterizing the first binding site are selected from any of the following listed in Table 1:

[0013] [Table 1]

[0014] In a fifth aspect of the present invention, the first antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of any of SEQ ID NOs: 104 or 105, or an amino acid sequence having the same three light chain CDRs as any of SEQ ID NOs: 104 or 105 and having at least 90% sequence identity to any of SEQ ID NOs: 104 or 105. In a more specific embodiment of the fifth aspect, the first antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of any of SEQ ID NOs: 104 or 105. In an alternative embodiment of the fifth aspect, the first antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 106 to 114, or an amino acid sequence having the same three light chain CDRs as any one of SEQ ID NOs: 106 to 114 and having at least 90% sequence identity to any one of SEQ ID NOs: 106 to 114.

[0015] In a sixth aspect of the present invention, the first antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 97 and having at least 90% sequence identity to SEQ ID NO: 97. In a more specific embodiment of the sixth aspect, the first antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97. In an alternative embodiment of the sixth aspect, the first antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 98 to 103, or an amino acid sequence having the same three heavy chain CDRs as any one of SEQ ID NOs: 98 to 103 and having at least 90% sequence identity to any one of SEQ ID NOs: 98 to 103.

[0016] In a seventh aspect of the invention, the first antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of either SEQ ID NO: 104 or 105, or an amino acid sequence having the same three light chain CDRs as either SEQ ID NO: 104 or 105 and having at least 90% sequence identity to either SEQ ID NO: 104 or 105; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 97 and having at least 90% sequence identity to SEQ ID NO: 97. In a more specific embodiment of the seventh aspect, the first antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of either SEQ ID NO: 104 or 105; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97.

[0017] In an alternative embodiment of the seventh aspect, the first antigen-binding domain comprises a combination of heavy and light chain variable regions selected from any of the following combinations shown in Table 2:

[0018] [Table 2]

[0019] In an eighth aspect of the present invention, the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 115 to 118, or a heavy chain comprising the same three heavy chain CDRs as any one of SEQ ID NOs: 115 to 118 and comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 115 to 118. In some embodiments of this aspect, the heavy chain comprises the amino acid sequence of any one of SEQ ID NOs: 115 to 118.

[0020] In a ninth aspect of the invention, the first antigen-binding domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence having the same three light chain CDRs as SEQ ID NO: 119 or SEQ ID NO: 120 and at least 90% sequence identity to SEQ ID NO: 119 or SEQ ID NO: 120. In some embodiments of this aspect, the light chain comprises the amino acid sequence of any one of SEQ ID NOs: 119-120.

[0021] In a tenth aspect of the present invention, the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 115 to 118, or a heavy chain comprising an amino acid sequence having the same three heavy chain CDRs as any one of SEQ ID NOs: 115 to 118 and having at least 90% sequence identity to any one of SEQ ID NOs: 115 to 118; and a light chain comprising the amino acid sequence of SEQ ID NO: 119 or the amino acid sequence of SEQ ID NO: 120, or a light chain having the same three light chain CDRs as SEQ ID NO: 119 or SEQ ID NO: 120 and having at least 90% sequence identity to SEQ ID NO: 119 or SEQ ID NO: 120. In a more specific embodiment of the tenth aspect, the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 115 to 118; and a light chain comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120.

[0022] In an eleventh aspect of the present invention, the first antigen-binding domain described in any of the first to tenth aspects comprises two heavy chains and two light chains. In some embodiments of the eleventh aspect, each of the two heavy chains has an identical amino acid sequence, and each of the two light chains has an identical amino acid sequence.

[0023] In a twelfth aspect of the invention, the second antigen-binding domain that binds to human DR5 has DR5 binding competed by a) one or more of the antibodies conatumumab, drozitumab, lexatumumab, LBY135, tigatuzumab, and DS-8273a; or b) TRAIL, or a DR5-binding fragment of TRAIL.

[0024] In a thirteenth aspect of the present invention, the second antigen-binding domain comprises six specific heavy and light chain CDR sequences (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In one embodiment of the thirteenth aspect, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 121; CDR-H2 comprises the amino acid sequence of SEQ ID NO: 122; CDR2-H3 comprises the amino acid sequence of SEQ ID NO: 123; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 124; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 125; and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 126. The above CDR sequences follow the Kabat rules for determining CDRs.

[0025] In an alternative embodiment of the thirteenth aspect, the set of six CDR sequences (and their corresponding CDR rules) characterizing the first binding site are selected from any of the following listed in Table 3:

[0026] [Table 3]

[0027] In a fourteenth aspect of the present invention, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 158 and having at least 90% sequence identity to SEQ ID NO: 158. In a more specific embodiment of the fourteenth aspect, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158. In an alternative embodiment of the fourteenth aspect, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 159 to 164, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NOs: 159 to 164 and having at least 90% sequence identity to SEQ ID NOs: 159 to 164. In an alternative embodiment of the fourteenth aspect, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 218 to 220, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NOs: 218 to 220 and having at least 90% sequence identity to SEQ ID NOs: 218 to 220. In a more specific embodiment, the heavy chain variable region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 218. In another more specific embodiment, the heavy chain variable region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 219. In yet another more specific embodiment, the heavy chain variable region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 220.

[0028] In a fifteenth aspect of the present invention, the second antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 165 and having at least 90% sequence identity to SEQ ID NO: 165. In a more specific embodiment of the fifteenth aspect, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 165. In an alternative embodiment of the fifteenth aspect, the second antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 166 to 171, or an amino acid sequence having the same three light chain CDRs as SEQ ID NOs: 166 to 171 and having at least 90% sequence identity to SEQ ID NOs: 166 to 171. In another alternative embodiment of the fifteenth aspect, the second antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 215 to 217, or an amino acid sequence having the same three light chain CDRs as SEQ ID NOs: 215 to 217 and having at least 90% sequence identity to SEQ ID NOs: 215 to 217. In a more specific embodiment, the light chain variable region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 215. In a more specific embodiment, the light chain variable region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 216. In yet another more specific embodiment, the light chain variable region of the second antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 217.

[0029] In a sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 158 and having at least 90% sequence identity to SEQ ID NO: 158; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three light chain CDRs as SEQ ID NO: 165 and having at least 90% sequence identity to SEQ ID NO: 165. In a more specific embodiment of the sixteenth aspect, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 158; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 165.

[0030] In an alternative embodiment of the sixteenth aspect, the second antigen-binding domain comprises a combination of heavy and light chain variable regions selected from any of the following combinations shown in Table 4:

[0031] [Table 4]

[0032] In yet another alternative embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 218; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 215. In yet another alternative embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 219; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 215. In yet another alternative embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 219; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 216. In yet another alternative embodiment of the sixteenth aspect of the invention, the second antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 220; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 217.

[0033] In a seventeenth aspect of the present invention, the second antigen-binding domain is an scFv fragment of an antibody characterized by any one of the twelfth to sixteenth aspects above. The scFv fragment comprises a heavy chain variable region and a light chain variable region linked to each other via a peptide linker. In some embodiments of this aspect, the scFv fragment comprises, from N-terminus to C-terminus, a light chain variable region, a peptide linker, and a heavy chain variable region. In some embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of any one of SEQ ID NO: 172 and SEQ ID NOs: 210-214; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as SEQ ID NO: 172 or any one of SEQ ID NOs: 210-214 and at least 90% sequence identity to SEQ ID NO: 172 or any one of SEQ ID NOs: 210-214. In a more specific embodiment of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as SEQ ID NO: 172 and having at least 90% sequence identity to SEQ ID NO: 172. In an even more specific embodiment of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 210. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 211. In other specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 212. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 213. In other more specific embodiments of the seventeenth aspect, the scFv fragment comprises the amino acid sequence of SEQ ID NO: 214.

[0034] In view of the above, it will be readily apparent to one skilled in the art that the bispecific binding molecules disclosed herein may comprise a first antigen-binding domain characterized by any of the aforementioned aspects 1 to 11 and a second antigen-binding domain characterized by any of the aforementioned aspects 12 to 17. All possible combinations of such first and second antigen-binding domains are within the scope of the present disclosure.

[0035] In an eighteenth aspect of the present invention, the N-terminus of the second antigen-binding domain is fused to the C-terminus of one of the heavy chains of the first antigen-binding domain directly or via a peptide linker 4 to 20 amino acids in length. In some embodiments of the eighteenth aspect, the N-terminus of the second antigen-binding domain is fused to the C-terminus of one of the heavy chains of the first antigen-binding domain via a peptide linker 4 to 20 amino acids in length. In a more specific embodiment of the eighteenth aspect, the peptide linker has the amino acid sequence of SEQ ID NO: 173. In another more specific embodiment of the eighteenth aspect, the second antigen-binding domain is an scFv fragment of the seventeenth aspect. In yet another specific embodiment of the eighteenth aspect, the bispecific antigen-binding molecule comprises two scFv fragments of the seventeenth aspect that specifically bind to human DR5. In some even more specific embodiments of the eighteenth aspect, the bispecific antigen-binding molecule comprises two scFv fragments of the seventeenth aspect that specifically bind to human DR5, each binding to the C-terminus of a different heavy chain of the first antigen-binding domain. In some further embodiments, each of the two scFv fragments has the same amino acid sequence.

[0036] In a nineteenth aspect of the invention, the bispecific binding molecule comprises: a) two antibody light chains, each light chain independently having an amino acid sequence selected from SEQ ID NO: 119 and SEQ ID NO: 120; and b) two antibody heavy chain fusions, each heavy chain fusion independently selected from the following formula: XLY, where X is the amino acid sequence of any one of SEQ ID NOs: 115-118; L is the amino acid sequence of SEQ ID NO: 173; and Y is the amino acid sequence of SEQ ID NO: 172 or any one of SEQ ID NOs: 210-214. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 172. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 210. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 211. In some embodiments of the nineteenth aspect, Y is the amino acid sequence of SEQ ID NO: 212. In some embodiments of the 19th aspect, Y is the amino acid sequence of SEQ ID NO: 213. In some embodiments of the 19th aspect, Y is the amino acid sequence of SEQ ID NO: 214. In some embodiments of the 19th aspect, each light chain has an identical amino acid sequence and each heavy chain fusion has an identical amino acid sequence. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 203. In some embodiments of the nineteenth aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 204.In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 206. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 119, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 207. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 187. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 203. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 206. In some embodiments of the 19th aspect, each light chain comprises the amino acid sequence of SEQ ID NO: 120, and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO: 207.

[0037] In a twentieth aspect of the invention, there is provided a pharmaceutical composition comprising a bispecific binding molecule as described herein and a pharmaceutically acceptable carrier.

[0038] In a twenty-first aspect of the present invention, there is provided an isolated nucleic acid sequence encoding a heavy chain fusion having an amino acid sequence of the formula XLY, where X is the amino acid sequence of any one of SEQ ID NOs:115-118; L is the amino acid sequence of SEQ ID NO:173; and Y is the amino acid sequence of SEQ ID NO:172 or any one of SEQ ID NOs:210-214. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO:172. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO:210. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO:211. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO:212. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO:213. In some embodiments of the twenty-first aspect, Y is the amino acid sequence of SEQ ID NO:214. In some embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:174. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 175. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 186. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 187. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 203. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 204. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 205. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 206. In other embodiments of this aspect, the isolated nucleic acid sequence encodes the amino acid sequence of SEQ ID NO: 207. In yet other embodiments of this aspect, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 182, or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 182 and encoding the same amino acid sequence as SEQ ID NO: 182.In yet other embodiments of this aspect, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:183, or a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO:183 and encodes the same amino acid sequence as SEQ ID NO:183.

[0039] In a 22nd aspect of the invention, there is provided an expression vector comprising the nucleic acid sequence of the 21st aspect or any embodiment thereof. In some embodiments of the 22nd aspect, the expression vector further comprises a first promoter operably linked to the nucleic acid sequence and capable of driving expression thereof.

[0040] In a 23rd aspect of the invention, there is provided an expression vector of the 22nd aspect, further comprising a nucleic acid sequence encoding a light chain of the first antigen-binding domain of the bispecific binding molecule according to any of the preceding aspects of the invention; and a second promoter operably linked to the nucleic acid sequence encoding the light chain and capable of driving its expression. In a more specific embodiment of this aspect, the nucleic acid sequence encoding the light chain of the first antigen-binding domain of the bispecific binding molecule comprises the nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 184 and encodes the same amino acid sequence as SEQ ID NO: 184. In another more specific embodiment of this aspect, the nucleic acid sequence encoding the light chain of the first antigen-binding domain of the bispecific binding molecule comprises the nucleotide sequence of SEQ ID NO: 185, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 185 and encodes the same amino acid sequence as SEQ ID NO: 185. In an even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 182, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 182 and encodes the same amino acid sequence as SEQ ID NO: 182; the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 184 and encodes the same amino acid sequence as SEQ ID NO: 184. In another even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 183, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 183 and encodes the same amino acid sequence as SEQ ID NO: 183; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 185 and encodes the same amino acid sequence as SEQ ID NO: 185.

[0041] In a 24th aspect of the invention, there is provided a host cell comprising: a) the expression vector of the 22nd aspect or any embodiment thereof; and b) an expression vector comprising a nucleic acid sequence encoding the light chain of the first antigen-binding domain of the bispecific binding molecule according to any of the preceding aspects of the invention. In some embodiments of the 24th aspect, the expression vector comprising the nucleic acid sequence encoding the light chain further comprises a promoter operably linked to the nucleic acid sequence encoding the light chain and capable of driving expression of the nucleic acid sequence encoding the light chain. In a more specific embodiment of this aspect, the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 184 and encoding the same amino acid sequence as SEQ ID NO: 184. In another more specific embodiment of this aspect, the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185, or a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 185 and encoding the same amino acid sequence as SEQ ID NO: 185. In an even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 182, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 182 and encodes the same amino acid sequence as SEQ ID NO: 182; the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 184, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 184 and encodes the same amino acid sequence as SEQ ID NO: 184. In another even more specific embodiment of this aspect, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleotide sequence of SEQ ID NO: 183, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 183 and encodes the same amino acid sequence as SEQ ID NO: 183; and the nucleic acid sequence encoding the light chain comprises the nucleotide sequence of SEQ ID NO: 185, or a nucleotide sequence which has at least 70% sequence identity to SEQ ID NO: 185 and encodes the same amino acid sequence as SEQ ID NO: 185.

[0042] In a twenty-fifth aspect of the invention there is provided a host cell comprising an expression vector of the twenty-third aspect or any embodiment thereof.

[0043] In a 26th aspect of the present invention, there is provided a method for producing a bispecific binding molecule disclosed herein, comprising: a) culturing a host cell of the 24th or 25th aspect or any embodiment thereof under conditions that allow expression of a nucleic acid molecule encoding the heavy chain fusion and expression of a nucleic acid molecule encoding the light chain, and association of the expressed heavy chain fusion and the expressed light chain into a bispecific binding molecule; and b) recovering the bispecific binding molecule from the culture medium. In some embodiments of the 26th aspect, the recovered bispecific binding molecule is further purified and / or modified and / or formulated. In a 27th aspect, the present invention provides a method for treating a MUC16-associated fibrotic, inflammatory, immune, or autoimmune disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific binding molecule of any of the preceding aspects or embodiments thereof, or a pharmaceutical composition of the 20th aspect. In some embodiments of the twenty-seventh aspect, the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, hepatic fibrosis, liver cirrhosis, renal fibrosis, glial scar, myocardial fibrosis, arteriosclerosis, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, or adhesive capsulitis.

[0044] In a 28th aspect, the invention provides a method of treating a cancer or malignancy characterized by overexpression of MUC16, comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any of the preceding aspects or embodiments thereof, or the pharmaceutical composition of the 20th aspect. In some embodiments of the 28th aspect, the cancer or malignancy is gynecological cancer (cancer of the female reproductive organs), including cervical, endometrial, fallopian tube, ovarian, uterus, and vaginal; pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, or lung cancer. [Brief explanation of the drawings]

