Multi-specific binding proteins that bind dectin-1 and trop-2 and methods of use thereof

Abstract

The present disclosure relates to bispecific binding molecules comprising a first arm comprising a first antigen binding domain that binds to human Dectin-1 and a second arm comprising a second antigen binding domain that binds to human Trop-2, as well as methods, polynucleotides, vectors, host cells, compositions and uses related thereto.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 530,018, filed July 31, 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to the electronic sequence list The contents of the electronic sequence list (186542001040seqlist.xml; size: 111,873 bytes; and creation date: July 26, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to a bispecific binding molecule comprising a first arm and a second arm, the first arm comprising a first antigen-binding domain binding to human Dectin-1, the second arm comprising a second antigen-binding domain binding to human Trop-2, and related methods, polynucleotides, vectors, host cells, compositions, and uses. Background Technology

[0004] Phagocytosis is the primary mechanism for removing pathogens and cellular debris. Specialized phagocytes, such as monocytes, macrophages, dendritic cells, and granulocytes, specifically recognize and engulf abnormal or disease-causing host or foreign factors. Engulfed material is destroyed via the lysosomal pathway within phagocytes. Furthermore, dendritic cells and macrophages can present antigens to cells of the adaptive immune system to further promote pathogen elimination. Dectin-1 is a C-type lectin receptor that recognizes β-glucan and promotes antifungal phagocytic activity. It is expressed on phagocytes and has been clearly shown to be sufficient to activate phagocytosis. Dectin-1 can be used to target phagocytosis and eliminate pathogens with antibodies.

[0005] Trop-2 (tumor-associated calcium signaling transducer 2, also known as epithelial glycoprotein-1) is a transmembrane glycoprotein known to be overexpressed in many cancers (see, for example, Shvartsur, A. and Bonavida, B. (2015)). Genes Cancer6(3-4):84-105). Trop-2 overexpression has been found to drive cancer growth through several pathways, including activation of ERK / MAPK. In many cancers, Trop-2 overexpression is associated with decreased survival, increased tumor invasiveness, and metastasis. Cancers with observed Trop-2 overexpression include breast cancer, cervical cancer, colorectal cancer, endometrioid carcinoma, esophageal cancer, gastric cancer, glioma, cholangiocarcinoma, chronic lymphocytic lymphoma (CLL), extranodal NK / T-cell lymphoma, non-Hodgkin's lymphoma, ovarian cancer, prostate cancer, pancreatic cancer, thyroid cancer, bladder cancer, and uterine cancer. Antibodies and antibody-drug conjugates targeting Trop-2 are in clinical testing, including gavitecane sacetuzumab, detrastuzumab datopramumab, FDA018-ADC, JS108, and SKB264 (Shastry, M. et al., (2022)). Breast 66:169-177).

[0006] Therefore, there is still a need for enhanced therapies that can promote immune infiltration and eliminate cancers expressing Trop-2.

[0007] All references cited in this article, including patent applications, patent publications, and scientific literature, are incorporated into this article as a whole, as if each individual reference were specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] This disclosure relates to a multispecific (e.g., bispecific) binding molecule comprising a first arm and a second arm, the first arm comprising a first antigen-binding domain binding to human Dectin-1, and the second arm comprising a second antigen-binding domain binding to human Trop-2, and related methods, polynucleotides, vectors, host cells, compositions, and uses. The multispecific (e.g., bispecific) binding molecule allows phagocytes to bind to and form synapses with Trop-2-expressing target cells using the Trop-2 binding arm, and to promote the accumulation of Dectin-1 on the phagocyte using the Dectin-1 binding arm. This stimulates phagocytosis of the target cells, while simultaneously stimulating the phagocytes to secrete cytokines via the Dectin-1 / Syk / NfkB pathway. Furthermore, antigens from phagocytosed material are presented on the surface of dendritic cells / macrophages to enhance adaptive immune responses against pathogens expressing Trop-2. Overall, the multispecific (e.g., bispecific) binding molecules of this disclosure are believed to promote immune stimulation, targeted phagocytosis, and neoantigen presentation / activation of the adaptive immune system to eliminate pathogenic cells reaching Trop-2.

[0009] Therefore, this disclosure specifically describes the generation and functional characterization of anti-Dectin-1 x anti-Trop-2 bispecific antibodies. These antibodies were found to bind to human Trop-2 and Dectin-1 with high affinity and to activate human Dectin-1 signaling. Various formulations were tested, demonstrating their specific binding to both targets, induction of phagocytosis, and depletion of Trop-2-expressing target cells (such as cancer cell lines), and inhibition of tumor growth in a variety of preclinical mouse models, including both “hot” and “cold” tumor types in terms of immune cell infiltration and responsiveness to immunotherapy (e.g., monotherapy).