[0045] [Figure 1]Schematic diagram of an exemplary bispecific binding molecule of the invention. Depicted is a binding molecule comprising: (i) an Ig molecule comprising two heavy chain fusions, each comprising a variable region that specifically binds the extracellular domain of MUC16 ("VHMUC16"), a constant region ("CH1," "CH2," and "CH3"), a first peptide linker ("Linker 1"), and a single-chain variable fragment ("scFv") comprising, from N- to C-terminus, a variable light chain region that specifically binds DR5 ("VLDR5"), a second peptide linker ("Linker 2"), and a variable heavy chain region that specifically binds DR5 ("VHDR5"); and (ii) two light chains, each comprising a variable region that specifically binds the extracellular domain of MUC16 ("VLMUC16") and a constant region ("CL"). The resulting molecule is a symmetric, bispecific, tetravalent antibody-like molecule. [Figure 2A] Panel A (Figure 2A) shows an SDS-PAGE gel of antibody IMV-18 expressed in CHO cells and then purified from the culture supernatant by Protein A affinity chromatography and further purified by preparative size-exclusion chromatography. Lane R - reduced gel, lane NR - non-reduced gel, lane M - molecular weight marker. [Figure 2B] Panel B (FIG. 2B) shows the SEC-HPLC chromatogram of purified IMV-18 measured at 214 nm and 280 nm. [Figure 3A] Panel A (FIG. 3A) is a series of plots comparing the cytotoxicity of IMV-18 and the anti-DR5 antibody lexatumumab using the CellTiter-Glo® assay on NCI-H292, CAOV-3, and OVCAR-3 cells, each of which express both MUC16 and DR5, after 2 days of exposure to either IMV-18 (circles) or lexatumumab (triangles). [Figure 3B]Panel B (FIG. 3B) is a series of plots comparing the cytotoxicity of a bispecific antibody targeting human CD38 and DR5 ("IMV-15"; circles), a bispecific antibody targeting human LIV-1 and DR5 ("IMV-20"; squares), or lexatumumab (triangles) using the CellTiter-Glo® assay against MM.1S, SU-DHL-8, Ramos, RPMI-8226, and MOLP-8 cell lines, which express CD38, LIV-1, and DR5, respectively (Table 11), after 2 days of exposure to various agents. [Figure 3C] Panel C (FIG. 3C) is a series of plots comparing the cytotoxicity of a bispecific antibody targeting human MUC16 and DR5 ("MCLX-SE," squares), a bispecific antibody targeting fluorescein and DR5 ("FLX-SE," triangles), and a monospecific antibody targeting human MUC16 ("MC-SE," circles) against various cell lines. Cytotoxicity was assessed using the CellTiter-Glo® assay and was performed on the following cell lines: HPAC, PK-59, NCI-H1975, OVCAR-3, NCC-StC-K140, and HDQ-P1, each expressing MUC16 and DR5 (Table 11). [Figure 4A] Panel A (FIG. 4A) is a plot showing the mean tumor volume (Y-axis; mm) after the indicated days (X-axis) in a HPAC xenograft model in BALB / c nude mice (n=5 per group) intravenously administered a single dose of vehicle alone (PBS) (Group 1 (G1)), a bispecific antibody targeting CD74 and DR5 ("IMV-21"; 5 mg / kg in PBS, Group 2 (G2)), or IMV-18 (5 mg / kg in PBS, Group 3 (G3)). HPAC cells express both MUC16 and DR5. Intravenous administration of test agents was performed when the mean tumor volume reached approximately 145 mm. [Figure 4B] Panel B (FIG. 4B) shows the mean body weight (X-axis; grams) after the indicated number of days (Y-axis) following administration of each of the drugs administered in FIG. 4A. [Figure 4C]Panel C (FIG. 4C) shows tumor volume (Y-axis; mm 3 ) in individual mice at the indicated number of days (X-axis) after drug administration. [Figure 5A] Panel A (Figure 5A) is a series of plots comparing the growth inhibitory effects of sequential exposure of PK-59 cells to various concentrations of a bispecific antibody targeting human MUC16 and DR5 ("MCLX-SE"), a bispecific antibody targeting fluorescein and DR5 ("FLX-SE"), and a monospecific antibody targeting human MUC16 ("MC-SE"). Cells were stained with Incucyte® Nuclight Rapid Red Dye for nuclear labeling and counted as red fluorescent objects every 2 hours using an Incucyte S3 (Sartorius AG, sartorius.com). The y-axis is plotted on a logarithmic (base 10) scale. For each condition, the average value from three wells was plotted. Each plot (except the control plot "medium") compares the time course of proliferation of cells sequentially exposed to the same concentration of either MC-SE (circles), FLX-SE (squares), or MCLX-SE (triangles). Cells exposed to medium alone (plot "Medium") continued to grow at the same rate (same doubling time) throughout the experiment, forming a straight line on this semi-log plot. At every concentration between 41.2 pM and 10 nM, cells exposed to the parent monospecific anti-MUC16 antibody MC-SE continued to grow at a similar rate to cells exposed to medium alone, demonstrating cell viability. [Figure 5B]Panel B (FIG. 5B) shows a time course of apoptotic events in PK-59 cells exposed sequentially to a bispecific antibody targeting human MUC16 and DR5 ("MCLX-SE"), a bispecific antibody targeting fluorescein and DR5 ("FLX-SE"), or a monospecific antibody targeting human MUC16 ("MC-SE"), all at 41.2 pM, or to medium alone (control). Cells were stained with Incucyte® Caspase-3 / 7 Green Dye for Apoptosis, catalog number 4440, Sartorius (green fluorescence). Caspase-3 / 7 Green Dye is a fluorescent substrate for caspase 3 and caspase 7, two caspases activated late in apoptosis. [Figure 5C] Panel C (FIG. 5C) shows another data set from the same experiment, plotting the number of apoptotic events per well in PK-59 cells exposed to various concentrations of these three antibodies for 8 hours. [Figure 6A] Panel A (FIG. 6A) shows a plot of tumor volume over time in a HPAC xenograft model in BALB / c nude mice (n=5 per group) intravenously treated with vehicle only (PBS) on day 1 (circles); the bispecific antibody "MCLX-SE" targeting MUC16 and DR5 at 5 mg / kg on day 1, followed by 5 mg / kg on day 13 (upward triangles); 5 mg / kg fluorescein and the bispecific antibody "FLX-SE" targeting DR5 at 5 mg / kg on day 1 (downward triangles); or the monospecific antibody "MC-SE" targeting MUC16 at 5 mg / kg on day 1 (squares). Mean and standard error (SEM) values ​​are shown (n=5). HPAC cells express both MUC16 and DR5 (Table 11). [Figure 6B]Panel B (Figure 6B) shows a plot of tumor volume over time in an HCC827 xenograft model in BALB / c nude mice (n = 5 per group) intravenously treated with vehicle only (PBS) on day 1 (circles); the bispecific antibody "MCLX-SE" targeting MUC16 and DR5 at 5 mg / kg on day 1, followed by 5 mg / kg on day 15 (upward triangles); 5 mg / kg fluorescein and the bispecific antibody "FLX-SE" targeting DR5 at 5 mg / kg on day 1 (downward triangles); or the monospecific antibody "MC-SE" targeting MUC16 at 5 mg / kg (squares). HCC827 cells express both MUC16 and DR5 on day 1. Mean and standard error of the mean (SEM) values ​​are shown (n = 5). Note that in Figure 6B, some error bars overlap at the same time point, obscuring others (see, e.g., some time points for vehicle, FLX-SE, and MC-SE). [Figure 6C] Panel C (FIG. 6C) is a plot of tumor volume over time in a PK-59 xenograft model in BALB / c nude mice (n=4 per group) intravenously administered vehicle alone (PBS) (circles) or the bispecific antibody "MCLX-SE" targeting MUC16 and DR5 at 5 mg / kg (squares) on day 1. PK-59 and NCI-H1975 cells express both MUC16 and DR5. Mean and standard error (SEM) values ​​are shown (n=4). [Figure 6D] Panel D (Figure 6D) shows a time plot of tumor volume in the NCI-H1975 xenograft model in BALB / c nude mice (n = 4 per group) intravenously treated with vehicle alone (PBS) (circles) or the bispecific antibody "MCLX-SE" targeting MUC16 and DR5 at 5 mg / kg (squares) on day 1. PK-59 and NCI-H1975 cells express both MUC16 and DR5. Mean and standard error (SEM) values ​​are shown (n = 4). Note that for some data points in each of Figures 6A-6D, the error bars are obscured by the data points themselves. [Figure 7]FIG. 1 shows the effect of various anti-MUC16-anti-DR5 bispecific antibodies (IMV-18, MCLX-SE, IMV-AA, or MC-AA) administered alone or in combination with excess non-targeting murine IgG1 antibody (muIgG1) on tumor growth in PK-59 pancreatic cancer xenograft mice over time. [Figure 8] 1 is a plot of the effect of varying amounts of anti-MUC16-anti-DR5 bispecific antibody IMV-M over time on tumor growth in PK-59 pancreatic cancer xenograft mice. DETAILED DESCRIPTION OF THE INVENTION

[0046] definition Terms not specifically defined herein shall be given the meaning that would be given to them by one of ordinary skill in the art in light of this disclosure and the context. However, as used herein, unless otherwise indicated, the following terms have the meanings indicated and are subject to the following rules:

[0047] As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0048] As used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0049] As used herein, the terms "about" and "approximately" when referring to a measurable value such as a polypeptide amount, dosage, time, temperature, enzyme activity, or other biological activity, are meant to encompass variations of ±20%, ±10%, ±5%, ±1%, +0.5%, or even ±0.1% of the stated amount.

[0050] The transitional phrase "consisting essentially of" means that the scope of a claim should be construed to include the specific materials or steps recited in the claim, as well as those "which do not materially affect the basic and novel characteristics" of the claimed invention. See In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03.

[0051] The term "consisting essentially of" (and grammatical variations) as applied to polynucleotide or polypeptide sequences of the present disclosure means a polynucleotide or polypeptide that consists of both a recited sequence (e.g., SEQ ID NO:) and a total of 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids at the N-terminus and / or C-terminus of the recited sequence, such that the function of the polypeptide is not substantially altered. A total of 10 or fewer additional amino acids can include the total number of additional amino acids at both termini added together.

[0052] The term "human MUC16," as used herein, includes any variants, isoforms, and species homologs of human MUC16 (mucin 16, NCBI Entrez Gene:94025) expressed by cells on their surface. The term "recombinant human MUC16," as used herein, includes human MUC16 (NCBI Entrez Gene:94025; UniProtKB-Q8WXI7), available on the World Wide Web at uniprot.org, and the nucleic acid sequence encoding the protein. The term "MUC16" is also intended to include variants (e.g., allelic variants) and derivatives thereof. Representative human MUC16 cDNA and human MUC16 protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). Human MUC16 variants include mucin-16 precursor (NM_001401501.2 and NP_001388430.1) and mucin-16 (NM_024690.2 and NP_078966.2). The nucleic acid and polypeptide sequences of MUC16 orthologs in organisms other than humans are known.

[0053] The term "death receptor 5" is also known as DR5; tumor necrosis factor receptor superfamily member 10B, TNFRSF10B, TNF-related apoptosis-inducing ligand receptor 2, TRAIL receptor 2, TRAIL-R2, and CD262, TNF receptor superfamily member 10b, KILLER, KILLER / DR5, TRICK2, TRICK2A, TRICK2B, TRICKB, ZTNFR9. The term includes any variants, isoforms, and species homologs of the human protein provided in UniProt O14763, available on the World Wide Web at uniprot.org / uniprot / O14763, and the nucleic acid sequences encoding the proteins.

[0054] As used herein, the term "analog" includes any compound discovered in structure-activity relationship studies (e.g., Schnecke V. and Bostroem J. Drug Discovery Today. 11(1-2): 43-50 (2006)) that has a similar structure to another compound but differs with respect to a specific component (e.g., Willett P. et al. Informat. Comp. Sci. 38: 983-996 (1998), Johnson AM and Maggiora GM. Concepts and Applications of Molecular Similarity. New York: John Willey & Sons. ISBN 978-0-471-62175-1 (1990), Nikolova N. and Jaworska J. QSAR & Combinatorial Science. 22: 1006-1026 (2003)); specifically, for example, those compounds can be used in combination with compounds depicted in Scheme 1 (Sijbrandi NJ et al. Cancer Res;77;257-67(2017)) or depicted in Scheme 2 and described in (Drake PM et al. Bioconjug. Chem. 25:1331-1341(2014)).

[0055] The term "variant," as used herein in reference to a reference amino acid sequence (e.g., a particular SEQ ID NO:), means an amino acid sequence in which one or more amino acids have been inserted, deleted, or substituted compared to the reference sequence.

[0056] As used herein in reference to a reference antibody, the term "derivative" refers to an immunoglobulin that has been chemically or recombinantly modified to include a moiety with a different activity from that of the reference antibody, without destroying the ability of the reference antibody to bind to its intended antigen. For example, the moiety may be a second antibody or antigen-binding portion thereof that recognizes a different antibody; a peptide or chemical "tag" that allows the antibody to be recognized (e.g., for purification) or identified (e.g., a fluorescent molecule); a small molecule (e.g., an anti-cancer drug) that has activity against cells targeted by the antibody; a radioisotope; etc. As used herein in reference to a compound or small peptide, the term "derivative" includes any compound that is derived from a similar compound by chemical reaction and / or that can, at least theoretically, be formed from a precursor compound (see, e.g., Oxford Dictionary of Biochemistry and Molecular Biology, Oxford University Press, ISBN 0-19-850673-2).

[0057] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light chains and one pair of heavy chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized (see, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. Each light chain typically consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions, which may be hypervariable in sequence and / or in the form of structurally defined loops) interspersed with more conserved regions, also called complementarity-determining regions (CDRs), also called framework regions (FRs). VH and VL each typically consist of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk See also J. Mol. Biol. 196, 901-917 (1987).

[0058] The term "expression vector" in the context of this invention refers to a plasmid or virus designed for expressing genes in cells. Various types of expression vectors are well known in the art.

[0059] The term "antibody" in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof that is capable of specifically binding to an antigen under typical physiological conditions. An antibody is the source of each of the two distinct binding sites present in the bispecific binding molecules disclosed herein. Thus, the terms "antibody" and "immunoglobulin" are used interchangeably and refer to: 1) molecules that have all the structural features of an immunoglobulin (e.g., a pair of light chains and a pair of heavy chains linked together by disulfide bonds) and specifically bind to a particular antigen or its epitope, such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, recombinantly produced antibodies, veneered antibodies, humanized antibodies, and murine antibodies; 2) molecules that have only a portion of the immunoglobulin structure but retain the CDRs of three heavy chains and three light chains and retain the ability to specifically bind to an antigen or its epitope, such as Fab, Fab', F(ab'), These include F(ab)3, scFv, scFv-Fc, diabodies, triabodies, tetrabodies, and minibodies (individually and collectively referred to as "antigen-binding fragments"); 3) heavy-chain antibodies, i.e., molecules composed of heavy chains but lacking light chains, and single-domain antibodies; and 4) molecules of 1)-3) that retain the ability to specifically bind to an antigen or its epitope and have been modified (chemically or recombinantly) to contain additional moieties with different activities than the reference antibody or antigen-binding fragment, such as bispecific antibodies, multispecific antibodies, and antibodies conjugated with toxins, radioisotopes, therapeutic agents, fluorescent tags, or peptide tags (individually and collectively referred to as "antibody derivatives"). The moieties of antibody derivatives may be attached to the antibody directly or via a linker, depending on the nature and desired function of the moieties. Each of the above antigen-binding fragments is well known in the art. The term "antibody" includes monoclonal antibodies (full-length four-chain antibodies or full-length heavy-chain antibodies having an immunoglobulin Fc region, antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab'), and Fv)). Antibodies contemplated herein include single-domain antibodies such as heavy-chain antibodies.

[0060] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L chains) and two identical heavy chains (H chains). IgM antibodies consist of five basic heterotetrameric units along with an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies consist of two to five basic four-chain units, which can polymerize and combine with the J chain to form multivalent assemblies. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The term "heavy-chain antibody" (HCAb) refers to a functional antibody composed of heavy chains and lacking the light chains typically found in four-chain antibodies. Camelids (such as camels, llamas, or alpacas) and sharks are known to produce HCAbs. The term "single domain antibody" or "dAb" refers to a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). sdAbs can bind to antigens alone without pairing with a polypeptide containing the corresponding CDRs. In some cases, single domain antibodies are produced from camelids or sharks, and their heavy chain variable domains are referred to herein as "VHHs." Some VHHs may also be known as nanobodies. Camelid sdAbs are among the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)).A basic VHH has the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1, CDR2, and CDR3 refer to complementarity-determining regions 1, 2, and 3, respectively.

[0061] Antibodies present in the bispecific binding molecules of the invention that comprise a heavy chain constant region can be of any isotype. As used herein, "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region genes.

[0062] The term "chimeric antibody" in the context of the present invention refers to an antibody created by fusing an antigen-binding region (heavy and light chain variable domains, VH and VL) derived from one species, such as a mouse, with a constant domain (effector region) derived from a human antibody (see, for example, Morrison SLet et al. Proc. Natl. Acad. Sci. USA 81, 6851-6855 (1984)).

[0063] The term "murine antibody", as used herein, is intended to include antibodies having variable and constant regions derived from mouse germline immunoglobulin sequences.

[0064] As used herein, the term "humanized antibody" refers to murine antibodies in which the variable framework and constant regions have been replaced with the corresponding regions from human germline immunoglobulin sequences.

[0065] A "humanized" antibody refers to an antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the HVRs (e.g., complementarity-determining regions (CDRs)) corresponding to those of a non-human antibody and all or substantially all of the entire framework regions (FRs) corresponding to those of a human antibody. In this context, "substantially" means that both the heavy and light chains of the humanized antibody share at least 80% sequence identity with the top human germline V-gene hits. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0066] Murine, chimeric, and humanized antibodies that may be present in the bispecific binding molecules of the invention may contain amino acid residues not encoded by their respective germline immunoglobulin sequences, such as by mutations, substitutions, deletions, or insertions introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. These antibodies are referred to as "mutants" because of these amino acid changes.

[0067] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds MUC16 is substantially free of antibodies that specifically bind to antigens other than MUC16; an isolated bispecific antibody that specifically binds MUC16 and DR5 is substantially free of antibodies that specifically bind to antigens other than MUC16 and DR5). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of human MUC16 may have cross-reactivity to other related antigens, for example, from other species (such as MUC16 species homologs). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0068] As used herein, the term "bispecific binding molecule" refers to a molecule that can specifically bind to two different antigens (e.g., MUC16 and DR5), i.e., a molecule that has two different "antigen-binding sites."

[0069] The term "antigen-binding site" refers to the portion of an antibody or bispecific binding molecule that contains the amino acids responsible for specific binding between the antibody or bispecific binding molecule and a particular antigen. If the antigen is large, the antigen-binding domain may only bind to a portion of the antigen. The portion of the antigen molecule responsible for specific interaction with the antigen-binding domain is called the "epitope" or "antigenic determinant."

[0070] An antigen-binding site typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not necessarily comprise both. For example, the second antigen-binding site of a bispecific binding molecule of the present invention may consist of only the VH domain of an anti-DR5 antibody, while still retaining the ability to bind and agonize DR5. Each variable region or domain comprises three CDRs. The generalized structure of an antibody or immunoglobulin molecule is well known to those skilled in the art.

[0071] As used herein, the term "bispecific antibody" refers to a bispecific binding molecule in which each antigen-binding site is derived from an antibody. The term "bispecific antibody" is intended to include any antibody with two different binding specificities, including, inter alia, tetravalent antibody molecules containing IgG1 or fragments or variants thereof with one antigen specificity, as well as antigen-binding fragments of antibodies with another antigen specificity, such as VHHs, Fabs, Fab's, F(ab')2, F(ab)3, scFvs, scFv-Fc, diabodies, triabodies, tetrabodies, and minibodies. The term "bispecific antibody" also includes diabodies (see, e.g., Holliger, P. et al., PNAS USA 90, 6444-6448 (1993); Poljak, R.J. et al., Structure 2, 1121-1123 (1994)).

[0072] As used herein, the term "binding" in the context of binding of an antibody to a given antigen typically refers to a binding activity that is greater than or equal to the K, as measured by, for example, surface plasmon resonance (SPR) technology on a BIAcore or Octet instrument, using the antigen as the ligand and the antibody as the analyte. D and binding with an affinity (dissociation constant) of about 10,000 nM or less, such as about 1,000 nM or less, for example about 100 nM or less, such as about 10 nM or less, for example about 1 nM or less, about 0.1 nM or less, or about 10 pM or less, and further having a K that is at least 10-fold lower, such as at least 100-fold lower, for example at least 1,000-fold lower, such as at least 10,000-fold lower, for example at least 100,000-fold lower, such as at least 100,000-fold lower, compared to the binding affinity for a non-specific antigen other than the predetermined antigen (e.g., bovine serum albumin, casein, etc.), a closely related antigen, or, in the case of a bispecific or multispecific antibody, an antigen other than the antigen targeted by such other specificity. D The amount at which the affinity decreases is the antibody's K D Because it depends on the antibody's K D is very low (i.e., the antibody is highly specific), the amount by which affinity for the antigen is reduced below affinity for a nonspecific antigen may be at least 10,000-fold.

[0073] As used herein, the term "k D " refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is k off Also called value.

[0074] As used herein, the term "k A (M -1 ·Seconds -1 )" refers to the dissociation rate constant of a particular antibody-antigen interaction.

[0075] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0076] As used herein, the term "EC 50" (M) refers to the concentration of antibody that binds to and targets cells expressing an antigen, such as MUC16, such that the cells reach 50% of maximum fluorescence as measured by flow cytometry.

[0077] The term "percent sequence identity" as used herein refers to the percentage of amino acids or nucleotides in a candidate sequence that are identical to amino acids in a reference amino acid or nucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be determined using any mathematical algorithm known in the art for determining amino acid sequence homology. One such algorithm is the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Another is the GAP program of the GCG software package (available on the World Wide Web at the GCG corporate website), which uses either a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Still other algorithms that can be used to determine sequence identity include ClustalW, Clustal Omega, BLAST, BLAST-2, ALIGN-2 or Megalign (DNASTAR).

[0078] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational epitopes are distinguished from nonconformational epitopes in that the binding of the former is lost in the presence of denaturing solvents, but not the latter. Epitopes may include amino acid residues directly involved in binding (also called immunodominant components of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by the specific antigen-binding peptide (in other words, amino acid residues within the footprint of the specific antigen-binding peptide).

[0079] As used herein, the terms "inhibit growth" or "inhibit proliferation," used interchangeably (e.g., referring to cells such as tumor cells), are intended to include any measurable decrease in cell growth (which means the same as cell proliferation) or decrease in cell number when contacted with an anti-MUC16 antibody, or with an antibody that binds another antigen, or with a non-targeting antibody, or with a bispecific antibody, or with any other molecule, compared to the growth of the same cells not contacted with the anti-MUC16 antibody, or with an antibody that binds another antigen, or with a non-targeting antibody, or with a bispecific antibody, or with any other molecule, such as, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% inhibition of cell culture growth. Such a decrease in cell proliferation can occur by various mechanisms, e.g., ADCC, ADCP, CDC, cell cycle arrest, and / or apoptosis, and / or a decrease in cell number.

[0080] As used herein, the term "cytotoxicity" or "cytotoxic effect" (e.g., referring to cells such as tumor cells) is intended to include any measurable decrease in cell growth or decrease in cell number when contacted with an anti-MUC16 antibody, or with an antibody that binds to another antigen, or with a non-targeting antibody, or with a bispecific antibody molecule disclosed herein, or with any other molecule, compared to the growth of the same cells in the absence of such antibody or molecule, such as a cytotoxic effect of, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of the cell culture medium. Such a cytotoxic effect can occur by various mechanisms, e.g., ADCC, CDC, ADCP, cell cycle arrest, and / or apoptosis, and / or a decrease in cell number. In some embodiments, the cytotoxicity or cytotoxic effect is observed in cells that are positive for MUC16 and DR5 ("MUC16"). + / DR5 + "), or a decrease in cell growth or a decrease in cell number of cells that are positive for another antigen and DR5.

[0081] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector has been introduced. Such terms are intended to refer not only to the subject cell but also to the progeny of such a cell. Because certain changes may occur in successive generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas such as CHO cells, HEK293 cells, NS / 0 cells, and lymphocytic cells.

[0082] In the context of the present invention, the terms "solid tumor" and "solid cancer", which are used interchangeably, include, but are not limited to, malignant tumors, i.e., epithelial ovarian cancer or any other ovarian cancer, pancreatic ductal adenocarcinoma or any other pancreatic cancer, esophageal adenocarcinoma or any other esophageal cancer, gastric adenocarcinoma or any other gastric cancer, colon cancer, colorectal cancer, invasive papillary carcinoma of the breast, any other breast cancer, non-small cell lung cancer, or any other lung cancer.

[0083] The term "hematological cancer" in the context of the present invention includes acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, lymphoma, Hodgkin's lymphoma (all four subtypes), non-Hodgkin's lymphoma (all subtypes), small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, B-cell chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse large intestinal ... These include, but are not limited to, malignant hematological diseases such as idiopathic large B-cell lymphoma, MM, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytoma, e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, MALT lymphoma, nodal marginal zone B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, hairy cell leukemia, and primary effusion lymphoma.

[0084] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the present invention with the purpose of alleviating, ameliorating, preventing, or eradicating (curing) the symptoms or condition.

[0085] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a bispecific binding molecule disclosed herein may vary depending on factors such as the individual's condition, age, sex, and weight, and the molecule's ability to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. In some embodiments, an "effective amount" refers to an amount effective to administer MUC16 + / DR5 + The amount of ATP that causes cytotoxicity in cells that are resistant to ATP.

[0086] The antigen-binding domain of an antibody is located in the so-called variable domain, or variable region (Fv) of the antibody. The variable domain contains three so-called complementarity-determining regions (CDRs) separated by framework regions (FRs). The CDRs are the regions of an antibody responsible for the specificity (binding) of the antibody for a particular antigen.

[0087] In the context of the present invention, references to the CDRs of the MUC16-binding domain and the DR5-binding domain are based on the CDRs reported in the prior art disclosing various MUC16 and DR5 antibodies. These CDRs typically include Kabat (EAKabat, et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda (1983), IMGT (Lefranc MP, et al., Dev Comp Immunol. 2003 January;27(1):55-77; Giudicelli V et al., Cold Spring Harb Protoc.2011;2011(6):695-715), Chothia(Chothia and Lesk,J.Mol.Biol.1987,196:901-917), AbM(Martin and Allen,2007,Bioinformatics tools for antibody engineering.In:Duebel S,editor.Handbook of Therapeutic Antibodies.Weinheim:Wiley-VCH Verlag GmbH(2008).p.95-117;Abhinandan and Martin, 2008, Protein Eng Des Sel. (2010) 23:689-97), or based on the CDR definition of Contact (Padlan EA, et al., FASEB J Off Publ Fed Am Soc Exp Biol. (1995) 9:133-9).

[0088] As used herein, the expressions "variable domain" or "variable region" or Fv refer to each of a pair of light and heavy chains directly involved in binding of an antibody to an antigen. The light chain variable domain is abbreviated as "VL," and the heavy chain variable domain is abbreviated as "VH." The light and heavy chain variable domains have the same overall structure, with each domain consisting of four framework (FR) regions, the sequences of which are widely conserved, and connected by three HVRs (or CDRs). The framework regions adopt a β-sheet structure, and the CDRs may form loops connecting the β-sheet structure. The CDRs of each chain are held in a three-dimensional structure by the framework regions and, together with the CDRs of the other chain, form an antigen-binding site.

[0089] As used herein, the term "constant domain" or "constant region" refers to the collection of antibody domains other than the variable region. Such constant domains and regions are well known in the art and are described, for example, by Kabat et al. ("Sequence of proteins of immunological interest", U.S. Public Health Services, NIH, Bethesda, Md., Publication No. 91).

[0090] The "Fc portion" or "Fc region" of an antibody is not directly involved in binding of the antibody to an antigen but exhibits various effector functions. The term "Fc portion of an antibody" is well known to those skilled in the art and is defined based on papain cleavage of an antibody. Antibodies or immunoglobulins are classified into classes IgA, IgD, IgE, IgG, and IgM based on the amino acid sequence of the constant region of their heavy chains. The different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively, based on the constant region of the heavy chain. Some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The Fc portion of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, Clq binding, and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor Clq to the Fc portion of most IgG antibody subclasses. Although the effect of antibodies on the complement system depends on the specific conditions, binding to Clq is mediated by a defined binding site in the Fc portion. Such binding sites are known in the art and are described, for example, in Boakle et al., Nature 282 (1975) 742-743; Lukas et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse and Cebra, Mol. Immunol. 16 (1979) 907-917; Burton et al., Nature 288 (1980) 338-344; Thommesen et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie et al., J. Immunol. 164 (2000) 4178-4184; Hezareh et al., J. Virology 75 (2001) 12161-12168; Morgan et al., Immunology 86 (1995) 319-324, and EP 0 307 434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat, see below).Among these residues, the most important for mediating Clq and Fcγ receptor binding in IgG1 are L234 and L235 (Hezareh et al., J. Virology 75 (2001) 12161-12168). Antibodies of the subclasses IgG1 and IgG3 typically exhibit complement activation and Clq and C3 binding, whereas IgG2 and IgG4 do not activate the complement system or bind Clq or C3.

[0091] The art has further developed antibodies, making them versatile tools in medicine and technology. Thus, in the context of the present invention, the terms "antibody molecule" or "antibody" (used interchangeably herein) not only include antibodies that may be found in nature, e.g., comprising two light chains and two heavy chains, or comprising only two heavy chains, such as in camelid species, but also encompass any molecule comprising at least one paratope with binding specificity for an antigen and structural similarity to the variable domain of an immunoglobulin.

[0092] Thus, the antibody portion of the bispecific binding molecules provided herein may include monoclonal antibodies, multispecific antibodies, bispecific antibodies, antibody derivatives, human antibodies, recombinant antibodies, veneered antibodies, humanized antibodies, chimeric antibodies, antibody fragments, particularly Fv, Fab, Fab', and F(ab')2 fragments, single-chain antibodies, particularly scFv, small modular immunopharmaceuticals (SMIPs), domain antibodies, nanobodies, and diabodies. Antibodies may have effector functions, such as ADCC or CDC, which are typically mediated by the Fc portion (antibody constant region) of the antibody, or may lack effector functions, for example, by lacking an Fc portion or by having a blocked or masked Fc portion, i.e., an Fc portion that is not recognized or is poorly recognized by immune system components such as immune cells or the complement system. Monoclonal antibodies (mAbs) are monospecific antibodies with identical amino acid sequences. Monoclonal antibodies are produced by hybridoma technology from hybrid cell lines (called hybridomas) that represent the fusion of specific antibody-producing B cells with myeloma (B-cell cancer) cells (Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:495-7). Alternatively, monoclonal antibodies may be produced by recombinant expression in host cells (Norderhaug L, Olafsen T, Michaelsen TE, Sandlie I. (1997) J Immunol Methods 204(1):77-87; see also below). A "recombinant antibody" or "recombinant binding molecule" is an antibody or binding molecule produced by a host cell engineered by recombinant techniques, which is optionally isolated or purified.

[0093] For human application, it is often desirable to reduce the immunogenicity of antibodies originally derived from other species, such as mice. This can be achieved by constructing chimeric antibodies, or a process called "humanization." In this context, a "chimeric antibody" is understood to be an antibody containing a sequence portion (e.g., a variable domain) from a different species (e.g., mouse) fused with a sequence portion (e.g., a constant domain) from one species (e.g., human). A "humanized antibody" is an antibody containing a variable domain originally derived from a non-human species, in which certain amino acids have been mutated to more closely resemble the overall sequence of the variable domain of a human. Methods for chimerizing and humanizing antibodies are well known in the art (Billetta R, Lobuglio AF. Int. Immunol. 1993;10:165-76; Riechmann L, Clark M, Waldmann H, Winter G (1988). Nature:332:323).

[0094] Furthermore, techniques for producing antibodies based on sequences derived from the human genome have been developed, for example, by using phage display methods or transgenic animals (WO 90 / 05144; D. Marks, H.R. Hoogenboom, T.P. Bonnert, J. McCafferty, A.D. Griffiths and G. Winter (1991) J. Mol. Biol., 222, 581-597; Knappik et al., J. Mol. Biol. 296:57-86, 2000; S. Carmen and L. Jermutus, Briefings in Functional Genomics and Proteomics 2002 1(2):189-203; Lonberg N, Huszar D. Int Rev Immunol. 1995; 13:65-93; Bruggemann M, Taussig M. J. Curr Opin Biotechnol. 1997 August;8:455-458). Such antibodies are "human antibodies" in the context of the present invention.

[0095] Antibodies can also include immunoglobulin fragments that retain antigen-binding properties, such as Fab, Fab', or F(ab')2 fragments. Such fragments can be obtained, for example, by fragmenting immunoglobulins by proteolytic digestion or by recombinant expression of such fragments. For example, immunoglobulin digestion can be performed by conventional techniques, for example, using papain or pepsin (WO 94 / 29348). Papain digestion of an antibody typically yields two identical antigen-binding fragments, so-called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment. Pepsin treatment yields F(ab')2. In Fab molecules, the variable domains are each fused to an immunoglobulin constant domain, preferably of human origin. Thus, the heavy chain variable domain may be fused to a CH1 domain (so-called Fd fragment), and the light chain variable domain may be fused to a CL domain. Fab molecules may also be produced by recombinant expression of the respective nucleic acid in a host cell. See below.

[0096] Several techniques have been developed to place immunoglobulin variable domains, or molecules derived from such variable domains, in different molecular contexts. These should also be considered "antibodies" in accordance with the present invention. Generally, these antibody molecules are small in size compared to immunoglobulins and may contain one or several amino acid chains. For example, single-chain variable fragments (scFvs) are fusions of the variable regions of the heavy and light chains of immunoglobulins, linked by a short linker, usually serine (S) or glycine (G) (WO 88 / 01649; WO 91 / 17271; Huston et al.; International Immunol. 10, 1993, 195-217). "VHH," or "single-domain antibody," or "nanobody" has an antigen-binding site in a single Ig-like domain (WO 94 / 04678; WO 03 / 050531; Ward et al., Nature. 1989 Oct. 12; 341:544-546; Revets et al., Expert Opin Biol Ther. 5:111-24, 2005). One or more single-domain antibodies with binding specificities for the same or different antigens can be linked together. Diabodies are bivalent antibody molecules consisting of two amino acid chains containing two variable domains (WO 94 / 13804; Holliger et al., Proc. Natl. Acad. Sci. USA. 1993; 90:6444-8). Other examples of antibody-like molecules are immunoglobulin superfamily antibodies (IgSF; Srinivasan and Roeske, Current Protein Pept. Sci. 2005, 6:185-96). A different concept leads to the so-called small modular immunopharmaceuticals (SMIPs), which contain an Fv domain linked to a single hinge and effector domain without a constant domain CH1 (WO 02 / 056910).

[0097] bispecific binding molecules The present disclosure provides bispecific binding molecules having at least one antigen-binding domain (first antigen-binding domain) that specifically binds to the extracellular domain of MUC16 and at least one antigen-binding domain (second antigen-binding domain) that specifically binds to DR5. The provided bispecific binding molecules can induce apoptosis of MUC16-expressing cells that also express DR5. The second antigen-binding domain stimulates the apoptotic activity of DR5 upon binding to DR5. The first antigen-binding domain creates specificity that enhances the stimulated apoptotic activity of DR5 on MUC16-expressing cells. Without wishing to be bound by theory, the applicant believes that the function of the first antigen-binding domain to bind to the extracellular domain of MUC16, and thus enhances the agonist effect caused by the second, anti-DR5 antigen-binding domain, is important. MUC16 is a highly glycosylated protein with 14,507 amino acids. MUC16 consists of a 14,451-amino acid extracellular domain, a 21-amino acid transmembrane domain, and a 35-amino acid C-terminal cytoplasmic domain. As disclosed herein, applicants have demonstrated that bispecific antibodies comprising anti-MUC16 antibodies targeting multiple MUC16 epitopes present in the extracellular domain of MUC16 within its TR domain are effective in enhancing the agonistic effect of a second anti-DR5-binding domain. Applicants believe this enhancement is due to the clustering of DR5 on the cell surface by multiple bispecific antibody molecules bound to the same MUC16 molecule. Therefore, applicants believe that bispecific antibodies comprising anti-MUC16 antibodies targeting unique MUC16 epitopes outside the TR / SEA domain of MUC16 (i.e., epitopes present only once per MUC16 molecule) are inefficient in enhancing the agonistic effect of a second anti-DR5-binding domain. Furthermore, the ability of MUC16 to enhance the agonist effect by binding of the second antigen-binding domain to DR5 is not shared by other cell surface proteins.For example, an antibody targeting CD44v6 (a splice variant of CD44 known as a tumor-associated antigen with a preferential expression pattern in tumors over normal tissues) did not enhance the agonistic effect of death receptor-binding molecules when fused to an anti-DR5 antibody (see U.S. Patent No. 10,858,438).

[0098] Furthermore, applicants' own experiments have shown that: a) bispecific binding molecules that bind to both CD38 and DR5 are unable to kill cells expressing both antigens; and b) bispecific binding molecules that bind to the cell surface antigen LIV-1 and DR5 binding sites are ineffective at killing cells expressing both LIV-1 and DR5. See Example 3. Based on these results, the utility of MUC16 as an antigen capable of enhancing DR5-mediated apoptosis by the anti-MUC16 / anti-DR5 bispecific binding molecules disclosed herein was not reasonably predictable. As noted above, until the present invention, applicants were unaware of, much less suggested, any binding molecules capable of specifically binding to both DR5 and MUC16. Nevertheless, each protein and its associated gene, as well as antibodies specific for each, are known in the art and are well represented in biological databases.

[0099] In the context of the present invention, in some embodiments, bispecific binding molecules are derived from antibodies. Techniques for producing bispecific binding molecules include, but are not limited to, recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Bispecific binding molecules of the invention can also be prepared by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., Immunol., 148:1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., al., Immunol., 152:5368 (1994); and, for example, by preparing trispecific antibodies as described in Tutt et al., Immunol., 147:60 (1991).

[0100] As described above, the first antigen-binding domain binds to the extracellular domain of MUC16. In some embodiments, the first antigen-binding domain binds to an epitope present in one or more tandem repeat / SEA segments within the extracellular domain of MUC16. In some embodiments, the first antigen-binding domain is derived from an antibody known to bind to the extracellular domain of MUC16. In some embodiments, the first antigen-binding domain has the same heavy and light chain CDRs as those present in the antibody that binds to the extracellular domain of MUC16. In some embodiments, the first antigen-binding domain has the same heavy and / or light chain variable regions as those present in the antibody that binds to the extracellular domain of MUC16. In some embodiments, the first antigen-binding domain has the same heavy and / or light chains as those present in the antibody that binds to the extracellular domain of MUC16. Antibodies that bind to the extracellular domain of MUC16 are known in the art and include, but are not limited to, OC125, H185 (Invitrogen catalog no. MA5-11579), M11 (American Tissue Culture Collection accession no. PTA-6206), OV197 (Fujirebio Diagnostic), 5E11 (Millipore Sigma catalog no. MABC1608-25UG), AR9.6, H1H8794, VK-8, B43.13 (also known as oregovomab), or 3A5 (sofituzumab).These antibodies have been reported in Aithal et al. Exp. Opin. Therapeutic Targets 2018:22,675-686; Bressan et al., Disease Markers 34(2013)257-267; White et al., Proteins, 2022;90:1210-1218; Argueso et al. Investigat. Ophthalmol. Visual Sci. 2003;44,2487-2495; Eric Nunez Aguilar “HARNESSING ANTIBODIES FOR TREATING PANCREATIC CANCER: AR9.6-A MUC16 SPECIFIC MONOCLONAL ANTIBODY”, a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology and Mathematics, California State University, Fresno, May 2021; Marcos-Silva et al. al. Glycobiology, 2015, vol. 25, no. 11, 1172-1182; U.S. Patent No. 7,989,595; and Chen et al. Cancer Res. 2007:67, 4924-4932.

[0101] In other embodiments, antibodies to MUC16 can be generated using standard monoclonal antibody techniques. For these newly generated antibodies, determining whether they bind to the TR / SEA portion of the extracellular domain can be accomplished by methods known in the art. One way to make such a determination is to determine whether the new MUC16 antibody competes for binding to MUC16 or its extracellular fragment by one or more of the known antibodies described above that bind to the TR / SEA portion of the extracellular domain of MUC16. Another method for determining whether an anti-MUC16 antibody binds to the TR / SEA portion of the extracellular domain is by ELISA or another well-known antigen-antibody binding assay using a peptide or polypeptide from the TR / SEA portion of the MUC16 extracellular domain or a derivative thereof as the antigen. These antigens include at least one amino acid sequence selected from SEQ ID NOs: 176-181, or a peptide or polypeptide consisting essentially of an extracellular fragment of MUC16 isolated from the cell culture medium of OVCAR-3 cells.

[0102] In certain embodiments, the first antigen-binding domain comprises a heavy chain and a light chain, the amino acid sequences of which are derived from an antibody that binds to the extracellular domain of MUC16, and the specificity for MUC16 is determined by the CDRs present in each of the heavy and light chains. In certain embodiments, the amino acid sequences of the heavy and / or light chains differ from those of the antibody from which they are derived to improve one or more properties for use in humans. Typically, these differences include conservative substitutions of fewer than five amino acids. See Tables 5-8.

[0103] As described above, the second antigen-binding domain binds to DR5. In certain embodiments, the second antigen-binding domain is an amino acid sequence derived from an antibody that binds to DR5. In some embodiments, the parent antibody also agonizes DR5. In some embodiments, the second antigen-binding domain has the same heavy and light chain CDRs as those present in the antibody that binds to DR5. In some embodiments, the second antigen-binding domain has the same heavy and / or light chain variable regions as those present in the antibody that binds to DR5.

[0104] Antibodies that bind to DR5 are known in the art and include, but are not limited to, conatumumab (AMG655), drozitumab (Apomab or PRO955780), lexatumumab (HGS-TR2), LBY135, tigatuzumab (CS-1008 or TRA-8), and DS-8273a. In certain embodiments, the amino acid sequence of the second antigen-binding domain is derived from an scFv that binds to and agonizes DR5, and the specificity for DR5 is determined by the CDRs present in the scFv.