[0010] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the second antigen-binding domain comprises a second heavy chain variable (VH) domain and a second light chain variable (VL) domain.

[0011] In some embodiments according to any of the embodiments described herein, the second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 48, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 49, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 50, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 51, CDR-L2 comprising the amino acid sequence of GAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 45, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, CDR-L2 comprising the amino acid sequence of GAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the second VH domain contains the amino acid sequence of SEQ ID NO: 74, and the second VL domain contains the amino acid sequence of SEQ ID NO: 75.

[0012] In some embodiments according to any of the embodiments described herein, the second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 56, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 58. In some embodiments, the second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-L2 comprising the amino acid sequence of AAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 59, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 60, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 61, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 62, CDR-L2 comprising the amino acid sequence of AAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 68. In some embodiments, the second VH domain contains the amino acid sequence of SEQ ID NO: 79, and the second VL domain contains the amino acid sequence of SEQ ID NO: 80.

[0013] In some embodiments according to any of the embodiments described herein, the second arm comprises: an antibody heavy chain polypeptide comprising a second VH domain and an antibody light chain polypeptide comprising a second VL domain. In some embodiments, the antibody heavy chain polypeptide of the second arm comprises an Fc region. In some embodiments, the Fc region of the second arm is a human IgG Fc region. In some embodiments, the Fc region of the second arm is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG1 Fc region substituted with S239D and I332E according to EU designations. In some embodiments, the Fc region is a human IgG1 Fc region substituted with S239D, A330L, and I332E according to EU designations. In some embodiments, the Fc region is a human IgG1 Fc region substituted with G236A, S239D, A330L, and I332E according to EU designations. In some embodiments, the Fc region of the second arm is a human IgG4 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region substituted with S228P according to EU designations. In some embodiments, the first arm includes an Fc region; wherein the Fc region of the first arm contains one or more mutations forming a pestle, and the Fc region of the second arm contains one or more paired mutations forming a mortar. In some embodiments, according to EU designations, the Fc region of the first arm contains a T366W substitution, and wherein the Fc region of the second arm contains T366S, L368A, and Y407V substitutions. In some embodiments, the first arm includes an Fc region; wherein the Fc region of the second arm contains one or more mutations forming a pestle, and the Fc region of the first arm contains one or more paired mutations forming a mortar. In some embodiments, according to EU designations, the Fc region of the second arm contains a T366W substitution, and wherein the Fc region of the first arm contains T366S, L368A, and Y407V substitutions.

[0014] In some embodiments according to any of the embodiments described herein, the second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 76 or 77 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 81 or 82 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 83.

[0015] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 71 or 72 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 73.

[0016] In some embodiments according to any of the embodiments described herein, the first antigen-binding domain comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain. In some embodiments, the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, CDR-L2 comprising the amino acid sequence of GAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first VH domain contains the amino acid sequence of SEQ ID NO: 24, and the first VL domain contains the amino acid sequence of SEQ ID NO: 25.

[0017] In some embodiments according to any of the embodiments described herein, the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 22, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, CDR-L2 comprising the amino acid sequence of GAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first VH domain comprises the amino acid sequence of SEQ ID NO: 26, and the first VL domain comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the first VH domain comprises the amino acid sequence of SEQ ID NO: 30, and the first VL domain comprises the amino acid sequence of SEQ ID NO: 31.

[0018] In some embodiments according to any of the embodiments described herein, the first arm comprises: an antibody heavy chain polypeptide comprising a first VH domain and an antibody light chain polypeptide comprising a first VL domain. In some embodiments, the antibody heavy chain polypeptide of the first arm comprises an Fc region. In some embodiments, the first arm comprises: a single-chain variable fragment (scFv) comprising a first VH domain and a first VL domain. In some embodiments, the scFv comprises a linker between the first VH domain and the first VL domain. In some embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:34. In some embodiments, the first arm further comprises an Fc region linked to the scFv. In some embodiments, the Fc region of the first arm is a human IgG Fc region. In some embodiments, the Fc region of the first arm is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG1 Fc region comprising S239D and I332E substitutions according to EU designations. In some embodiments, the Fc region is a human IgG1 Fc region comprising S239D, A330L, and I332E substitutions according to EU designations. In some embodiments, the Fc region is a human IgG1 Fc region containing G236A, S239D, A330L, and I332E substitutions, according to EU designations. In some embodiments, the Fc region of the first arm is a human IgG4 Fc region. In some embodiments, the Fc region is a human IgG4 Fc region containing S228P substitutions, according to EU designations. In some embodiments, the second arm contains an Fc region; wherein the Fc region of the first arm contains one or more club-forming mutations, and the Fc region of the second arm contains one or more paired mortise-forming mutations. In some embodiments, the Fc region of the first arm contains T366W substitutions, and wherein the Fc region of the second arm contains T366S, L368A, and Y407V substitutions. In some embodiments, the second arm contains an Fc region; wherein the Fc region of the second arm contains one or more club-forming mutations, and the Fc region of the first arm contains one or more paired mortise-forming mutations. In some implementations, according to EU designations, the Fc region of the second arm contains T366W substitutions, and the Fc region of the first arm contains T366S, L368A, and Y407V substitutions.

[0019] In some embodiments according to any of the embodiments described herein, the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 87 or 88 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 111 or 112 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 28 or 90 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 99 or 100 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments, the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 or 91 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36. In some embodiments, the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 101 or 102 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104.

[0020] In some embodiments according to any of the embodiments described herein, at least one or both of the first and second arms are unfucosylated or contain reduced fucosylation. In some embodiments, human Dectin-1 comprises the amino acid sequence of SEQ ID NO: 84 or 85. In some embodiments, human Trop-2 comprises the amino acid sequence of SEQ ID NO: 86.

[0021] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0022] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0023] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0024] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0025] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0026] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0027] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0028] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0029] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0030] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0031] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0032] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0033] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0034] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0035] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0036] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0037] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0038] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0039] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a single-chain variable fragment (scFv) linked to an Fc region, and the second arm comprises an antibody heavy chain polypeptide and an antibody light chain polypeptide; and wherein the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0040] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a single-chain variable fragment (scFv) linked to an Fc region, and the second arm comprises an antibody heavy chain polypeptide and an antibody light chain polypeptide; and wherein the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.

[0041] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a single-chain variable fragment (scFv) linked to an Fc region, and the second arm comprises an antibody heavy chain polypeptide and an antibody light chain polypeptide; and wherein the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0042] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a single-chain variable fragment (scFv) linked to an Fc region, and the second arm comprises an antibody heavy chain polypeptide and an antibody light chain polypeptide; and wherein the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78.

[0043] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a single-chain variable fragment (scFv) linked to an Fc region, and the second arm comprises an antibody heavy chain polypeptide and an antibody light chain polypeptide; and wherein the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0044] In some embodiments, this document provides a bispecific binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2; wherein the first arm comprises a single-chain variable fragment (scFv) linked to an Fc region, and the second arm comprises an antibody heavy chain polypeptide and an antibody light chain polypeptide; and wherein the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0045] This document also provides a polynucleotide encoding a bispecific binding molecule according to any of the above embodiments. This document also provides a vector comprising the polynucleotide according to any of the above embodiments, such as an expression vector. This document also provides a host cell (e.g., an isolated host cell) comprising the polynucleotide or vector according to any of the above embodiments. In some embodiments, the cell is a yeast cell, insect cell, plant cell, or prokaryotic cell. In some embodiments, the host cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell comprises α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) knockout. In some embodiments, the host cell overexpresses β-1,4-N-acetylglucosamine transferase III (GnT-III) and optionally also overexpresses Golgi μ-mannosidase II (ManII). This document also provides a method for generating a bispecific binding molecule, the method comprising culturing a host cell according to any of the above embodiments under conditions suitable for generating the bispecific binding molecule. In some embodiments, the method further includes recovering the bispecific binding molecule from the host cell molecule. In some embodiments, the host cell is treated with a chiffonine prior to generating the bispecific binding molecule. This document also provides a bispecific binding molecule generated by any of the methods described in the above embodiments. This document also provides a pharmaceutical composition comprising the bispecific binding molecule according to any of the above embodiments and a pharmaceutically acceptable carrier.