[0105] In other embodiments, antibodies against DR5 can be generated using standard monoclonal antibody technology. For these newly generated antibodies, determining whether they bind to DR5 can be accomplished by methods known in the art. One way to make such a determination is whether the new DR5 antibody is competed for binding to DR5 by one or more of the known antibodies listed above that bind to DR5. Another way to determine whether an anti-DR5 antibody binds to DR5 is whether binding to DR5 is competed by TRAIL or a fragment or derivative of TRAIL that binds to DR5. Yet another way to determine whether an antibody binds to DR5 is by ELISA or another well-known antigen-antibody binding assay using DR5 or a peptide or polypeptide fragment of DR5 as the antigen.

[0106] In some embodiments, a bispecific binding molecule comprises two polypeptide chains, each comprising a variable heavy chain region and a heavy chain CDR, and two polypeptide chains, each comprising a variable light chain region and a light chain CDR; this combination of polypeptide chains enables the bispecific molecule to specifically bind to the TR / SEA region of MUC16. In some embodiments, the two polypeptide chains, each comprising a variable heavy chain region, have the same amino acid sequence. In some embodiments, the two polypeptide chains, each comprising a variable light chain region, have the same amino acid sequence. In some embodiments, the combination of the two variable heavy chain regions (or CDRs therein) and the two variable light chain regions (or CDRs therein) defines a first antigen-binding domain. In some embodiments, the polypeptide chain comprising the variable heavy chain region is derived from the heavy chain of an antibody that binds to the TR / SEA region of MUC16. In some embodiments, the polypeptide chain comprising the variable light chain region is derived from the light chain of an antibody that binds to the TR / SEA region of MUC16. In some embodiments, both the polypeptide chain comprising the variable heavy chain region and the polypeptide chain comprising the light chain region are derived from the same MUC16 antibody.

[0107] In some embodiments, the polypeptide chain comprising the MUC16-specific variable heavy chain region further comprises a second antigen-binding domain. In some embodiments, the polypeptide chain comprising the MUC16-specific variable light chain region further comprises a second antigen-binding domain.

[0108] The location of the second antigen-binding domain relative to the first antigen-binding domain can vary, including all of the following: 1) the N-terminus of the second antigen-binding domain is linked to the C-terminus of the heavy chain of the first antigen-binding domain, either directly or via a peptide linker; 2) the N-terminus of the second antigen-binding domain is linked to the C-terminus of the light chain of the first antigen-binding domain, either directly or via a peptide linker; 3) the N-terminus of the heavy chain of the first antigen-binding domain is linked to the C-terminus of the second antigen-binding domain, either directly or via a peptide linker; 4) the N-terminus of the heavy chain of the first antigen-binding domain is linked to the C-terminus of the second antigen-binding domain, either directly or via a peptide linker; 5) a second antigen-binding domain bound directly or via a linker to an amino acid other than the C- or N-terminal amino acid of the heavy chain of the first antigen-binding domain; or 6) a second antigen-binding domain bound directly or via a linker to an amino acid other than the C- or N-terminal amino acid of the light chain of the first antigen-binding domain (e.g., by reaction with a side chain group of such a light chain amino acid). In some specific embodiments, the N-terminus of the second antigen-binding domain is bound directly or via a peptide linker of 4 to 20 amino acids to the C-terminus of the heavy chain of the first antigen-binding domain.

[0109] While detailed amino acid sequences are provided herein for the variable light chain region, variable heavy chain region, light chain, and heavy chain of the first antigen-binding site, and the scFv of the second antigen-binding site, the present disclosure further encompasses variants thereof. Such variants differ from the specific portions of the bispecific binding molecules described herein, for example, by modification, i.e., substitution, deletion, insertion, and / or addition of terminal sequences, of one or more appropriate amino acid residues in the constant domain and / or variable region (or any one or more CDRs thereof). Variants of the VL, VH, HC, LC, or scFv regions of the bispecific binding molecules of the invention may have a K that is greater than or equal to the affinity / activity and / or specificity / selectivity (i.e., up to 100-fold greater, up to 50-fold greater, up to 40-fold greater, up to 30-fold greater, up to 20-fold greater, up to 10-fold greater, or less than 10-fold greater) of the parent antibody for either MUC16 or DR5. D) at least a significant fraction (up to about 100 times greater or 10 times greater) of D In some cases, the sequences of such variants may possess higher affinity, selectivity and / or specificity (i.e., lower K) than the parent antibody for any of the antigens. D ) may be related to

[0110] Such functional variants have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity at the amino acid level to the reference portion of the particular sequence.

[0111] The sequence of the variant may differ from that of the parent antibody sequence mostly by conservative substitutions: for example, at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more (e.g., about 65-99%) of the substitutions in the variant are conservative amino acid residue substitutions. In the context of the present invention, conservative substitutions may be defined by substitutions within classes of amino acids reflected in one or more of the following tables:

[0112] [Table 5]

[0113] [Table 6]

[0114] [Table 7]

[0115] [Table 8]

[0116] As explained above, amino acid sequence modifications should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should not be liable to disrupt the secondary structure that characterizes the function of the parent sequence), but may be associated with advantageous properties such as altered functional or pharmacokinetic properties of the antibody, e.g., increased half-life, altered immunogenicity, provision of sites for covalent or non-covalent binding to other molecules, decreased susceptibility to proteolysis, decreased susceptibility to oxidation, decreased susceptibility to deamidation, or altered glycosylation patterns. In most embodiments, amino acid changes will not occur in CDR regions unless the changes result in the maintenance of at least one CDR as defined by the rules.

[0117] In some embodiments, the CDRs of the bispecific binding molecules described herein are based on CDRs defined in the prior art that discloses the anti-MUC16 or DR5 antibodies from which the binding sites are derived. Applicants indicate which specific CDR rules were used to define these CDRs (see, e.g., Tables 1 and 3). One skilled in the art will understand that, given the amino acid sequences of the variable regions of the bispecific binding molecules described herein, one can readily determine the CDR sequences therein using available web-based tools for determining CDRs within such variable regions based on any known rules, e.g., IMGT, Kabat, Chothia, or Contact, using these or any other rules used to define CDRs. Such tools include those found at www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi and www.novoprolabs.com / tools / cdr. Thus, the disclosure herein of a set of three CDRs in a heavy or light chain variable region based on one CDR rule is considered equivalent to the same set of CDRs determined by any other rule.

[0118] In certain embodiments, the bispecific binding molecules disclosed herein comprise the constant region of an anti-MUC16 antibody or a portion thereof. For example, a VL domain may be linked at its C-terminus to an antibody light chain constant domain comprising a human Cκ chain or Cλ chain. Similarly, a specific antigen-binding domain based on a VH domain may be linked to all or part of an immunoglobulin heavy chain from any antibody isotype, for example, IgG, IgA, IgE, and IgM, and any of the isotype subclasses, including, but not limited to, IgG1 and IgG4. The amino acid sequences of C-terminal fragments of the constant region are well known in the art, as are the DNA sequences encoding such amino acids.

[0119] In a more specific embodiment, the first antigen-binding domain that specifically binds to MUC16 is an immunoglobulin (Ig) molecule (having the traditional Y-shaped structure of a full-length antibody, comprising two heavy chains and two light chains), and the second antigen-binding domain that specifically binds to DR5 comprises at least one or more scFv binding elements.

[0120] In other more specific embodiments, one or more scFvs that specifically bind to DR5 are fused by a peptide linker to an Ig molecule (e.g., human IgG1) that specifically binds to MUC16. In some embodiments, the peptide linker is about 4-20 amino acids in length. In some more specific embodiments, the N-terminus of the scFv is fused to the C-terminus of the heavy chain of the Ig molecule or the C-terminus of the light chain of the Ig molecule. In some embodiments, the Ig molecule is an IgG.

[0121] Methods for linking scFv molecules to the C-terminus of the heavy or light chain of an IgG molecule are well known in the art. Typically, a small linker sequence containing glycine and serine (called a GS minilinker) is used. The number of amino acids in the linker can vary from 4 (GGGS, SEQ ID NO: 221), 6 (GGSGGS, SEQ ID NO: 222), 10 (GGGGSGGGGS, SEQ ID NO: 223), 12 (GGGSGGGSGGGS, SEQ ID NO: 173), 15 (GGGGSGGGGSGGGGS, SEQ ID NO: 224), or more. In a more specific embodiment, the GS minilinker between the scFv molecule and the C-terminus of the heavy chain of the IgG molecule is GGGSGGGGSGGGS (SEQ ID NO: 173).

[0122] In some embodiments, the invention provides bispecific binding molecules comprising: (i) an Ig molecule that specifically binds to MUC16, the Ig molecule comprising two heavy chains and two light chains, and (ii) two scFv molecules, each of which specifically binds to DR5. In more specific embodiments, each heavy chain of the Ig molecule has one scFv molecule fused to its C-terminus, thereby forming a bispecific tetravalent binding protein.

[0123] In some embodiments, the present invention provides: (i) two heavy chains, each comprising, in order from N-terminus to C-terminus: a heavy chain variable domain specific for MUC16 (e.g., a murine, humanized, or human VH domain); a constant domain of IgG (e.g., a human IgG1); a peptide linker (e.g., a GS minilinker); and an scFv specific for DR5; (ii) a bispecific binding molecule comprising two light chains, each comprising, in N- to C-terminal order: a light chain variable domain specific for MUC16 (e.g., a murine, humanized, or human VL domain); and a light chain constant domain (e.g., a human κ chain).

[0124] Methods for producing bispecific binding molecules It would be routine for one of ordinary skill in the art to prepare the bispecific binding molecules described herein using methods known in the art and described herein. Isolation of binding domains from antibodies (e.g., anti-MUC16 or anti-DR5 antibodies) is routine, and indeed, further information regarding methods that can be used to generate the antibodies and binding molecules described herein is provided in the accompanying Examples.

[0125] Bispecific Molecule Activities and Assays Therefor The binding activity of the bispecific binding molecules disclosed herein (and the parent MUC16 and DR5 antibodies from which these bispecific molecules can be derived) can be measured using a variety of methods. One method is enzyme-linked immunosorbent assay (ELISA). ELISA is a biochemical assay that uses a solid-phase enzyme immunoassay to detect the presence of a substance, usually an antigen, in a liquid or wet sample. The antigen in the sample is attached to a surface. An additional specific antibody or bispecific binding molecule is then applied to the surface so that it can bind to the antigen. This antibody is conjugated to an enzyme, and in a final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate. Fluorescence-activated cell sorting (FACS), also known as flow cytometry, provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell type at a time, based on the specific light scattering and fluorescence properties of each cell. These assays involve the use of EC 50 is the concentration at which an antibody or bispecific binding molecule induces a response halfway between baseline and maximum after a specified time of exposure to a given concentration of antigen by ELISA (enzyme-linked immunosorbent assay) or to cells expressing the antigen by FACS. Surface plasmon resonance is a label-free method for determining binding activity that directly measures the binding of a molecule in the soluble phase (the "analyte") to a "ligand" molecule immobilized on a sensor surface. In the sensor device, ligand binding is monitored by an optical phenomenon called surface plasmons. Specifically, a decrease in the SPR signal (expressed in resonance units, RU) is observed when the "analyte" molecule dissociates from the "ligand" molecule. The association ("on-rate", ka ) and dissociation rate ("off rate", k D ) is obtained from the signals obtained during association and dissociation and is given by the equilibrium dissociation constant ("binding constant", K D ) can be calculated from these. The signal given in resonance units (RU) depends on the size of the ligand present in the analyte, but when the experimental conditions are the same, i.e., when the ligand is the same molecule under the same conditions, the obtained RU can indicate the affinity; the larger the signal obtained in RU, the stronger the binding.

[0126] Affinity can be measured, for example, by the 50% effective concentration (EC 50 ) or equilibrium dissociation constant (K D ) can be expressed as "EC 50 "Median Effective Concentration," also referred to as "EC," refers to the concentration of a drug, antibody, or toxin that induces a response, such as a binding or cytotoxic effect, halfway between baseline and maximum after a specified exposure time. 50 and affinity are inversely proportional, 50 The lower the value, the higher the affinity of the antibody. In one aspect, a binding molecule of the invention has a K in the range of 1 pM to 100 μM, preferably 1 pM to 1 μM, as measured, for example, by ELISA or surface plasmon resonance analysis. D The affinity of an antibody or bispecific binding molecule can also be measured using the technique of equilibrium exclusion assay (KinExA) (Darling, RJ, and Brault PA. Assay and Drug Development Technologies. 2004, 2:647-657).

[0127] MUC16 + / DR5 +The ability of the bispecific binding molecules disclosed herein to cause cell death or apoptosis in cells can be detected by inducing growth inhibition of MUC16-expressing cells using a LIVE / DEAD Cell Viability Assay (Thermo Fisher Scientific) or similar assay, or a CellTiter-Glo assay (Promega) or similar assay, or AlamarBlue, and / or concentrations ranging from 1 μM to 1 pM; typically, 10 nM to 0.01 nM.

[0128] In some embodiments, the bispecific molecules of the invention induce cell death and inhibit MUC16 in a sample or a patient. + / DR5 + At least 30% of the cells undergo cell death. In some aspects of these embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells undergo cell death.

[0129] In one embodiment, the bispecific binding molecule of the invention is directed to a MUC16 + / DR5 + and can induce DR5-mediated apoptosis in one or more cancer cell types, inhibiting cell viability by more than 50% at concentrations of 3 nM or less.

[0130] The bispecific binding molecules described herein induce apoptosis in cancer cells and can therefore be used to treat cancers that express both MUC16 and DR5. Methods for identifying whether a particular tumor expresses MUC16 and DR5 are well known in the art. For example, immunohistochemistry can be used to determine whether tumor tissue expresses MUC16 and DR5 (e.g., using the bispecific binding molecules described herein or any known anti-MUC16 and / or anti-DR5 antibodies) and therefore would be suitable for treatment with the binding molecules of the invention.

[0131] Nucleic acids, cloning, and expression systems Bispecific binding molecules disclosed herein may be produced through the use of nucleic acid sequences that encode their amino acid sequences. The nucleic acid sequences can comprise DNA or RNA and can be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence defined herein encompasses DNA molecules having the specified sequence, unless the context requires otherwise, and encompasses RNA molecules having the specified sequence in which U is substituted with T. In some aspects, the nucleotide sequence is an RNA sequence that includes one or more modified ribonucleic acids (e.g., N6-methyladenosine (m6A), pseudouridine (ψ), N1-methylpseudouridine (m1ψ), or 5-methoxyuridine (5moU)). In some embodiments, the bispecific binding molecules disclosed herein are composed of four polypeptide chains. In some embodiments, bispecific binding molecules disclosed herein are composed of two identical heavy chain fusions and two identical light chains, each heavy chain fusion comprising a heavy chain of a first antigen-binding domain (e.g., a MUC16 antibody that specifically binds to the extracellular domain of MUC16) linked by a short amino acid linker to an scFv of a second antigen-binding domain (e.g., an scFv that specifically binds to DR5); and each light chain comprising a light chain of a first binding site (e.g., a MUC16 antibody that specifically binds to the extracellular domain of MUC16). In some embodiments, bispecific binding molecules disclosed herein are composed of two identical heavy chain and two identical light chain fusions, each light chain fusion comprising a light chain of a first antigen-binding domain linked by a short amino acid linker to an scFv of a second antigen-binding domain; and each heavy chain comprising a heavy chain of a first antigen-binding domain. In this section, the term "fusion" refers to the fusion of either the light or heavy chain of a first binding site, either directly or via a small peptide linker, to the scFv of a second antigen-binding domain, and the term "chain" refers to either the heavy or light chain of the first binding domain.

[0132] In some embodiments of the present invention, an isolated nucleic acid sequence is provided encoding a fusion of a heavy chain of a first antigen-binding domain linked by a short amino acid linker to an scFv of a second antigen-binding domain, wherein the heavy chain of the first antigen-binding domain has the amino acid sequence of any one of SEQ ID NOs: 115-118, the linker has the amino acid sequence of SEQ ID NO: 173, and the scFv of the second antigen-binding domain has the amino acid sequence of SEQ ID NO: 172. In certain embodiments, the fusion has the amino acid sequence of SEQ ID NO: 174. In other embodiments, the fusion has the amino acid sequence of SEQ ID NO: 175. In other embodiments, the fusion has the amino acid sequence of SEQ ID NO: 186. In other embodiments, the fusion has the amino acid sequence of SEQ ID NO: 187. In some embodiments, the nucleic acid sequence encoding the heavy chain fusion comprises the nucleic acid sequence of SEQ ID NO: 182, or a sequence having at least 70% identity thereto and encoding the same amino acid sequence encoded by SEQ ID NO: 182. In some more specific embodiments, the nucleic acid sequence encoding the fusion comprises a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 182 and encodes the same amino acid sequence encoded by SEQ ID NO: 182. In some embodiments, the nucleic acid sequence encoding the fusion comprises the nucleic acid sequence of SEQ ID NO: 183, or a sequence that has at least 70% identity thereto and encodes the same amino acid sequence encoded by SEQ ID NO: 183. In some more specific embodiments, the nucleic acid sequence encoding the fusion comprises a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identity thereto and encodes the same amino acid sequence encoded by SEQ ID NO: 183.

[0133] In some embodiments, nucleic acid sequences encoding the chains and / or fusion polypeptides of a bispecific binding molecule are constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired chain or fusion polypeptide and codon optimization based on host cell preferences. Standard methods can be routinely applied to synthesize and isolate polynucleotide sequences encoding the chains or fusion polypeptides of a bispecific binding molecule.

[0134] In embodiments where the bispecific binding molecule comprises two identical copies of chains and two identical copies of fusions as described above (e.g., two identical copies of heavy chain fusions and two identical copies of light chains, or two identical copies of light chain fusions and two identical copies of heavy chains), the chains and fusions must be expressed separately in the host cell. This is achieved by inserting the nucleotide sequences encoding each of the chains and fusions into one or more expression vectors such that the sequences are operably linked to transcriptional and translational control sequences. Such expression vectors comprising the isolated nucleic acid sequences disclosed herein are also part of the present invention. In some embodiments, the nucleotide sequences encoding the fusion and the chains are present in the same expression vector. In these embodiments, the nucleic acid sequences encoding the fusion and the chains may be operably linked to the same or different transcriptional and translational control sequences. In some embodiments, the nucleotide sequences encoding the fusion and the chains are present in different expression vectors. In these embodiments, the nucleic acid sequences encoding the fusion and the chains may also be operably linked to the same or different transcriptional and translational control sequences.

[0135] To produce a binding molecule or antibody of the invention, one skilled in the art can select from a wide variety of expression systems known in the art, such as those reviewed by Kipriyanov and Le Gall, Curr. Opin. Drug Discov. Devel. 2004;7:233-242.

[0136] Expression vectors include plasmids, retroviruses, cosmids, EBV-derived episomes, and the like. Expression vectors and expression control sequences are selected to be compatible with the host cell. Convenient vectors encode functionally complete human CH or CL immunoglobulin sequences and have appropriate restriction sites engineered into them so that nucleotide sequences encoding the VH or VL disclosed herein can be easily inserted and expressed as described above. The constant chain is usually a kappa or lambda chain for antibody light chains, and can be any IgG isotype (IgG1, IgG2, IgG3, IgG4) or other immunoglobulin, including allelic variants, for antibody heavy chains, without limitation.

[0137] The recombinant expression vector can also encode a signal peptide that facilitates secretion of antibody chains (e.g., the heavy and light chains of a binding molecule or antibody described herein) from a host cell. An isolated nucleic acid sequence encoding a heavy or light chain can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the mature chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous peptide derived from a non-immunoglobulin protein. Alternatively, the nucleic acid sequence encoding the heavy or light chain of a binding molecule described herein may already contain a signal peptide sequence.