[0046] This document also provides a method of treating cancer, comprising administering to an individual in need an effective amount of a bispecific binding molecule or pharmaceutical composition according to any of the above embodiments. This document also provides the use of a bispecific binding molecule or pharmaceutical composition according to any of the above embodiments in a method of treating cancer, said method comprising administering to an individual in need an effective amount of the bispecific binding molecule or pharmaceutical composition. This document also provides the use of a bispecific binding molecule or pharmaceutical composition according to any of the above embodiments in the manufacture of a medicament for treating an individual's cancer. In some embodiments, the individual has or has been diagnosed with cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is breast cancer, prostate cancer, urothelial carcinoma, cervical cancer, bladder cancer, pancreatic cancer, thyroid cancer, head and neck cancer, esophageal cancer, endometrial cancer, gastric cancer, liver cancer, colorectal cancer, skin cancer (e.g., melanoma), lung cancer, ovarian cancer, or uterine cancer. In some implementation schemes, the cancers are cervical squamous cell carcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma, cervical endometrial adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, papillary thyroid carcinoma, mucinous carcinoma, endometrioid carcinoma, pancreatic adenocarcinoma, invasive lobular carcinoma of the breast, lung adenocarcinoma, uterine endometrioid carcinoma, serous ovarian cancer, invasive breast cancer (NOS), and invasive ductal carcinoma of the breast. Serous carcinoma of the uterus, metaplastic breast cancer, mixed endometrial carcinoma of the uterus, invasive mixed mucinous carcinoma of the breast, papillary cell carcinoma of the kidney, esophageal adenocarcinoma, gastric adenocarcinoma, invasive breast cancer, intrahepatic bile duct carcinoma, cholangiocarcinoma, tubular gastric adenocarcinoma, intestinal-type gastric adenocarcinoma, mucinous gastric adenocarcinoma, papillary gastric adenocarcinoma, diffuse gastric adenocarcinoma, teratoma, gastric signet ring cell carcinoma, colorectal / rectal mucinous adenocarcinoma, uterine carcinosarcoma, rectal adenocarcinoma, thymoma, or perihepatic hilar bile duct carcinoma. In some embodiments, the cancer cells express Trop-2. In some embodiments, the individual is human. In some embodiments, the cancer responds to single-agent immunotherapy (e.g., single-agent treatment with an immune checkpoint inhibitor), for example, before the administration of a bispecific binding molecule. In some embodiments, the cancer does not respond to single-agent immunotherapy (e.g., single-agent treatment with an immune checkpoint inhibitor), for example, before the administration of a bispecific binding molecule.

[0047] This document also provides a kit comprising a bispecific binding molecule or pharmaceutical composition according to any of the embodiments described above. In some embodiments, the kit further comprises instructions for treating an individual cancer using the bispecific binding molecule or pharmaceutical composition, such as any of the methods disclosed herein.

[0048] It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of this disclosure. These and other aspects of this disclosure will become apparent to those skilled in the art. These and other embodiments of this disclosure are further described in the following detailed description. Attached Figure Description

[0049] Figures 1A to 1C An exemplary format of an anti-Dectin-1 x anti-Trop-2 bispecific antibody according to some embodiments is shown. Figure 1A One embodiment is shown where both arms contain Fab (Fab / Fab). Engineered disulfide bonds can also be used in this format to drive the correct pairing of heavy and light chain variable domains. For example, cysteine, which forms a native disulfide bond between the heavy and light chains, can be removed from one arm, and cysteine ​​substitution can be introduced to produce a non-native disulfide bond. The other arm can have a native disulfide bond, thus distinguishing the variable domains of each arm by the position of the disulfide bond. Figure 1B An embodiment is shown in which interchain disulfide bonds are engineered in the Dectin-1 binding arm between the VH and VL domains, while the Trop-2 binding arm has natural interchain disulfide bonds between the CH1 and CL domains (interchain disulfide bonds are indicated by arrows). Figure 1C One embodiment is shown, in which one arm (in this case, the anti-Dectin-1 arm) contains a single-chain variable fragment (scFv), while the other arm (in this case, the anti-Trop-2 arm) contains Fab (scFv / Fab). In all examples, the bispecific antibody also contains an Fc region (in these examples, human IgG1).

[0050] Figures 2A to 2D The binding assay is shown, in which cells expressing human Trop-2 are incubated with an anti-Dectin-1 x anti-Trop-2 bispecific antibody having human IgG1 Fc (as indicated) or an isotype control, and binding is measured by flow cytometry using a fluorescently labeled anti-human secondary antibody. The antibody's effect on human epidermoid carcinoma A-431 cells is also shown. Figure 2A ), human gastric cancer NCI-N87 cells ( Figure 2B ), human breast cancer SKBR3 cells ( Figure 2C ) or CHO-K1 cells expressing human Trop-2 ( Figure 2D The combination of ).

[0051] Figures 3A to 3DThe activation of the Dectin-1 pathway by the anti-Dectin-1 x anti-Trop-2 bispecific antibody with human IgG1 Fc (as indicated) or an isotype control is shown. Human cancer cell lines expressing hTrop-2 were treated with serial dilutions of the indicated bispecific antibody or isotype control in the presence of HEK-Blue hDectin-1a cells engineered to express Dectin-1 isotype A and genes involved in the Dectin-1 / NF-κB / SEAP signaling pathway (and thus express secretory alkaline phosphatase (SEAP) in response to stimulation by Dectin-1 ligands). The activation of the Dectin-1 pathway by the anti-Dectin-1 x anti-Trop-2 bispecific antibody with human IgG1 Fc (as indicated) or an isotype control is shown. Figure 3A ), NCI-N87 cells ( Figure 3B ), SKBR3 cells ( Figure 3C ) or CHO-KI cells expressing human Trop-2 ( Figure 3D In the case of incubation with the indicated antibody, activation of Dectin-1 signaling in HEK-Blue hDectin-1a cells (as measured by SEAP).