[0138] In addition to nucleic acid sequences encoding antibody chains (e.g., the heavy and light chains of a binding molecule or bispecific antibody described herein), recombinant expression vectors can carry regulatory sequences including promoters, enhancers, termination and polyadenylation signals, and other expression control elements that control the expression of antibody chains in host cells. The choice of expression control sequence and expression vector will depend on the choice of host. Exemplary promoter sequences (exemplified for expression in mammalian cells) are promoters and / or enhancers derived from strong mammalian promoters such as CMV (e.g., CMV Simian Virus 40 (SV40) (e.g., the SV40 promoter / enhancer), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), polyoma, bovine papillomavirus, cytomegalovirus, and the native immunoglobulin promoter and actin promoter. Exemplary polyadenylation signals are BGH polyA, SV40 late or early polyA; or the SV40 promoter and / or enhancer sequences of immunoglobulin genes. 3'UTR, etc., can be used. Expression vectors useful for bacterial hosts include known bacterial plasmids such as E. coli-derived plasmids, including pCR1, pBR322, pMB9, and their derivatives, as well as broader host range plasmids such as M13 and filamentous single-stranded DNA phages. The recombinant expression vector may have sequences that control replication of the vector in host cells (e.g., origins of replication) and a selectable marker gene. The recombinant expression vector may also be a viral vector known in the art, including, but not limited to, AAV, lentivirus, or other retroviral vectors.

[0139] Nucleic acid molecules encoding the heavy chain or antigen-binding portion thereof and / or the light chain or antigen-binding portion thereof of a binding molecule or antibody described herein, and vectors containing these nucleic acid molecules, can be introduced into host cells, e.g., bacterial cells or higher eukaryotic cells, e.g., mammalian cells, according to transfection methods well known in the art, including, but not limited to, liposome-mediated transfection, polycation-mediated transfection, protoplast fusion, microinjection, calcium phosphate precipitation, electroporation, or introduction by viral vectors. In some embodiments, one or both of the heavy and light chains are fused to a second antigen-binding site, either directly or via an amino acid linker.

[0140] In some embodiments, the nucleic acid molecules encoding the chains of a binding molecule and the fusion polypeptide described herein are present on two separate expression vectors that are co-transfected into a host cell, preferably a mammalian cell.

[0141] Thus, a further embodiment provides a host cell comprising an expression vector comprising a nucleic acid molecule encoding a chain and an expression vector comprising a nucleic acid molecule encoding a fusion polypeptide of a binding molecule described herein.

[0142] Mammalian cell lines available as hosts for expression are well known in the art, including, inter alia, Chinese hamster ovary (CHO, CHO-DG44) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human cancer cells (e.g., HepG2), A549 cells, 3T3 cells, HEK-293 and HEK-293T cells, the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175 (1981)), or derivatives / progeny of such cell lines. Other mammalian cells, including, but not limited to, human, mouse, rat, monkey, and rodent cell lines, or other eukaryotic cells, including, but not limited to, yeast, insect, and plant cells, or prokaryotic cells such as bacteria, may also be used. The binding molecules of the present invention are produced by culturing host cells for a period sufficient to allow expression of the binding molecule in the host cells. In some embodiments, the present invention relates to recombinant eukaryotic or prokaryotic host cells harboring the above-described expression vectors. In some aspects of these embodiments, the host cell is eukaryotic. In some aspects, the host cell is a mammalian host cell. In some aspects, the mammalian host cell is an NS0 mouse myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell. In some aspects, the host cell is a hybridoma.

[0143] The present disclosure also provides methods for making anti-MUC16 / anti-DR5 bispecific binding molecules, comprising culturing host cells (e.g., hybridomas or transformed mammalian host cells) capable of expressing both the chains of the bispecific binding molecule and the fusion polypeptide under appropriate conditions, and, optionally, isolating the resulting expression product that is secreted from the host cells. The present disclosure further provides bispecific binding molecules isolated using the disclosed methods. The bispecific binding molecules are preferably recovered from the culture medium as secreted polypeptides or, for example, from host cell lysates if expressed without a secretory signal. To obtain substantially homogeneous preparations of the binding molecules or antibodies described herein, it is necessary to purify the bispecific binding molecules described herein using standard protein purification methods used for recombinant proteins and host cell proteins. By way of example, state-of-the-art purification methods useful for obtaining the binding molecules and antibodies of the present invention include, as a first step, the removal of cells and / or particulate cell debris from the culture medium or lysate. The binding molecules or antibodies are then purified from contaminating soluble proteins, polypeptides, and nucleic acids by, for example, fractionation on immunoaffinity or ion exchange columns, ethanol precipitation, reverse-phase HPLC, Sephadex chromatography, chromatography on silica or on cation exchange resins. As a final step in the process to obtain the bispecific binding molecules described herein, the purified binding molecules may be dried, e.g., lyophilized, as described below for therapeutic uses.

[0144] The present disclosure also optionally provides bispecific binding molecule antibodies produced using this method, as well as pharmaceutical compositions comprising the bispecific binding molecule antibodies and a pharmaceutically acceptable carrier.

[0145] Pharmaceutical Composition The present disclosure further provides pharmaceutical compositions comprising a bispecific binding molecule disclosed herein; and a pharmaceutically acceptable carrier, including any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents that are physiologically compatible with the compounds of the invention.

[0146] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, glucose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like) and suitable mixtures thereof, vegetable oils, carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical art.

[0147] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0148] The pharmaceutical compositions of the present invention may further comprise isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride.

[0149] The pharmaceutical compositions of the present invention may also contain one or more adjuvants suitable for the selected administration route, such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives, or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition.The compounds of the present invention may be prepared with carriers that protect the compounds from rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems.Methods for preparing such formulations are generally known to those skilled in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0150] The bispecific binding molecules provided herein can be administered to humans or other subjects in an amount sufficient to produce a therapeutic effect. Such a "therapeutically effective amount" is the minimum amount necessary to prevent, alleviate, or treat the clinical symptoms of any of the diseases or conditions described below, and in particular the minimum amount effective to ameliorate or treat these disorders. The actual dosage level of the bispecific binding molecule in the pharmaceutical compositions of the invention may be varied to obtain an amount of bispecific binding molecule effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the invention or its amide used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and factors well known in the medical arts.

[0151] The therapeutically effective daily dose of the applicable bispecific binding molecule of the present invention is generally 0.001 mg / kg to 100 mg / kg, preferably 0.1 mg / kg to 20 mg / kg.

[0152] Generally, for the treatment and / or amelioration of the diseases, disorders, and conditions described herein, and depending on the particular disease, disorder, or condition being treated, the potency of the bispecific binding molecule of the invention used, the particular route of administration, and the particular pharmaceutical formulation or composition used, antibody molecules of the invention are generally administered in the range of 0.005-20.0 mg per kg body weight and dose, preferably 0.05-10.0 mg / kg / dose, more preferably 0.5-10 mg / kg / dose, continuously (e.g., by infusion) or, more preferably, as a single dose. The dosing interval can be, for example, twice weekly, once weekly, or once monthly, but can vary significantly, depending, inter alia, on the aforementioned parameters. Thus, in some cases, use of less than the minimum dose stated above may be sufficient, while in other cases the upper limit may have to be exceeded.

[0153] When administering large amounts, it may be desirable to administer them in smaller doses several times a day. Administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, and may be administered, for example, proximal to the target site. If desired, the pharmaceutical composition may be administered as two, three, four, five, six, or more small doses of the effective daily dose administered separately at appropriate intervals throughout the day, optionally in unit dosage form.

[0154] Depending on the particular binding molecule of the invention and its particular pharmacokinetic and other properties, the pharmaceutical composition may be administered daily, every other day, every third day, every fourth day, or every fifth day, weekly, monthly, etc. The dosing regimen may include weekly treatment over a long period of time. "Long-term" means a period of at least two weeks, preferably several months or years.

[0155] The actual pharmaceutically effective amount or therapeutic dosage will, of course, depend on factors known to those skilled in the art, such as the age and weight of the patient, the route of administration, and the severity of the disease, etc. In any event, the binding molecules of the present invention will be administered in a dosage and manner that will deliver a pharmaceutically effective amount based on the patient's unique condition.

[0156] The pharmaceutical composition may be administered by any suitable route and mode. Suitable routes for administering the compounds of the present invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art. In some embodiments, the application mode is parenteral administration by infusion or injection (intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal), although other application modes such as inhalation, transdermal, intranasal, buccal, oral, etc. may also be applicable. In some embodiments, the pharmaceutical composition of the present invention is administered parenterally.

[0157] In a further aspect, the bispecific binding molecules of the invention are used in combination with a device useful for their administration, such as a syringe, injection pen, micropump, or another device. In a further aspect, the binding molecules of the invention are comprised in a kit of parts, which also includes, for example, a package insert with instructions for use of the binding molecule.

[0158] The efficacy of the binding molecules of the invention and compositions comprising them can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and / or animal model known per se, or any combination thereof, depending on the particular disease involved. Suitable assays and animal models will be apparent to those skilled in the art and include, for example, the assays and animal models used in the Examples below.

[0159] The binding molecules of the invention may be used alone or in combination with other pharmacologically active ingredients, such as cytostatic or cytotoxic agents, cytostatic agents, antiangiogenic agents, steroids, immune modulators / checkpoint inhibitors, and other state-of-the-art or standard of care compounds.

[0160] Therefore, a further aspect of the invention provides a pharmaceutical composition comprising a binding molecule of the invention together with a pharmaceutically acceptable carrier and optionally one or more further active ingredients.

[0161] therapeutic use In some embodiments, the present invention provides methods for treating a disease or disorder involving cells that co-express MUC16 and DR5, comprising administering to a subject in need thereof a bispecific binding molecule of the present invention or a pharmaceutical composition containing the bispecific binding molecule. The present disclosure also provides methods for treating and / or ameliorating a condition associated with MUC16-mediated activity in a subject, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a bispecific binding molecule provided herein. In some embodiments, the bispecific binding molecule is administered alone. In other embodiments, the bispecific binding molecule is administered as a combination therapy. Also provided is a method for reducing MUC16 activity in a subject, comprising administering to a subject in need thereof an effective amount of a bispecific binding molecule.

[0162] In some embodiments, the methods are used to treat a fibrotic, inflammatory, immune, or autoimmune disorder associated with MUC16. In some embodiments, the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, liver fibrosis, cirrhosis, renal fibrosis, glial scarring, myocardial fibrosis, arteriosclerosis, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, or adhesive capsulitis. In some aspects of these embodiments, the disorder being treated is idiopathic pulmonary fibrosis.

[0163] In some embodiments, the above methods are used to treat cancers or malignancies characterized by overexpression of MUC16. In some embodiments, such cancers or malignancies are gynecological cancers (cancers of the female reproductive organs), including cervical, endometrial, fallopian tube, ovarian, uterine, and vaginal cancers; pancreatic, esophageal, gastric, colorectal, breast, or lung cancers (e.g., non-small cell lung cancer). In some embodiments, the solid tumors treated are selected from ovarian and pancreatic tumors.

[0164] As used herein, the term "subject" is intended to include human and non-human animals that respond to the bispecific antigen-binding molecule. A subject may include, for example, a human patient suffering from a disorder that can be corrected or ameliorated by modulating MUC16 function, such as enzyme activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and / or induction of lysis, and / or elimination / reduction of the number of MUC16-expressing cells.

[0165] For example, the bispecific binding molecules may be used to induce one or more of the following biological activities in vivo or in vitro: modulation of MUC16 function (e.g., apoptosis, permeabilization of cell membranes, reduction of cell number, enzymatic activity, signal transduction, induction of cytokine expression, induction of proliferation or differentiation, and / or induction of lysis), killing of cells expressing MUC16, mediating phagocytosis or ADCC of cells expressing MUC16 in the presence of human effector cells, and mediating CDC of cells expressing MUC16 in the presence of complement, or killing of cells expressing MUC16 by apoptosis.

[0166] Dosage regimens in the above-described therapeutic methods and uses are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0167] An "effective amount" for treating cancer is an amount sufficient to stabilize the progression of the disease for a period of time. A therapeutically effective amount of an antibody of the invention may reduce tumor size, or otherwise ameliorate symptoms in a subject.

[0168] An "effective amount" for idiopathic alveolar fibrosis may result in improvement in any one or more of the following observed test results: change from baseline in St. George's respiratory questionnaire (SGRQ), dyspnea score, cough score, and 6-minute walk test (grade and distance); change in pulmonary function (forced vital capacity (FVC); diffusing capacity of the lungs for carbon monoxide (DLCO)) compared to baseline; change in abnormal values ​​of routine safety tests (blood routine, urine routine, blood chemistry, electrocardiogram (ECG), etc.) compared to baseline; change in chest high-resolution computed tomography (HRCT) score compared to baseline; change in lung tumor markers from baseline; frequency and severity of acute exacerbations of IPF.

[0169] The bispecific binding molecules disclosed herein may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when the cancer is in remission. This may be particularly useful in patients where other biological factors make it difficult to locate a known tumor.

[0170] The bispecific binding molecules disclosed herein may also be administered prophylactically to reduce the risk of developing a fibrotic disease or disorder.

[0171] The bispecific binding molecules disclosed herein may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition to be treated. Such administration may be simultaneous, separate, or sequential. In the case of simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0172] Kit containing anti-MUC16 / anti-DR5 antibodies The present disclosure further provides kits comprising suitably packaged bispecific binding molecules (including variants and derivatives thereof) disclosed herein and written materials that can be used to practice the methods described herein. The written materials can include any of the following information: instructions for use, clinical trial discussions, side effect tables, scientific references, package inserts, clinical trial results, and / or summaries thereof. The written materials can indicate or demonstrate the activity and / or benefits of the composition and / or can describe dosage, administration, side effects, drug interactions, or other information useful to healthcare providers. Such information can be based on the results of various studies, for example, studies using experimental animals including in vivo models, and / or studies based on human clinical trials. The kits can further include written materials, as described above, that serve to provide information regarding alternative therapies (e.g., additional agents) and / or additional treatments (e.g., additional agents).

[0173] In certain embodiments, the kit comprises at least one purified bispecific binding molecule disclosed herein in one or more containers. In some embodiments, the kit contains all of the necessary and / or sufficient components to perform a detection assay, including all controls, instructions for performing the assay, and / or any necessary software for analysis and presentation of results.

[0174] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art.