[0052] Figures 4A to 4C It shows the use of Figure 1C Cell binding and Dectin-1 activation of anti-Dectin-1 x anti-Trop-2 bispecific antibodies with human IgG1 Fc (as indicated) or isotype control, as shown in scFv format. Figure 4A The binding of the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody to HEK cells expressing hDectin-1, as measured by flow cytometry, is shown. Figure 4B The binding of the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody to A431 cells expressing hTrop-2, as measured by flow cytometry, is shown. Figure 4C The activation of Dectin-1 by the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody, as measured by SEAP secretion, is shown; EC50 values ​​are also shown.

[0053] Figures 5A to 5C It shows the use of Figure 1B Cell binding and Dectin-1 activation of an anti-Dectin-1 x anti-Trop-2 bispecific antibody with human IgG1 Fc (as indicated) or isotype control in the Fab format shown, with engineered disulfide bonds in the anti-Dectin-1 arm. Figure 5A The binding of the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody to HEK cells expressing hDectin-1, as measured by flow cytometry, is shown. Figure 5B The binding of the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody to CHO-K1 cells expressing hTrop-2, as measured by flow cytometry, is shown. Figure 5C The activation of Dectin-1 by the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody, as measured by SEAP secretion, is shown; EC50 values ​​are also shown.

[0054] Figures 6A to 6C It shows the use of Figure 1A Phagocytosis induced by the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody in Fab format with human IgG1 Fc (as indicated) or an isotype control. Human macrophages and calcein AM-labeled human cancer cells (3:1 ratio) were co-cultured at 37°C for 24 hours with the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody, followed by staining with PE-labeled anti-CD206 antibody to label macrophages. Phagocytosis was quantified as the percentage of FITC+ cancer cells phagocytosed by macrophages in a single phylum. The antibody was tested in serial dilutions. Figure 6A The phagocytosis of A431 cells by human macrophages induced by the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody or isotype control is shown. Figure 6B The phagocytic activity of human macrophages induced by the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody or isotype control on NCI-N87 cells is shown. Figure 6C The phagocytosis of SKBR3 cells by human macrophages induced by the indicated anti-Dectin-1 x anti-Trop-2 bispecific antibody or isotype control is shown.

[0055] Figures 7A to 7D This demonstrates the effect of co-culturing PBMCs with CHO-K1 cells expressing hTrop-2 in response to the use of Figure 1A Cytokine secretion produced by treatment with anti-Dectin-1 x anti-Trop-2 bispecific antibodies in Fab format, as indicated (with human IgG1 Fc as shown) or isotype control. Figure 7A The dose-response of TNF-α secretion to treatment with an anti-Dectin-1 x anti-Trop-2 bispecific antibody with human IgG1 Fc (as indicated) is shown. Figure 7B The dose-response of IL-6 secretion to treatment with an anti-Dectin-1 x anti-Trop-2 bispecific antibody with human IgG1 Fc (as indicated) is shown. Figure 7CThe dose-response of IFN-γ secretion to treatment with an anti-Dectin-1 x anti-Trop-2 bispecific antibody having human IgG1 Fc (as indicated) is shown. Figure 7D A summary of all cytokines used from PBMCs from two donors is shown.

[0056] Figure 8A and Figure 8B The activity of the anti-Dectin-1 x anti-Trop-2 bispecific antibody (using the anti-mouse Dectin-1 arm and mouse IgG2a Fc) in an endogeneic mouse tumor xenograft model was demonstrated. Eight-week-old female Balb / c or C57BL6 mice were subcutaneously inoculated with 2 million human Trop2-overexpressing mouse colon cancer cells (hTrop2-CT26) or 500,000 human Trop2-overexpressing mouse melanoma cancer cells (hTrop2-B16F10), respectively. When the tumor reached 80 mm... 3 Mice were randomly divided into four groups of 12 each and received six doses of anti-Dectin-1 x anti-Trop-2 bispecific antibody containing anti-mDectin-1 Fab (“Dectin-1 surr”), anti-Trop-2 clones A, B, or C, and mIgG2a Fc (or isotype control) via intraperitoneal injection at a dose of 10 mg / kg twice weekly. Tumor volume and body weight were measured 2 to 3 times weekly. Figure 8A The study showed the median tumor volume increase in mice inoculated with hTrop-2-CT26 cells and treated with either anti-Dectin-1 x anti-Trop-2 bispecific antibody or mIgG2a isotype control. Figure 8B The study showed the median tumor volume increase in mice inoculated with hTrop-2-B16F10 cells and treated with either anti-Dectin-1 x anti-Trop-2 bispecific antibody or mIgG2a isotype control.

[0057] Figure 9 The activity of the anti-Dectin-1 x anti-Trop-2 bispecific antibody (using a disulfide-engineered anti-hDectin-1 arm and mouse IgG2a Fc) against hTrop2-B16F10 tumor xenografts in an syngeneic mouse model expressing hDectin-1 is shown. Tumor volume changes over time.

[0058] Figures 10A to 10D This demonstrates the use of anti-Dectin-1 x anti-Trop-2 bispecific antibodies to deplete cancer cells from single-cell suspensions derived from tissue biopsies. Figures 10A to 10C It shows the origin of lung cancer ( Figure 10A ovarian cancer Figure 10B ) or uterine cancer ( Figure 10C The percentage of remaining cancer cells (as %) after incubating a single-cell suspension of biopsy tissue with anti-Dectin-1 x anti-Trop-2 bispecific antibodies (as indicated) for 24 hours. Figure 10D The average expression (mRNA) of Trop-2 in the indicated cancer types is shown from data from the TCGAPanCancer Atlas study. Detailed Implementation

[0059] The following description, using example applications, illustrates several aspects. It should be understood that numerous specific details, relationships, and methods are presented to provide a comprehensive understanding of the features described herein. However, those skilled in the art will readily recognize that the features described herein can be practiced without one or more specific details or using other methods. The features described herein are not limited to the order of the actions or events shown, as some actions may occur in a different order and / or simultaneously with other actions or events. Furthermore, not all actions or events described are necessary for implementing the methods according to the features described herein.

[0060] As used herein, the singular forms “an” and “the” are intended to also include the plural forms, unless the context clearly indicates otherwise. Furthermore, with regard to the terms “including / includes,” “having / has / with,” or variations thereof used in the detailed description and / or claims, these terms are intended to be included in a manner similar to the term “comprising.” As used herein, the term “comprising” is synonymous with “including” or “containing” and is inclusive or open-ended.

[0061] Unless otherwise stated, any reference to “or” in this document is intended to cover “and / or”. As used herein, the term “about” with respect to numbers means the number plus or minus 10% of the number. The term “about” with respect to ranges means the range minus 10% of its lowest value and plus 10% of its highest value.

[0062] In some implementations, antibodies and immunoglobulins are used interchangeably and are used in the broadest sense herein, covering a variety of antibody structures, including but not limited to monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and single-domain antibodies (as described in more detail herein), provided they exhibit the desired antigen-binding activity.

[0063] In some implementations, an antibody (immunoglobulin) refers to a protein whose structure is substantially similar to that of a natural antibody, or a protein having variable regions in both the heavy and light chains that are substantially similar in structure to the variable regions of the natural heavy and light chains. A natural antibody is a naturally occurring immunoglobulin molecule with a different structure. For example, IgG-type natural immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant regions. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or light chain variable domain, followed by a constant light chain (CL) domain, also called a light chain constant region. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and broadly described, for example, in Abbas et al., 2000, Cellular and Mol and Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). Antibodies (immunoglobulins) can be designated as different classes depending on the amino acid sequence of the heavy chain constant domain. Antibodies are primarily classified into five classes: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Based on the amino acid sequence of their constant domain, the light chain of an antibody can be designated as one of two types (called kappa (κ) and lambda (λ)). Immunoglobulins are essentially composed of two Fab molecules linked via an immunoglobulin hinge region and an Fc domain.

[0064] In some implementations, Fc, Fc region, or Fc domain refers to the C-terminal region of an antibody heavy chain containing at least a portion of a constant region. The terminology includes native sequence Fc regions and variant Fc regions. Fc may refer to the last two constant immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG; the last three constant immunoglobulin domains of IgE and IgM; and optionally, all or part of a flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J-chain. The IgG Fc region contains the IgG CH2 and IgG CH3 domains and, in some cases, includes a hinge. Unless otherwise stated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991. In this disclosure, the human IgG Fc domain has a specific purpose and may be an Fc domain derived from human IgG1, IgG2 or IgG4.

[0065] As is known in the art, the C-terminal lysine of some antibody heavy chain peptides may be cleaved in certain moieties. Therefore, in some embodiments, the arms or multispecific binding molecules of this disclosure may be present (e.g., in a composition), including mixtures of types in which some peptides retain the C-terminal lysine while others do not.

[0066] As is known in the art, the variable domains (VH and VL, respectively) of the heavy and light chains of antibodies typically have similar structures, with each domain containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al.) Kuby Immunology , 6th edition, WH Freeman and Co., page 91 (2007)). A frame (or “FR” as used herein) can refer to a variable domain residue other than a CDR residue. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences in a VH (or VL) typically appear in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In some embodiments, FR1, FR2, FR3, and / or FR4 of this disclosure refer to the human frame region, i.e., the human frame region of the VH or VL domain.