[0175] array: (SEQ ID NO: 1) NDYAWN (SEQ ID NO: 2) YISYSGYTTYNPSLKS (SEQ ID NO: 3) YINYSGYTTYNPSLKS (SEQ ID NO: 4) YINYAGYTTYNPSLKS (SEQ ID NO: 5) YISYAGYTTYNPSLKS (SEQ ID NO: 6) WTSGLDY (SEQ ID NO: 7) WDGGLTY (SEQ ID NO: 8) WAAGLTN (SEQ ID NO: 9) WDAGLSY (SEQ ID NO: 10) WDAGLTY (SEQ ID NO: 11) WEAGLNH (SEQ ID NO: 12) WEAGLNY (SEQ ID NO: 13) WMAGLSD (SEQ ID NO: 14) WSAGLDH (SEQ ID NO: 15) WTAGLDY (SEQ ID NO: 16) WTAGLTH (SEQ ID NO: 17) WVAGLTN (SEQ ID NO: 18) WAGGLEN (SEQ ID NO: 19) WDGGLSY (SEQ ID NO: 20) WDRGLTY (SEQ ID NO: 21) WASGLSH (SEQ ID NO: 22) WASGLSN (SEQ ID NO: 23) WASGLSY (SEQ ID NO: 24) WASGLTH (SEQ ID NO: 25) WASGLTN (SEQ ID NO: 26) WDSGLKY (SEQ ID NO: 27) WDSGLNY (SEQ ID NO: 28) WDSGLSS (SEQ ID NO: 29) WDSGLSV (SEQ ID NO: 30) WDSGLSY (SEQ ID NO: 31) WDSGLTY (SEQ ID NO: 32) WESGLSH (SEQ ID NO: 33) WESGLSV (SEQ ID NO: 34) WKSGLDS (SEQ ID NO: 35) WKSGLEY (SEQ ID NO: 36) WLSGLDF (SEQ ID NO: 37) WLSGLDS (SEQ ID NO: 38) WLSGLES (SEQ ID NO: 39) WLSGLSD (SEQ ID NO: 40) WRSGLEH (SEQ ID NO: 41) WSSGLNY (SEQ ID NO: 42) WSSGLTY (SEQ ID NO: 43) WTSGMDS (SEQ ID NO: 44) WTSGLTY (SEQ ID NO: 45) WDTGLTY (SEQ ID NO: 46) WAAGLDH (SEQ ID NO: 47) WAAGLDS (SEQ ID NO: 48) WLAGLSN (SEQ ID NO: 49) WTAGLDQ (SEQ ID NO: 50) WASGLDH (SEQ ID NO: 51) WASGLDN (SEQ ID NO: 52) WASGLDS (SEQ ID NO: 53) WASGLDY (SEQ ID NO: 54) WKSGLDT (SEQ ID NO: 55) WKSGLGP (SEQ ID NO: 56) WMSGLDS (SEQ ID NO: 57) WRSGLES (SEQ ID NO: 58) WRSGLEY (SEQ ID NO: 59) WTSGLDS (SEQ ID NO: 60) WTSGLDT (SEQ ID NO: 61) WTSGLDV (SEQ ID NO: 62) KASDLIHNWLA (SEQ ID NO: 63) GATSLET (SEQ ID NO: 64) GATSLET (SEQ ID NO: 65) QQYWTTPFT (SEQ ID NO: 66) [VK-8 CDR1 (from the pamphlet of International Publication No. 2003076465)] DYNMH (SEQ ID NO: 67) [VK-8 H-CDR2] YIYPYNGDTGYNQKFRN (SEQ ID NO: 68) [VK-8 H-CDR3] SGGFWYFDV (SEQ ID NO: 69) [VK-8 L-CDR1] RATPSVSYMH (SEQ ID NO: 70) [VK-8 L-CDR2] TTSNLAS (SEQ ID NO: 71) [VK-8 L-CDR3] QQWSRSPPT (SEQ ID NO: 72) [OC125 CDR H-1 (from the pamphlet of International Publication No. 2003076465)] SYWMH (SEQ ID NO: 73) [OC125 CDR H-2] AIYPGNSDTSYNQKFKG (SEQ ID NO: 74) [OC125 CDR H-3] SYDWYFDV (SEQ ID NO: 75) [OC125 CDR L-1] RASQSIGTDMH (SEQ ID NO: 76) [OC125 CDR L-2] YASESIS (SEQ ID NO: 77) [OC125 CDR L-3] QQSYSWPLT (SEQ ID NO: 78) [AR9.6 CDR H-1 (from Eric Nunez Aguilar, “HARNESSING ANTIBODIES FOR TREATING PANCREATIC CANCER: AR9.6-A MUC16 Specific Monoclonal antibody,” a thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Biology and Mathematics, California State University, Fresno, May 2021)] GFTFSTF (SEQ ID NO: 79) [AR9.6 CDR H-2] SSGSST (SEQ ID NO: 80) [AR9.6 CDR H-3] SGYDYDPIYYALDY (SEQ ID NO: 81) [AR9.6 CDR L-1] RASESVDNYGISFMN (SEQ ID NO: 82) [AR9.6 CDR L-2] GASNQGS (SEQ ID NO: 83) [AR9.6 CDR L-3] QQTKEVPWT (SEQ ID NO: 84) [H1H8794 CDR H-1 (U.S. Patent No. 10,738,130)] GFTFRDYS (SEQ ID NO: 85) [H1H8794 CDR H-2] VTFFNSAI (SEQ ID NO: 86) [H1H8794 CDR H-3] AREREPIVGGFDY (SEQ ID NO: 87) [H1H8794 CDR L-1] QSINSY [H1H8794 CDR L-2] AAS (SEQ ID NO: 89) [H1H8794 CDR L-3] QQSYSSPPIT (SEQ ID NO: 90) [B43.13 CDR-H1 (derived from Sharm et al. 2014 Protein Expression and Purification 102:27-37] NYWMN (SEQ ID NO: 91) [B43.13 CDR-H2] QIVPGGGDPNYNGKFKG (SEQ ID NO: 92) [B43.13 CDR-H3] WAHSYAMDY (SEQ ID NO: 93) [B43.13 CDR-L1 v1] KSSQSLLNSSTQKNYLA (SEQ ID NO: 94) [B43.13 CDR-L1 v2] KSSQSLLYSSTQKNYLA (SEQ ID NO: 95) [B43.13 CDR-L2] WASTRES (SEQ ID NO: 96) [B43.13 CDR-L3] QQYYSYPWT (SEQ ID NO: 97) [ka] (SEQ ID NO: 98) [VK-8 VH v1] [ka] (SEQ ID NO. 99)[VK-8 vH v2] [ka] (SEQ ID NO: 100) [OC125 VH] [ka] (SEQ ID NO: 101) [AR9.6 VH] [ka] (SEQ ID NO: 102) [H1H8794 VH] [ka] (SEQ ID NO: 103) [B43.13 VH] [ka] (SEQ ID NO: 104) DIQMTQSPSSLSASVGDRVTITCKASDLIHNWLAWYQQKPGKAFKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWTTPFTFGQGTKVEIK (SEQ ID NO: 105) DIQMTQSPSSLSASVGDRVTITCKASDLIHNWLAWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYWTTPFTFGQGTKVEIK (SEQ ID NO: 106) [VK-8 VL v1] DIQMTQSPAILSASPGEKVTTMTCRATPSVSYMHWYQQKPGSSPKPWIYTTSNLASGVPARFSGGGSGTSYSLTVSRVEAEDAATYYCQQWSRSPPTFGAGTKLEIK (SEQ ID NO: 107) [VK-8 VL v2] DIQMTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPWIYTTSNLASGVPARFSGGGSGTSYSLTVSRVEVEDAATYYYCQQWSRSPPTFGAGTKLEIK (SEQ ID NO: 108) [VK-8 VL v3] DIQMTQSPAILSASPGEKVTMTCRATPSVSYMHWYQQKPGSSPKPLIYTTSNLASGVPARFSGGGSGTSYSLTVSRVEAEDAATYYCQQWSRSPPTFGAGTKLEIK (SEQ ID NO. 109)[VK-8 VL v4] DIQMTQSPAILSASPGEKVTTMTCRATPSVSYMHWYQQKPGSSPKPLIYTTSNLASGVPARFSGGGSGTSYSLTVSRVEVEDAATYYCQQWSRSPPTFGAGTKLEIK (SEQ ID NO: 110) [OC125 VL] DIELTQSPAILSVSPGERVSFSCRASQSIGTDMHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGGTDFTLSINSVESEDIADYYCQQSYSWPLTFGAGTKLEIK (SEQ ID NO: 111) [AR9.6 VL] [ka] (SEQ ID NO: 112) [H1H8794 VL] DIQMTQSPSSLSASVGDRVTITCRASQSINSYLNWYQQKPGKAPKLLIYAASSLQGGVPSRFSGGGSGTDFTLTITSLQPEDFATFYCQQSYSSPPITFGQGTRLEIK (SEQ ID NO: 113) [B43.13 VL v1] [ka] (SEQ ID NO: 114) [B43.13 VL v2] [ka] (SEQ ID NO: 115) [ka] (SEQ ID NO: 116) [ka] (SEQ ID NO: 117) [ka] (SEQ ID NO: 118) [ka] (SEQ ID NO: 119) [ka] (SEQ ID NO: 120) [ka] (SEQ ID NO: 121) [Lexatumumab HC CDR1] DYGMS (SEQ ID NO: 122) [Lexatumumab HC CDR2] GINWNGGSTGYADSVKG (SEQ ID NO: 123) [Lexatumumab HC CDR3] ILGAGRGWYFDL (SEQ ID NO: 124) [Lexatumumab LC CDR1] QGDSLRSYYAS (SEQ ID NO: 125) [Lexatumumab LC CDR2] GKNNRPS (SEQ ID NO: 126) [Lexatumumab LC CDR3] NSRDSSGNHVV (SEQ ID NO: 127) [Conatumumab CDR-H1] SGDYFWS (SEQ ID NO: 128) [Conatumumab CDR-H2] HIHNSGTTYYNPSLKS (SEQ ID NO: 129) [Conatumumab CDR-H3] DRGGDYYYGMDV (SEQ ID NO: 130) [Conatumumab CDR-L1] RASQGISRSYLA (SEQ ID NO: 131) [Conatumumab CDR-L2] GASSRAT (SEQ ID NO: 132) [Conatumumab CDR-L3] QQFGSSPWT (SEQ ID NO: 133) [Drozitumab CDR-H1] DYAMS (SEQ ID NO: 134) [Drozitumab CDR-H2] GINWQGGSTGYADSVKG (SEQ ID NO: 135) [drozitumab CDR-H3] ILGAGRGWYFDY (SEQ ID NO: 136) [Drozitumab CDR-L1] SGDSLRSYYAS (SEQ ID NO: 137) [drozitumab CDR-L2] GANNRPS (SEQ ID NO: 138) [drozitumab CDR-L3] NSADSSGNHVV (SEQ ID NO: 139) [Tigatuzumab CDR-H1] SYVMS (SEQ ID NO: 140) [Tigatuzumab CDR-H2] TISSGGSYTYYPDSVKG (SEQ ID NO: 141) [Tigatuzumab CDR-H3] RGDSMITTDY (SEQ ID NO: 142) [Tigatuzumab CDR-L1] KASQDVGTAVA (SEQ ID NO: 143) [Tigatuzumab CDR-L2] WASTRHT (SEQ ID NO: 144) [Tigatuzumab CDR-L3] QQYSSYRT (SEQ ID NO: 145) [DS-8273a CDR-H1] GYFMN (SEQ ID NO: 146) [DS-8273a CDR-H2] RFNPYNEDTFYNQKFKG (SEQ ID NO: 147) [DS-8273a CDR-H3] SAYYFDSGGYFDY (SEQ ID NO: 148) [DS-8273a CDR-L1] RSSQSLVHSNKNTYLH (SEQ ID NO: 149) [DS-8273a CDR-L2] KVSNRFS (SEQ ID NO: 150) [DS-8273a CDR-L3] SQSTHVPWT (SEQ ID NO: 151) [LBY135 CDR-H1] DYTIH (SEQ ID NO: 152) [LBY135 CDR-H2] WFYPGGGYIKYNEKFKD (SEQ ID NO: 153) [LBY135 CDR-H3] HEEGIYFDY (SEQ ID NO: 154) [LBY135 CDR-L1] KASQDVNTAIA (SEQ ID NO: 155) [LBY135 CDR-L2] WASTRHT (SEQ ID NO: 156) [LBY135 CDR-L3 v1] QQWSSNPLT (SEQ ID NO: 157) [LBY135 CDR-L3 v2] QQHYTTPFT (SEQ ID NO: 158) [VH of anti-DR5 antibody: lexatumumab (IMV-14)] [ka] (SEQ ID NO: 159) [conatumumab VH] [ka] (SEQ ID NO: 160) [drozitumab VH] [ka] (SEQ ID NO: 161) [Tigatuzumab VH] [ka] (SEQ ID NO: 162) [DS-8273a VH] [ka] (SEQ ID NO: 163) [LBY135 VH v1] [ka] (SEQ ID NO: 164) [LBY135 VH v2] [ka] (SEQ ID NO: 165) [VL of anti-DR5 antibody: lexatumumab (IMV-14)] SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVL (SEQ ID NO: 166) [conatumumab VL] EIVLTQSPGTLSLSPGERATLSCRASQGISRSYLAWYQQKPGQAPSLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQFGSSPWTFGQGTKVEIK (SEQ ID NO: 167) [drozitumab VL] SELTQDPAVSVALGQTVRITCSGDSLRSYYASWYQQKPGQAPVLVIYGANNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSADSSGNHVVFGGGTKLTVL (SEQ ID NO: 168) [Tigatuzumab VL] DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYRTFGQGTKVEIK (SEQ ID NO: 169) [DS-8273a VL] [ka] (SEQ ID NO: 170) [LBY135 VL v1] DIAMTQSHKFMSTLVGDRVSITCKASQDVNTAIAWYQQKPGQSPKLLIYWASTRHTGVPDRFYGSGSGTDYTLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK (SEQ ID NO: 171) [LBY135 VL v2] DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAIAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYTTPFTFGSGTKL (SEQ ID NO: 172) [ka] (SEQ ID NO: 173) GGGSGGGSGGGS (SEQ ID NO: 174) [ka] (SEQ ID NO: 175) [ka] (SEQ ID NO: 176) [TR5, Uniprot code Q8WXI7, residues 12665 ​​to 12857 (or 12858) (including SEA5)] [ka] (SEQ ID NO: 177) [extracellular epitope for anti-MUC16 binding] FNTTER (SEQ ID NO: 178) [TR4-SEA5-TR5 (Uniprot code Q8WXI7, residues 12538-12692 fused to 12663-12818] [ka] (SEQ ID NO: 179) [SEA5, Uniprot code Q8WXI7, residues 12697-12818] [ka] (SEQ ID NO: 180) KHPGSRKFNTTERVLQGL (SEQ ID NO: 181) [synthetic peptide] [ka] (SEQ ID NO: 182) [R-25] [ka] [ka] (SEQ ID NO: 183) [ka] [ka] [ka] (SEQ ID NO: 184) [ka] (SEQ ID NO: 185) [ka] (SEQ ID NO: 186) [ka] (SEQ ID NO: 187) [ka] (SEQ ID NO: 188) [This is the HC of the anti-DR5 antibody (IMV-14): Lexatumumab HC] [ka] (SEQ ID NO: 189) [This is the LC of the anti-DR5 antibody (IMV-14): Lexatumumab LC] [ka] (SEQ ID NO: 190) [IMV-21 LC - This is the LC of a bispecific antibody that targets CD74 and DR5] [ka] (SEQ ID NO: 191) [IMV-21 HC - This is the HC of a bispecific antibody targeting CD74 and DR5] [ka] (SEQ ID NO: 192) [This is the HC of a non-targeting bispecific antibody] [ka] (SEQ ID NO: 193) [This is the LC of a non-targeting bispecific antibody] [ka] (SEQ ID NO: 194) [11D10-SE—This is the HC of the anti-MUC16 bispecific antibody] [ka] (SEQ ID NO: 195) [11D10-SE anti-MUC16 / anti-DR5 LC—this is the LC of an anti-MUC16 bispecific antibody] [ka] (SEQ ID NO: 196) [11D10 anti-MUC16 / anti-DR5 HC, which is the HC of the anti-MUC16 bispecific antibody] [ka] (SEQ ID NO: 197) [anti-LIV bispecific antibody HC] [ka] (SEQ ID NO: 198) [anti-LIV bispecific antibody LC] [ka] (SEQ ID NO: 199) [024-LEX bispecific antibody HC] [ka] (SEQ ID NO: 200) [024-LEX bispecific antibody LC] [ka] (SEQ ID NO: 201) [muIgG1 HC - non-targeting murine IgG1 heavy chain portion of antibody used to block FcG receptors in xenograft mouse studies] [ka] (SEQ ID NO: 202) [muIgG1 LC - non-targeting murine IgG1 light chain portion of an antibody used to block FcG receptors in xenograft mouse studies] [ka] (SEQ ID NO: 203) [IMV-Mv1 HC] [ka] (SEQ ID NO: 204) [IMV-Mv2 HC] [ka] (SEQ ID NO: 205) [IMV-Mv3 HC] [ka] (SEQ ID NO: 206) [IMV-Mv4 HC] [ka] (SEQ ID NO: 207) [IMV-M HC] [ka] (SEQ ID NO: 208) [linker between VL and VH of scFv] GGGGSGGGDSGGGGSGGGGS (SEQ ID NO: 209) [linker between VL and VH of scFv] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 210) [IMV-M scFv] [ka] (SEQ ID NO: 211) [IMV-Mv1 scFv] [ka] (SEQ ID NO: 212) [IMV-Mv2 scFv] [ka] (SEQ ID NO: 213) [IMV-Mv3 scFv] [ka] (SEQ ID NO: 214) [IMV-Mv4 scFv] [ka] (SEQ ID NO: 215) [Lexatumumab VL variant 1-A42C mutation] [ka] (SEQ ID NO: 216) [Lexatumumab VL variant 2-Q41C mutation] [ka] (SEQ ID NO: 217) [Lexatumumab VL variant 3 - G101C mutation] [ka] (SEQ ID NO: 218) [Lexatumumab VH variant 1 - G112C mutation] [ka] (SEQ ID NO: 219) [Lexatumumab VH variant 2 - K113C mutation] [ka] (SEQ ID NO: 220) [Lexatumumab VH variant 3 - G44C mutation] [ka] [Example]

[0176] The invention will now be illustrated by the following non-limiting examples, which describe the methods used to generate bispecific molecules that bind MUC16 and DR5, variations in their formats, and the biological activity of these molecules.

[0177] Example 1: Design of binding molecules that recognize MUC16 and DR5 The present applicant has developed a binding molecule that binds to both MUC16 and DR5 and induces cell death in cancer cells expressing both MUC16 and DR5. The molecular design used in these experiments has an IgG antibody with specificity for MUC16, and an scFv with specificity for DR5 attached to the C-terminus of the IgG heavy chain via a small peptide linker. A schematic diagram of the design is shown in Figure 1.

[0178] To test the feasibility of this concept, four bispecific antigen-binding molecules (IMV-18, MCLX-SE, IMV-AA, and MC-AA) based on the format depicted in Figure 1 were prepared using methods well known to those skilled in the art. Each of these bispecific antigen-binding molecules had the same anti-MUC16 CDRs and anti-DR5 CDRs. The anti-MUC16 CDRs were as follows: CDR-H1-SEQ ID NO:1; CDR-H2-SEQ ID NO:2; CDR-H3-SEQ ID NO:6; CDR-L1-SEQ ID NO:62; CDR-L2-SEQ ID NO:63; and CDR-L3-SEQ ID NO:65. The anti-DR5 CDRs were as follows: CDR-H1-SEQ ID NO:121; CDR2-H2-SEQ ID NO:122; CDR-H3-SEQ ID NO:123; CDR-L1-SEQ ID NO:124; CDR-L2-SEQ ID NO:125; and CDR-L3-SEQ ID NO:126.

[0179] The SEQ ID NOs for the light and heavy chains fused to the scFv of these molecules, as well as further sequence information, are shown in Table 9 below.

[0180] [Table 9]

[0181] [Table 10]

[0182] Applicants also designed bispecific molecules that target DR5 and cell surface antigens different from MUC16, as well as monospecific antibodies to compare activity. See Table 10. All of these other bispecific molecules contained the same anti-DR5 scFv.

[0183] [Table 11]

[0184] Example 2. Expression and purification of bispecific tetravalent molecules that recognize human MUC16 or other antigens and human DR5 Knowing the amino acid sequence of the desired sequence, one of skill in the art can readily prepare the antibody or immunoglobulin chain by standard techniques for producing polypeptides. For example, the antibody or immunoglobulin chain can be synthesized by the well-known solid-phase method using a commercially available peptide synthesizer (e.g., one manufactured by Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Alternatively, the antibodies, immunoglobulin chains, and antibody-like binding proteins of the invention can be synthesized by recombinant DNA techniques, as is well known in the art. For example, these fragments can be obtained as DNA expression products after incorporating a DNA sequence encoding the desired (poly)peptide into an expression vector and introducing such a vector into an appropriate eukaryotic or prokaryotic host that expresses the desired polypeptide, from which they can then be isolated using well-known techniques.

[0185] Expression vectors were constructed, cells were transfected, and antibodies were produced and purified using methods well known in the art. Genes for both the heavy chain-scFv fusion and light chain of the bispecific binding compound were designed and optimized for expression in CHO cells, and their sequences were introduced into protein expression vectors from BioIntron, Building 5, No. 388 Galileo Road, Zhangjiang High-Tech Park, Shanghai, China. CHO cells were transiently transfected with the expression vectors and cultured for 5 to 7 days. The culture medium was then harvested and subjected to two stages of purification. The recombinant binding molecules or antibodies were purified from the culture supernatant by protein A affinity chromatography and further purified by preparative size-exclusion chromatography (SEC) using methods well known in the art. After purification, the antibodies were analyzed for monomer purity and the presence of fragments or degradation products by methods well known in the art, including sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with protein staining and analytical SEC-HPLC, and their concentrations were measured by UV spectroscopy. Extinction coefficients were calculated as per Pace et al., Protein Sci. 1995;4:2411-23. All purified proteins were free of fragments and other impurities and were 97.5-100% monomeric. An example of this analysis is shown in Figure 2.

[0186] For IMV-M and its variants (IMV-Mv1, IMV-Mv2, IMV-Mv3, and IMV-Mv4), the scFv portion of the heavy chain fusion is stabilized by a disulfide bond between two cysteines introduced at specific positions in the VL and VH of the scFv (see Table 9), and some were purified using only Protein A affinity chromatography without the need for an additional SEC step. The measured final yields of each of these bispecific antibodies are listed below.

[0187] [Table 12]

[0188] Example 3. Cytotoxicity of bispecific molecules recognizing human MUC16 and human DR5. To assess the cytotoxic effects of the antibodies of the invention, the following cell lines obtained from the American Type Culture Collection (ATCC) (www.ATCC.org) have been used: adherent cell line NCI-H292 lung carcinoma (CRL-1848™), adherent cell line CAOV-3 ovarian adenocarcinoma (HTB-75), adherent cell line NIH:OVCAR-3, also known as OVCAR-3 ovarian adenocarcinoma (HTB-161), suspension cell line MM.1S multiple myeloma (CRL-2974™), suspension cell line SU-DHL-8 large cell lymphoma (CRL-2961™), suspension cell line Ramos Burkitt lymphoma (CRL-1596™), suspension cell line RPMI-8226 plasmacytoma (CCL-155™), suspension cell line, and adherent cell line HPAC pancreatic adenocarcinoma (CRL-2119™), as well as Leibniz MOLP-8 multiple myeloma (ACC 569), a suspension cell line, was obtained from the Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ) (https: / / www.dsmz.de). All of these cell lines express DR5 transcripts (Table 11). NCI-H292, CAOV-3, OVCAR-3, PK-59, HCC827, NCI-H1975, NCC-StC-K140, HDQ-P1, and HPAC express MUC16 transcripts and protein. All of these cell lines for which data on MUC16 protein expression are available express MUC16 protein. Proteomic data reporting that MUC16 protein is expressed by OVCAR-3, NCI-H292, HCC827, NCI-H1975, and NCC-StC-K140 are shown in Table 11, and further flow cytometry or Western blot data showing MUC16 protein expression by OVCAR3, HPAC, and CAOV-3 cells are reported in Chen et al, Cancer Res. 2007, 67, 4924-5998; Haridas et al, PLoS ONE 2011;6:e26839; and Kline et al., Oncotarget 2017;8,52045-5260.MM.1S, SU-DHL-8, Ramos, RPMI-8226, and Molp-8 express CD38 transcripts (Table 11) and CD38 protein (Deckert et al., Cancer Res. 2014, 20, 4574-4583; Moreno et al., Clin Cancer Res. 2019, 25, 3176-3187). MM.1S, SU-DHL-8, Ramos, RPMI-8226, and Molp-8 cell lines express LIV-1 transcripts (Table 11).

[0189] [Table 13]

[0190] a) Adherent cells. Cells were plated in 96-well tissue culture plates at 2.5 × 10 3 Cells were plated at 1000 kJ / well and allowed to adhere. The following day, test reagents were added, and cells were incubated with the test antibody for an additional 2 days. The CellTiter-Glo® (Promega) assay was performed according to the manufacturer's protocol. Each condition was tested in triplicate. Untreated cells (negative control) were exposed to culture medium only. Values ​​were normalized to the untreated control.

[0191] b) Suspension cells. On the day of the assay, cells were plated in a 96-well plate at 4 × 10 3 Cells / well were plated and test antibody was added. After 2 days of incubation with the test antibody, the CellTiter-Glo® (Promega) assay was performed according to the manufacturer's protocol. Each condition was tested in triplicate. Untreated cells (negative control) were exposed to culture medium only. Values ​​were normalized to the untreated control.

[0192] We investigated the cytotoxicity of the anti-MUC16 / anti-DR5 bispecific molecule 3A5 / LEX (IMV-18; Table 9) and the parent anti-DR5 antibody lexatumumab (Table 10) against three cell lines: NCI-H292, CAOV-3, and OVCAR-3. These three cell lines express both MUC16 and DR5 transcripts above the low expression threshold level of 2.0 (Table 11). The results of the cytotoxicity experiments are shown in Figure 3A. 3A5 / LEX anti-MUC16 / anti-DR5 produced a robust cytotoxic effect in all three cell lines at a concentration of 0.3 nM, and this cytotoxic effect gradually increased as the concentration increased to 1 nM and 3 nM. At 3 nM, the viabilities of NCI-H292, Caov-3, and OVCAR-3 cells were 0.48, 0.20, and 0.21, respectively. The parent anti-DR5 antibody, lexatumumab, had significantly lower killing activity against these three cell lines, with most cells surviving exposure to lexatumumab at concentrations as high as 10 nM, with viability rates of 0.94, 0.95, and 0.81, respectively. Thus, binding to DR5 by monospecific antibodies that do not additionally target MUC16 was not sufficient to induce potent DR5-mediated cytotoxic effects, whereas the anti-MUC16 / anti-DR5 bispecific antibody exhibited high cytotoxic potential.

[0193] We further generated two other bispecific molecules (IMV-15 and IMV-20; Table 10) targeting DR5 and antigens other than MUC16 and tested their ability to induce cytotoxic effects in five cell lines: MM.1S, SU-DHL-8, Ramos, RPMI-8226, and MOLP-8. The results are shown in Figure 3B. Neither the anti-CD38 / anti-DR5 antibody IMV-15 nor the anti-LIV-1 / anti-DR5 antibody IMV-20 was able to kill any of these cell lines, indicating that not all cell surface antigens are suitable for cotargeting DR5 with a bispecific antibody targeting DR5. This, together with previously reported results showing that anti-CD44v6 / anti-DR5 bispecifics did not enhance the agonistic effect of DR5 in CD44v6-expressing cells (U.S. Pat. No. 10,858,438), further demonstrates that the cytotoxicity of the bispecific antigen-binding molecules disclosed herein was unpredictable. To further evaluate the cytotoxic effects of the bispecific antigen-binding molecules disclosed herein, all adherent cell lines are obtained from the American Type Culture Collection (ATCC) (www.ATCC.org), the RIKEN BioResource Research Center (RIKEN) (https: / / cell.brc.riken.jp / en), or the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ) (https: / / www.dsmz.de). The following cell lines are used: HPAC pancreatic adenocarcinoma (CRL-2119™; ATCC), PK-59 pancreatic carcinoma (RCB1901; RIKEN); HCC827 lung adenocarcinoma (CRL-2868; ATCC), NCI-H1975 lung adenocarcinoma (CRL-5908; ATCC); NIH:OVCAR-3, also known as OVCAR-3 ovarian adenocarcinoma (HTB-161; ATCC); NCC-StC-K140 gastric carcinoma (RCB2224; RIKEN); and HDQ-P1 breast ductal carcinoma (ACC494; DSMZ).

[0194] Applicants also investigated the cytotoxicity of the anti-MUC16 / anti-DR5 bispecific molecule 3A5 / LEX (MCLX-SE; Table 9), the comparative bispecific antibody FLX-SE (Table 10), which targets only DR5 on the cell surface, and the parental monospecific anti-MUC16 antibody 3A5 (MC-SE, Table 10). Applicants compared the cytotoxicity of these three antibodies against six cell lines: HPAC, PK-59, NCI-H1975, OVCAR-3, HDQ-P1, and NCC-StC-K140. 3x10 cells per well were used. 3 HDQ-P1 cells, or 8 x 10 3 The protocol for adherent cells described above was used, except that NCC-StC-K140 cells were plated at 100 μg / ml. These six cell lines express both MUC16 and DR5 transcripts above the low expression threshold level of 2.0 (Table 11). The results of the cytotoxicity experiments are shown in Figure 3C. The anti-MUC16 / anti-DR5 bispecific antibody MCLX-SE exhibited robust cytotoxic effects against these six cell lines at concentrations below 1 nM, whereas the anti-fluorescein / anti-DR5 bispecific antibody FLX-SE produced little, if any, cytotoxic effect against these cell lines, even at 10 nM, the highest concentration tested. Monospecific anti-MUC16 antibodies were not cytotoxic to these cell lines within this concentration range. Thus, neither the binding of the antibody FLX-SE, which does not target MUC16, to DR5 nor the binding of the antibody MC-SE, which does not target DR5, to MUC16 was sufficient to induce a strong cytotoxic effect, whereas the anti-MUC16 / anti-DR5 bispecific antibody had high cytotoxic potential.

[0195] Cells were plated in 96-well tissue culture plates at 2.5 x 10 3Cells were plated at 1000 x g / well and allowed to adhere. The next day, test reagents (IMV-18 (Table 9), MCLX-SE (Table 9), 11D10X (Table 10), and 11D10-SE (Table 10), parental anti-MUC16 antibody 3A5 (SEQ ID NOs: 115 and 119), parental anti-MUC16 antibody MC-SE (SEQ ID NOs: 116 and 120), non-targeting bispecific antibody FLX-SE (Table 10), or IMV-15) were added to different culture plates, and the cells were cultured for an additional 2 days. The CellTiter-Glo® (Promega) assay was performed according to the manufacturer's protocol. Each condition was assayed in triplicate. Untreated cells (negative control) were exposed to culture medium only. Values ​​were normalized to the untreated control.

[0196] Applicant compares the cytotoxicity of the anti-MUC16 / anti-DR5 bispecific antibodies 3A5 / anti-DR5 and 11D10 / anti-DR5. The K values ​​of the parent antibodies 3A5 and 11D10 against MUC16 on the surface of OVCAR-3 cells reported in U.S. Pat. No. 7,989,595 and Chen et al. Cancer Res. 2007:67, 4924-4932 are shown. D The values ​​are 360 ​​pM and 52 pM, respectively, ie the affinity of 11D10 is 7 times higher than that of 3A5.

[0197] Example 4. Evaluation of the antitumor activity of IMV-18 in an HPAC xenograft model The antitumor activity of IMV-18, a bispecific antibody targeting MUC16 and DR5, was examined in a xenograft model of the HPAC pancreatic adenocarcinoma cell line (CRL-2119™, American Type Culture Collection (www.atcc.org)) in immunodeficient mice. HPAC cells express both MUC16 and DR5 (Table 11). For comparison, some mice were treated with IMV-21, a bispecific antibody using the same anti-DR5 scFv but targeting a different antigen, CD74. BALB / c nude female mice, 6-8 weeks old and weighing 18-21 g, were used in this study. Prior to inoculation into mice, HPAC cells were maintained in F12 / DMEM medium supplemented with 5% fetal bovine serum, 0.5 mM sodium pyruvate, 0.002 mg / ml insulin, 5 μg / ml transferrin, 40 ng / ml hydrocortisone, and 10 ng / ml epidermal growth factor at 37°C in a humidified atmosphere containing 5% CO2. Tumor cells were routinely subcultured for no more than four to five passages before reaching confluence by trypsin-EDTA treatment. For tumor inoculation, cells growing in exponential growth phase were harvested and counted. Each mouse received 5 x 10 cells in the presence of Corning® Matrigel® Matrix according to the manufacturer's protocol. 6 HPAC cells were inoculated subcutaneously into the right flank. The average tumor volume was approximately 145 mm 3 Once tumor-bearing mice reached tumor volume, treatment with test agents was initiated. Based on tumor volume, mice were randomly assigned to groups of five, so that the average starting tumor size was the same for each treatment group. The average weight of mice in each group was 18 g or more. Mice were intravenously injected once (day 1, Figure 4) with IMV-21 (5 mg / kg in phosphate-buffered saline (PBS)), IMV-18 (5 mg / kg in PBS), or vehicle alone (PBS). All animals were weighed daily throughout the study. Tumor size measurements were performed twice weekly using calipers to determine tumor volume (mm 3) is estimated using the following formula: tumor volume = a × b × b / 2, where "a" and "b" are the long and short diameters of the tumor, respectively. As shown in Figure 4A, treatment with IMV-18 caused a significant delay in the average tumor growth in mice compared with vehicle controls, whereas treatment with IMV-21 did not slow tumor growth. Mouse weights did not decrease, indicating the lack of systemic toxicity of these molecules to mice (Figure 4B). Plots of tumor growth in individual mice (Figure 4C) show that HPAC xenograft growth was robust in mice treated with vehicle alone or IMV-21; however, there was no tumor growth delay in mice treated with IMV-21. Two of the five mice treated with IMV-18 experienced significant tumor growth delay, and the remaining three mice experienced tumor disappearance and no tumor regrowth throughout the experimental period. IMV-21, a bispecific antibody composed of an IgG antibody targeting a different antigen (CD74) and the same anti-DR5 scFv as IMV-18, was inactive, indicating that the antitumor activity of IMV-18 is MUC16-dependent.

[0198] Example 5. Antiproliferative effect and apoptosis of PK-59 cells induced by a bispecific molecule recognizing human MUC16 and human DR5. Cells were plated at 1.5 x 10 per well. 3Cells were plated onto tissue culture plates at a density of 1000 x g. After 24 hours, test agents were added according to the manufacturer's protocol, along with Incucyte® Caspase-3 / 7 Green Dye for Apoptosis, Catalog No. 4440, Sartorius (green fluorescence) and Incucyte® Nuclight Rapid Red Dye for nuclear labeling, Catalog No. 4717, Sartorius (red fluorescence). Each condition was tested in triplicate. Caspase-3 / 7 Green Dye is a fluorescent substrate for caspase 3 and caspase 7, two caspases activated in the late stages of apoptosis [Elmore S., Toxicol Pathol. 2007;35(4):495-516]. In the presence of this dye, healthy cells do not fluoresce, while cells undergoing apoptosis fluoresce green. When cells are exposed to Nuclight Rapid Red Dye, their nuclei fluoresce red. Green-fluorescent (Green) and red-fluorescent (Red) objects per well were counted every 2 hours using an IncuCyte S3 (Sartorius AG, sartorius.com). Each condition was tested in triplicate. The growth-inhibitory effect of the bispecific anti-MUC16 / anti-DR5 antibody MCLX-SE is shown in Figure 5A. The mean number of cells with red-fluorescent nuclei per well (average of three wells) (Y-axis: Log10 scale) was plotted against time (X-axis: hours). In the absence of test agent (medium), cells proliferated exponentially without slowing (linear semi-exponential plot), with a doubling time of 35 hours. In the presence of MCLX-SE, at the lowest concentration tested, 41.2 pM, cell proliferation rapidly declined during the first 24 hours of exposure and almost completely stopped. In contrast, at this concentration, the monospecific parent anti-MUC16 antibody MC-SE had no effect on cell proliferation (doubling time remained constant at 35 h), and the comparative bispecific antibody FLX-SE only partially reduced cell proliferation.At higher concentrations (123 pM, 370 pM, 1.11 nM, and 10 nM), exposure to MCLX-SE resulted in a single doubling of cell number followed by an almost complete cessation of proliferation, whereas in the presence of MC-SE, cells continued to proliferate without slowing, with doubling times ranging from 35 to 40 h. FLX-SE, on the other hand, only partially slowed cell proliferation, even at the highest concentration tested, 10 nM.

[0199] Induction of cell apoptosis by the bispecific anti-MUC16 / anti-DR5 antibody MCLX-SE is shown in Figures 5B and 5C. As shown in Figure 5B, exposure to 41.2 pM of the bispecific anti-MUC16 / anti-DR5 antibody MCLX-SE rapidly led to apoptosis in the majority of cells. By 18 hours after exposure, approximately 1900 cells contained activated caspase-3 and / or caspase-7. In contrast, only a small proportion of cells underwent apoptosis during exposure to the comparative bispecific antibody FLX-SE, which targets cell surface DR5 but not MUC16. Another comparative antibody, the monospecific antibody MC-SE, which targets MUC16, did not induce significant apoptosis in the cells. Exposure of cells to high concentrations of MCLX-SE induced even more pronounced apoptosis (Fig. 5C), whereas the control antibody FLX-SE induced apoptosis in only a small proportion of cells, even at the highest concentration tested, 10 nM, and another control antibody, MC-SE, did not induce significant apoptosis at any concentration (Fig. 5C).

[0200] Example 6. Evaluation of the antitumor activity of MCLX-SE in a MUC16-positive / DR5-positive xenograft model The antitumor activity of MCLX-SE was investigated in immunodeficient mice using the following xenograft model using MUC16-positive cell lines expressing both MUC16 and DR5 above the low expression threshold (Table 11). The cell lines tested were: HPAC pancreatic adenocarcinoma cell line (CRL-2119™), HCC827 (HCC827 lung adenocarcinoma (CRL-2868)), PK-59 cells (pancreatic adenocarcinoma), and NCI-H1975 cells (lung adenocarcinoma). For comparison, some mice were treated with FLX-SE (Table 10) or MC-SE. BALB / c nude female mice were used in this study. The average tumor volume was approximately 100-150 mm. 3 Once tumor volume reached 100 mm, treatment of tumor-bearing mice with test agents began. Mice were randomly assigned to each group based on tumor volume so that the average starting tumor size was the same for each treatment group. Mice were intravenously injected with one of these agents or vehicle alone (PBS). Tumor size measurements were performed using calipers to determine tumor volume (mm). 3 ) is estimated using the following formula: tumor volume = a × b × b / 2, where "a" and "b" are the longest and shortest diameters of the tumor, respectively. As shown in Figures 6A-6D, treatment with the anti-MUC16 / anti-DR5 bispecific antibody MCLX-SE significantly delayed the mean tumor growth in mice in all four xenograft models compared with vehicle controls. Furthermore, in the HPAC and HCC827 xenograft studies, two additional controls were included: the monospecific antibody MC-SE, which targets MUC16, and the bispecific antibody FLX-SE, which targets only DR5. As shown in Figures 6A and 6B, treatment with either FLX-SE or MC-SE did not delay tumor growth in HPAC or HCC827 xenograft tumors, indicating that targeting of both antigens is necessary for antitumor activity at this dose and that the antitumor activity of the anti-MUC16 / anti-DR5 antibody is both MUC16- and DR5-dependent.

[0201] MUC16 protein expression in xenografts was examined by immunohistochemistry. The following reagents were used: rabbit monoclonal anti-MUC16 antibody, Abcam #110640MXB (primary antibody); polyclonal goat anti-rabbit immunoglobulin, Dako (Agilent Technologies), cat #4003 (secondary antibody); Dako antibody diluent, Dako #S2022; washing buffer, Dako #K8007; and citrate 6.0 (MXB Biotechnologies, Fuzhou, Fujian, China #MVS-0066). Paraffin-embedded tumor tissues were sectioned at a thickness of 4 μm using a microtome and processed according to protocols well known in the art. After heat-induced antigen unmasking in citrate pH 6.0, sections were immersed in 3% hydrogen peroxide solution for 5 minutes. To avoid nonspecific staining, sections were incubated in blocking serum containing normal goat serum for 30 minutes at room temperature, followed by overnight incubation with rabbit monoclonal anti-MUC16 antibody (Abcam #110640, 1:500 dilution). Sections were then exposed to HRP-conjugated secondary antibody. Nuclei were stained with hematoxylin. Slides were scanned at 200x magnification using an Aperio Scanner: Versa 8 (Leica). Images were then opened in HALO, and the annotation layer was selected using the pen tool. Necrotic areas were excluded in the annotation layer. The following xenografts were examined: Capan-1, PK-59, HCC827, NCI-H1975, and HPAC, all in Balb / c nude mice; and OVCAR-3 in SCID mice. OVCAR-3 showed strong MUC16 expression, PK-59, HCC827, and HPAC showed moderate MUC16 expression, NCI-H1975 showed weak MUC16 expression, and Capan-1 showed very weak to negative MUC16 expression. These data were in good agreement with Table 11.

[0202] Example 7. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5 and FLX-SE to human recombinant DR5. Human DR5 recombinant protein (TRAIL R2, catalog number TR2-H5229; amino acids 56–182) was obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86 400-682-2521, www.acrobiosystems.cn. High-binding ELISA plates were coated with 1 μg / mL human DR5 or blocking buffer (2% bovine serum albumin in PBS) overnight at 4°C. The solution was aspirated, and blocking buffer was added to all wells for 1 h. After aspirating the blocking buffer, various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer were added at different concentrations (serial dilutions). The plates were incubated at room temperature for 1 hour, after which the antibodies were aspirated and the wells were washed. The wells were then incubated with horseradish peroxidase-conjugated anti-human IgG Fc-γ polyclonal antibody, washed, and the bound polyclonal antibody was detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by absorbance measurement at 450 nm using stop solution (ThermoFisher Scientific). Normal polyclonal human IgG was used to detect nonspecific binding of human IgG to the recombinant human DR5-coated plate.

[0203] Example 8. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE to human recombinant MUC16. Human recombinant MUC16 (catalog number CA5-H52H6, amino acids 13810–14451) was obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86 400-682-2521, www.acrobiosystems.cn. High-binding ELISA plates were coated with 1 μg / mL human MUC16 or blocking buffer (2% bovine serum albumin in PBS) overnight at 4°C. The solution was aspirated, and blocking buffer was added to all wells for 1 h. After aspirating the blocking buffer, various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). The plate is incubated at room temperature for 1 hour, after which the antibody is aspirated and the wells are washed. The wells are then incubated with horseradish peroxidase-conjugated anti-human IgG Fc-γ polyclonal antibody, washed, and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by measuring absorbance at 450 nm using stop solution (ThermoFisher Scientific).

[0204] Example 9. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE to human recombinant CD74. Human recombinant CD74 (catalog number CA4-H524c, amino acids 73–232) was obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86 400-682-2521, www.acrobiosystems.cn. High-binding ELISA plates were coated with 1 μg / mL human CD74 or blocking buffer (2% bovine serum albumin in PBS) overnight at 4°C. The solution was aspirated, and blocking buffer was added to all wells for 1 h. After aspirating the blocking buffer, various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). The plate is incubated at room temperature for 1 hour, after which the antibody is aspirated and the wells are washed. The wells are then incubated with horseradish peroxidase-conjugated anti-human IgG Fc-γ polyclonal antibody, washed, and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by measuring absorbance at 450 nm using stop solution (ThermoFisher Scientific).