[0067] Various definitions of the CDR sequence of antibody variable domains are known in the art; see, for example, Kabat ( Sequences of Proteins of Immunological Interest(5th edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3) and Chothia. Unless otherwise stated, this paper describes CDR sequences according to the definition of IMGT. See, for example, imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html.

[0068] Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, Fab'-SH, F(ab')2, double-chain antibodies, linear antibodies, single-chain antibodies, nanobodies, scFv fragments, VH, and multispecific (e.g., bispecific) antibodies / fragments formed from antibody fragments.

[0069] "Fab" (antigen-binding fragment) is a part of an antibody that binds to an antigen and includes a variable region of the heavy chain linked to the light chain via interchain disulfide bonds and CH1.

[0070] I. Multispecific binding protein In some aspects, this disclosure provides a multispecific (e.g., bispecific) binding molecule comprising: a first arm comprising a first antigen-binding domain binding to human Dectin-1; and a second arm comprising a second antigen-binding domain binding to human Trop-2. In some embodiments, the first and / or second antigen-binding domains comprise a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the second arm comprises an antibody heavy chain polypeptide (i.e., the second antibody heavy chain polypeptide of this disclosure) and an antibody light chain polypeptide (i.e., the second antibody light chain polypeptide of this disclosure). In some embodiments, the first arm comprises an antibody heavy chain polypeptide (i.e., the first antibody heavy chain polypeptide of this disclosure) and an antibody light chain polypeptide (i.e., the first antibody light chain polypeptide of this disclosure). In some embodiments, the first arm comprises a single-chain variable fragment (scFv) linked to an Fc region.

[0071] Any of the anti-Dectin-1 antigen-binding domains or arms disclosed herein can be used in the binding molecules of this disclosure. Any of the anti-Trop-2 antigen-binding domains or arms disclosed herein can be used in the binding molecules of this disclosure. Various non-limiting embodiments of the multispecific (e.g., bispecific) binding molecules of this disclosure comprising specific combinations of anti-Dectin-1 binding arms and anti-Trop-2 binding arms are further described in detail below.

[0072] Anti-Dectin-1 antigen-binding domain Various anti-Dectin-1 antigen-binding domains are considered for use in the multispecific (e.g., bispecific) binding molecules of this disclosure. In some embodiments, the anti-Dectin-1 antigen-binding domain is the antigen-binding domain of anti-Dectin-1 antibody 2M24, described in International Publication No. WO2022077006, including variants thereof. In some embodiments, the anti-Dectin-1 antigen-binding domain is a human antigen-binding domain. In some embodiments, the anti-Dectin-1 antigen-binding domain is a humanized antigen-binding domain.

[0073] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence DYYI (SEQ ID NO: 1); CDR-H2 containing the amino acid sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 2); and CDR-H3 containing the amino acid sequence NSGSYSFGY (SEQ ID NO: 3); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence RASQGISSWLA (SEQ ID NO: 4); CDR-L2 containing the amino acid sequence GASSLQS (SEQ ID NO: 5); and CDR-L3 containing the amino acid sequence QQAYSFPFT (SEQ ID NO: 6).

[0074] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GYTFTDYY (SEQ ID NO: 7); CDR-H2 containing the amino acid sequence INPNSGDT (SEQ ID NO: 8); and CDR-H3 containing the amino acid sequence ARNSGSYSFGY (SEQ ID NO: 9); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence QGISSW (SEQ ID NO: 10); CDR-L2 containing the amino acid sequence GAS; and CDR-L3 containing the amino acid sequence QQAYSFPFT (SEQ ID NO: 12).

[0075] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GYTFTDY (SEQ ID NO: 13); CDR-H2 containing the amino acid sequence NPNSGD (SEQ ID NO: 14); and CDR-H3 containing the amino acid sequence NSGSYSFGY (SEQ ID NO: 15); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence RASQGISSWLA (SEQ ID NO: 4); CDR-L2 containing the amino acid sequence GASSLQS (SEQ ID NO: 5); and CDR-L3 containing the amino acid sequence QQAYSFPFT (SEQ ID NO: 6).

[0076] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence DYYM (SEQ ID NO: 16); CDR-H2 containing the amino acid sequence WINPNEGDTNYAQKFEG (SEQ ID NO: 17); and CDR-H3 containing the amino acid sequence NTGAYSFGY (SEQ ID NO: 18); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence RASQGISSWLA (SEQ ID NO: 4); CDR-L2 containing the amino acid sequence GASDLQS (SEQ ID NO: 19); and CDR-L3 containing the amino acid sequence QQAYGFPFT (SEQ ID NO: 20).