[0205] Example 10. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE to human recombinant CD38. Human CD38 (catalog number CA8-H5224, amino acids 43–300) was obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86 400-682-2521, www.acrobiosystems.cn. High-binding ELISA plates were coated with 1 μg / mL human CD38 or blocking buffer (2% bovine serum albumin in PBS) overnight at 4°C. The solution was aspirated, and blocking buffer was added to all wells for 1 h. After aspirating the blocking buffer, various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). The plate is incubated at room temperature for 1 hour, after which the antibody is aspirated and the wells are washed. The wells are then incubated with horseradish peroxidase-conjugated anti-human IgG Fc-γ polyclonal antibody, washed, and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by measuring absorbance at 450 nm using stop solution (ThermoFisher Scientific).

[0206] Example 11. Binding of IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE to human recombinant LIV-1 / SLC39A6. Human LIV-1 (catalog number LV1-H5223, amino acids 29–325) was obtained from ACROBiosystems China, Floor 4, Building 5, No. 8 Hongda North Road, BDA, Beijing, 100176, China, Phone: 86 400-682-2521, www.acrobiosystems.cn. High-binding ELISA plates were coated with 1 μg / mL human LIV-1 or blocking buffer (2% bovine serum albumin in PBS) overnight at 4°C. The solution was aspirated, and blocking buffer was added to all wells for 1 h. After aspirating the blocking buffer, various agents (IMV-14, IMV-15, IMV-18, IMV-20, IMV-21, MCLX-SE, 11D10 anti-MUC16 / anti-DR5, and FLX-SE; see Tables 9 and 10) in blocking buffer are added at different concentrations (two independent serial dilutions). The plate is incubated at room temperature for 1 hour, after which the antibody is aspirated and the wells are washed. The wells are then incubated with horseradish peroxidase-conjugated anti-human IgG Fc-γ polyclonal antibody, washed, and detected with 3,3',5,5'-tetramethylbenzidine (TMB), followed by measuring absorbance at 450 nm using stop solution (ThermoFisher Scientific).

[0207] Example 12. Anti-tumor activity of anti-MUC16-anti-DR5 bispecific antibodies is independent of interaction with Fc receptor gamma in a pancreatic tumor xenograft model. As described in Shivange et al. 2018; Cancer Cell 34, 331-345, the interaction of anti-DR5 antibodies with Fcγ receptors, specifically Fcγ receptor 2B, which is found primarily on B cells, is important for anti-tumor activity in mouse xenograft models. Antibodies that lack the ability to interact with these receptors did not exhibit anti-tumor activity in these models. Surprisingly, the anti-MUC16-anti-DR5 bispecific antibodies disclosed herein do not require interaction with these receptors.

[0208] We compared the antitumor activity of four variants of an anti-MUC16-anti-DR5 bispecific antibody (IMV-18, MCLX-SE, IMV-AA, and MC-AA) in a pancreatic cancer xenograft model. These variants (listed in Table 9) were nearly identical except for several point mutations in the Fc region that affected their affinity for Fc receptor γ on immune cells in nude mice, as described in [Shivange et al. 2018; Cancer Cell 34, 331-345]. IMV-18 contained wild-type human IgG1 Fc; MCLX-SE enhanced affinity for Fc receptor 2B; IMV-AA inhibited affinity for all Fc receptor γ; and MC-AA inhibited affinity for Fc receptor γ except for Fc receptor 2B, restoring affinity. In some mice, these antibodies were co-injected at 30 mg / kg with a nontargeting murine IgG1 antibody (muIgG1, Table 10) containing wild-type human IgG1 Fc. This co-injection was performed to allow muIgG1 to compete with the anti-MUC16-anti-DR5 bispecific antibody for binding to Fcγ receptors.

[0209] In this study, we used the MUC16-positive / DR5-positive xenograft pancreatic cancer model PK-59 (RCB1901, RIKEN BioResource Research Center (BRC) Cell Bank, 3-1-1 Takanodai, Tsukuba, Ibaraki 305-0074, Japan). PK-59 cells were injected subcutaneously into BALB / c nude mice, and the average tumor volume was approximately 100–150 mm. 3 Once tumor size reached 100 μg / kg, the resulting tumor-bearing mice were treated with one of the anti-MUC16-anti-DR5 bispecific antibodies (or an appropriate control). Mice were randomly assigned to each group based on tumor volume so that the average starting tumor size was the same for each treatment group. Four mice per group were intravenously injected once on day 1 with either the anti-MUC16-anti-DR5 bispecific antibody (5 mg / kg) or vehicle (PBS), or the anti-MUC16-anti-DR5 bispecific antibody (5 mg / kg) and muIgG1 (30 mg / kg).

[0210] Tumor size measurements were performed using calipers and tumor volume (mm 3 ) is estimated using the following formula: tumor volume = a x b x b / 2, where "a" and "b" are the longest and shortest diameters of the tumor, respectively. Data are presented as mean ± SEM (n = 4). At some time points, the SEM was too small to be visible in the plot. As shown in Figure 7, all anti-MUC16 / anti-DR5 bispecific antibodies were equally active in their ability to delay the growth of PK-59 xenografts, with or without excess murine IgG1 antibody.

[0211] Example 13. IVM-M exhibits dose-dependent antitumor activity in a pancreatic tumor xenograft model. BALB / c nude mice were subcutaneously injected with PK-59 pancreatic cancer cells as described in Example 12. IVM-M treatment resulted in tumors with a mean tumor volume of approximately 100-150 mm. 3 Treatment was initiated when tumor volume reached 100 μg / mL. Mice were randomly assigned to each treatment group based on tumor volume, so that the average starting tumor size was the same for each treatment group. IMV-M was intravenously injected at 1 mg / kg, 2.5 mg / kg, or 5 mg / kg, or vehicle alone (PBS), once on day 1, into four mice per group. Tumor size measurements were performed using calipers, and tumor volume (mm3) was estimated using the following formula: tumor volume = a × b × b / 2 (where "a" and "b" are the longest and shortest diameters of the tumor, respectively). Data are presented as mean ± SEM (n = 4). At some time points, the SEM was too small to be visible on the plot. As shown in Figure 8, treatment with the anti-MUC16 / anti-DR5 bispecific antibody IMV-M caused a significant delay in the average tumor growth in mice at all three doses, in a dose-dependent manner.

[0212] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0213] Also incorporated by reference in their entirety are polynucleotide and polypeptide sequences that reference accession numbers that correlate to entries in public databases, such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

[0214] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A bispecific binding molecule comprising: a. a first antigen-binding domain that specifically binds to the extracellular domain of human MUC16; b. a second antigen-binding domain that specifically binds to human DR5; 1. A bispecific binding molecule comprising:

2. The bispecific binding molecule of claim 1, wherein the first antigen-binding domain binds to an epitope present in two or more tandem repeat / SEA segments within the extracellular domain of MUC16.

3. 2. The bispecific binding molecule of claim 1, wherein the first antigen-binding domain has MUC16 binding competed for by one or more of the following antibodies: OC125, H185, M11, OV197, 5E11, AR9.6, H1H8794, VK-8, B43.13, or 3A5.

4. 2. The bispecific binding molecule of claim 1, wherein the first antigen-binding domain binds to a polypeptide or peptide consisting essentially of at least one amino acid sequence of SEQ ID NOs: 176-181, or an extracellular fragment of MUC16 isolated from cell culture medium of OVCAR-3 cells.

5. The first antigen-binding domain comprises: a. a heavy chain variable region comprising three heavy chain complementarity determining regions (CDR-H1, CDR-H2, and CDR-H3): CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1; CRD-H2 comprises any one of the amino acid sequences of SEQ ID NOs: 2 to 5; a heavy chain variable region, wherein CDR2-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 6 to 61 (CDR3-H3); b. A light chain variable region comprising three light chain complementarity determining regions (CDR-L1, CDR-L2, and CDR-L3): CDR-L1 comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 63; CDR-L3 is a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65; 5. The bispecific binding molecule of claim 1, comprising:

6. CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1; CDR-H2 comprises the amino acid sequence of SEQ ID NO:2; CDR-H3 comprises the amino acid sequence of SEQ ID NO:6; CDR-L1 comprises the amino acid sequence of SEQ ID NO: 62; CDR-L2 comprises the amino acid sequence of SEQ ID NO: 63; CDR-L3 comprises the amino acid sequence of SEQ ID NO: 65; The bispecific binding molecule of claim 5 .

7. 7. The bispecific binding molecule of claim 5 or 6, wherein the first antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 97 and having at least 90% sequence identity to SEQ ID NO:

97.

8. The bispecific binding molecule of claim 7 , wherein the first antigen-binding domain comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

97.

9. 9. The bispecific binding molecule of claim 5, wherein the first antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 104 or 105, or an amino acid sequence having the same three light chain CDRs as either SEQ ID NO: 104 or SEQ ID NO: 105 and at least 90% sequence identity to either SEQ ID NO: 104 or 105.

10. 10. The bispecific binding molecule of claim 9, wherein the first antigen-binding domain comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 104 or SEQ ID NO:

105.

11. The first antigen-binding domain comprises: a. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97, or an amino acid sequence having the same three light chain CDRs as SEQ ID NO: 97 and at least 90% sequence identity to SEQ ID NO: 97; b. a light chain variable region comprising the amino acid sequence of either SEQ ID NO: 104 or 105, or an amino acid sequence having the same three heavy chain CDRs as either SEQ ID NO: 104 or 105 and at least 90% sequence identity to either SEQ ID NO: 104 or 105; 11. The bispecific binding molecule of claim 7, comprising:

12. The first antigen-binding domain comprises: a. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 97; b. a light chain variable region comprising the amino acid sequence of either SEQ ID NO: 104 or 105; 12. The bispecific binding molecule of claim 11 , comprising:

13. 13. The bispecific binding molecule of claim 5, wherein the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 115-118, or a heavy chain comprising the same three heavy chain CDRs as any one of SEQ ID NOs: 115-118 and comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 115-118.

14. The bispecific binding molecule of claim 13, wherein the first antigen-binding domain comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 115 to 118.

15. 15. The bispecific binding molecule of any one of claims 5 to 14, wherein the first antigen-binding domain comprises a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as SEQ ID NO: 119 or SEQ ID NO: 120 and having at least 90% sequence identity to SEQ ID NO: 119 or SEQ ID NO:

120.

16. 16. The bispecific binding molecule of claim 15, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 119 or the amino acid sequence of SEQ ID NO:

120.

17. The first antigen-binding domain comprises: a. a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 115-118, or a heavy chain comprising the same three heavy chain CDRs as any one of SEQ ID NOs: 115-118 and an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 115-118; b. a light chain comprising the amino acid sequence of SEQ ID NO: 119, the amino acid sequence of SEQ ID NO: 120; or an amino acid sequence having the same three light chain CDRs as SEQ ID NO: 119 or SEQ ID NO: 120 and having at least 90% sequence identity to SEQ ID NO: 119 or SEQ ID NO: 120; 17. The bispecific binding molecule of any one of claims 13 to 16, comprising:

18. The first antigen-binding domain comprises: a. a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 115-118; b. a light chain comprising the amino acid sequence of SEQ ID NO: 119 or SEQ ID NO: 120; 18. The bispecific binding molecule of claim 17, comprising:

19. The bispecific binding molecule of any one of claims 1 to 18, wherein the first antigen-binding domain comprises two heavy chains and two light chains.

20. 20. The bispecific binding molecule of claim 19, wherein each of the two heavy chains has an identical amino acid sequence and each of the two light chains has an identical amino acid sequence.

21. 21. The bispecific binding molecule of any one of claims 1 to 20, wherein the first antigen-binding domain is competed for DR5 binding by one or more of the following antibodies: conatumumab, drozitumab, lexatumumab, LBY135, tigatuzumab, and DS-8273a.

22. The bispecific binding molecule of any one of claims 1 to 21, wherein the second antigen-binding domain is competed for DR5 binding by TRAIL or a fragment of TRAIL that binds to DR5.

23. 23. The dual specificity binding molecule of any one of claims 1 to 22, wherein the second antigen-binding domain comprises: a second heavy chain variable region comprising second heavy chain complementarity determining regions (CDRs) comprising the amino acid sequences of SEQ ID NO: 121 (second CDR-H1); SEQ ID NO: 122 (second CDR2-H2); and SEQ ID NO: 123 (second CDR3-H3); and a second light chain variable region comprising second light chain complementarity determining regions comprising the amino acid sequences of SEQ ID NO: 124 (second CDR-L1); SEQ ID NO: 125 (second CDR-L2); and SEQ ID NO: 126 (second CDR-L3).

24. 24. The bispecific binding molecule of claim 23, wherein the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158, any one of SEQ ID NOs: 218-220, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 158 or any one of SEQ ID NOs: 218-220 and at least 90% sequence identity to SEQ ID NO: 158 or any one of SEQ ID NOs: 218-220.

25. 25. The bispecific binding molecule of claim 24, wherein the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

158.

26. 25. The bispecific binding molecule of claim 24, wherein the second heavy chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 218-220.

27. 27. The bispecific binding molecule of any one of claims 23 to 26, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 165, any one of SEQ ID NOs: 215-217, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 165, any one of SEQ ID NOs: 215-217 and at least 90% sequence identity to any one of SEQ ID NO: 165, any one of SEQ ID NOs: 215-217.

28. 28. The bispecific binding molecule of claim 27, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO:

165.

29. 28. The bispecific binding molecule of claim 27, wherein the second light chain variable region comprises the amino acid sequence of any one of SEQ ID NOs: 215-217.

30. a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 158 and at least 90% sequence identity to SEQ ID NO: 158; b) The bispecific binding molecule of any one of claims 24-29, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence having the same three heavy chain CDRs as SEQ ID NO: 158 and having at least 90% sequence identity to SEQ ID NO:

165.

31. a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 158; b) The bispecific binding molecule of claim 30, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO:

165.

32. a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:218; b) The bispecific binding molecule of any one of claims 24-29, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO:

215.

33. a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:219; b) The bispecific binding molecule of any one of claims 24-29, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO:

215.

34. a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO:219; b) The bispecific binding molecule of any one of claims 24-29, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO:

216.

35. a. the second heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 220; b) The bispecific binding molecule of any one of claims 24 to 29, wherein the second light chain variable region comprises the amino acid sequence of SEQ ID NO:

217.

36. 36. The bispecific binding molecule of claim 1, wherein the second antigen-binding domain is an scFv fragment of an antibody.

37. 37. The bispecific binding molecule of claim 36, wherein the scFv fragment comprises, from N-terminus to C-terminus, a light chain variable region, a peptide linker, and a heavy chain variable region.

38. 38. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO: 172; or an amino acid sequence having the same three heavy chain CDRs and the same three light chain CDRs as SEQ ID NO: 172 and having at least 90% sequence identity with SEQ ID NO:

172.

39. 39. The bispecific binding molecule of claim 38, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:

172.

40. 38. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:

210.

41. 38. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:

211.

42. 38. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:

212.

43. 38. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:

213.

44. 38. The bispecific binding molecule of claim 37, wherein the scFv fragment comprises the amino acid sequence of SEQ ID NO:

214.

45. 45. The bispecific binding molecule of any one of claims 36 to 44, wherein the N-terminus of the second antigen-binding domain is fused to the C-terminus of one of the heavy chains of the first antigen-binding domain directly or via a peptide linker 4 to 20 amino acids in length.

46. 46. ​​The bispecific binding molecule of claim 45, wherein the peptide linker has the amino acid sequence of SEQ ID NO:

173.

47. 47. The bispecific binding molecule of any one of claims 36 to 46, comprising two scFv fragments that specifically bind to DR5, wherein each of the two scFv fragments binds to a different heavy chain of the first antigen-binding domain.

48. 48. The bispecific binding molecule of claim 47, wherein each of the two scFv fragments has an identical amino acid sequence.

49. 1. A bispecific binding molecule comprising: a) two antibody light chains, each light chain independently having an amino acid sequence selected from SEQ ID NO: 119 and SEQ ID NO: 120; b) two antibody heavy chain fusions, each heavy chain fusion independently having a structure of the formula X-LY, wherein: X is an amino acid sequence of any one of SEQ ID NOs: 115-118; L is the amino acid sequence of SEQ ID NO: 173; Y is the amino acid sequence of SEQ ID NO: 172 or any one of SEQ ID NOs: 210-214; and 1. A bispecific binding molecule comprising:

50. 50. The bispecific binding molecule of claim 49, wherein each light chain has the same amino acid sequence and each heavy chain fusion has the same amino acid sequence.

51. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

174.

52. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

175.

53. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

186.

54. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

187.

55. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

210.

56. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

211.

57. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

212.

58. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

213.

59. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 119 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

214.

60. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

174.

61. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

175.

62. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

186.

63. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

187.

64. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

210.

65. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

211.

66. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

212.

67. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

213.

68. 51. The bispecific binding molecule of claim 50, wherein each light chain comprises the amino acid sequence of SEQ ID NO: 120 and each heavy chain fusion comprises the amino acid sequence of SEQ ID NO:

214.

69. 69. A pharmaceutical composition comprising the bispecific binding molecule of any one of claims 1 to 68; and a pharmaceutically acceptable carrier.

70. An isolated nucleic acid sequence encoding a heavy chain fusion comprising the amino acid sequence of any one of SEQ ID NOs: 174, 175, 186, 187, or 210-214.

71. 71. The isolated nucleic acid sequence of claim 70, comprising SEQ ID NO: 182 or 183.

72. 72. An expression vector comprising the nucleic acid sequence of claim 70 or 71.

73. 73. The expression vector of claim 72, further comprising a nucleic acid sequence encoding a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119 or 120.

74. 74. The expression vector of claim 73, wherein the nucleic acid sequence encoding the light chain comprises SEQ ID NO: 184 or 185.

75. 73. A host cell comprising a first expression vector of claim 72; and a second expression vector comprising a nucleic acid sequence encoding a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 119 or 120.

76. 76. The host cell of claim 75, wherein the nucleic acid sequence encoding the light chain comprises SEQ ID NO: 184 or 185.

77. A host cell comprising the expression vector of claim 73 or 74.

78. 1. A method of making a bispecific binding molecule, comprising: a) culturing the host cell of any one of claims 75-77 under conditions that allow expression of the nucleic acid sequence encoding the heavy chain fusion and expression of the nucleic acid sequence encoding the light chain, and association of the expressed heavy chain fusion and the expressed light chain into the bispecific binding molecule; b. recovering the bispecific binding molecule from the culture medium; A method comprising:

79. 70. A method of treating a MUC16-associated fibrotic, inflammatory, immune, or autoimmune disorder, comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any one of claims 1 to 68, or the pharmaceutical composition of claim 69.

80. 80. The method of claim 79, wherein the MUC16-mediated fibrotic disorder is pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury, hepatic fibrosis, liver cirrhosis, renal fibrosis, glial scar, myocardial fibrosis, arteriosclerosis, arthrofibrosis, chronic kidney disease, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, or adhesive capsulitis.

81. 70. A method of treating cancer or malignant tumors characterized by overexpression of MUC16, comprising administering to a subject in need thereof a therapeutically effective amount of the bispecific binding molecule of any one of claims 1 to 68, or the pharmaceutical composition of claim 69.

82. 82. The method of claim 81, wherein the cancer or malignant tumor is gynecological cancer (cancer of the female reproductive organs), including cervical, endometrial, fallopian tube, ovarian, uterus, and vaginal cancer; pancreatic cancer, esophageal cancer, gastric cancer, colorectal cancer, breast cancer, or lung cancer.

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