[0077] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GYTFTDYY (SEQ ID NO: 7); CDR-H2 containing the amino acid sequence INPEGDT (SEQ ID NO: 21); and CDR-H3 containing the amino acid sequence ARNTGAYSFGY (SEQ ID NO: 22); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence QGISSW (SEQ ID NO: 10); CDR-L2 containing the amino acid sequence GAS; and CDR-L3 containing the amino acid sequence QQAYGFPFT (SEQ ID NO: 20).

[0078] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GYTFTDY (SEQ ID NO: 13); CDR-H2 containing the amino acid sequence NPNEGD (SEQ ID NO: 23); and CDR-H3 containing the amino acid sequence NTGAYSFGY (SEQ ID NO: 18); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence RASQGISSWLA (SEQ ID NO: 4); CDR-L2 containing the amino acid sequence GASDLQS (SEQ ID NO: 19); and CDR-L3 containing the amino acid sequence QQAYGFPFT (SEQ ID NO: 20).

[0079] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 24) and / or wherein the VL domain comprises the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 25). In some embodiments, the VH domain contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 24) and the VL domain contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 25).

[0080] In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGQGTLVTVSS (SEQ ID NO: 26) and / or wherein the VL domain comprises the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIK (SEQ ID NO: 27). In some embodiments, the VH domain contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGQGTLVTVSS (SEQ ID NO: 26) and the VL domain contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIK (SEQ ID NO: 27).

[0081] In some embodiments, the anti-Dectin-1 antigen-binding domain, the anti-Dectin-1 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSS (SEQ ID NO: 30) and / or wherein the VL domain comprises the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIK (SEQ ID NO: 31). In some embodiments, the VH domain contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSS (SEQ ID NO: 30) and the VL domain contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIK (SEQ ID NO: 31).

[0082] In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein of this disclosure comprises: a VH domain containing 1, 2, or 3 CDRs from a single VH domain listed in Table 5; and / or a VL domain containing 1, 2, or 3 CDRs from a single VL domain listed in Table 5. In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein of this disclosure comprises: a VH domain containing 1, 2, or 3 CDRs from a single VH domain listed in Table 6; and / or a VL domain containing 1, 2, or 3 CDRs from a single VL domain listed in Table 6. In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein of this disclosure comprises the VH and / or VL domains listed in Table 6. In some embodiments, any set of three VH CDRs shown in Table 5 may be combined with any set of three VL CDRs shown in Table 5 in the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein of this disclosure. In some embodiments, any set of three CDRs from the VH domain shown in Table 6 may be combined with any set of three CDRs from the VL domain shown in Table 6 in the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein of this disclosure. In some embodiments, any VH domain shown in Table 6 may be combined with any VL domain shown in Table 6 in the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein of this disclosure.

[0083] Table 5. Anti-Dectin-1 antibody CDR sequence

[0084] Table 6. Variable domain sequences of anti-Dectin-1 antibody

[0085] In some embodiments, the anti-Dectin-1 antigen-binding domain or arm comprises a single-chain variable fragment (scFv) containing a first VH domain and a first VL domain. In some embodiments, the scFv contains a linker between the first VH domain and the first VL domain. Linkers for generating antibody fusion proteins are known in the art. In some embodiments, the linker comprises, consists of, or is substantially composed of glycine and / or serine residues. In some embodiments, the linker length is 15-20 amino acids. In some embodiments, the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 92). In some embodiments, the linker comprises one or more repeats of the sequence GGGGS (SEQ ID NO: 93). In some embodiments, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 94) or GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 95). Additional linker sequences are described in Chen, X. et al. (2013). Adv. Drug Deliv. Rev. In 65:1357-1369, in some embodiments (e.g., in the scFv of this disclosure), the scFv comprises a linker of one type between the VH domain and the VL domain, and another type of linker connecting the VL domain to the remainder of the arm, such as to the Fc region. For example, in some embodiments, the linker between the VH domain and the VL domain comprises glycine and / or serine residues, such as GGGSGGGSGGGS (SEQ ID NO:92), GGGGSGGGGSGGGS (SEQ ID NO:94), GGGGSGGGGSGGGSGGGS (SEQ ID NO:95), or one or more repeating sequences of the sequence GGGGS (SEQ ID NO:96); and / or the linker connecting the VL domain to the Fc region comprises EPKRSDKTHTCPPC (SEQ ID NO:97) or SATHTCPPC (SEQ ID NO:98). In some embodiments, the linker between the VH domain and the VL domain contains glycine and / or serine residues and is 15-20 amino acids in length.

[0086] In some embodiments, scFv contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSSGGGGSGG GGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGAS DLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIKEPK (SEQ ID NO:34).

[0087] In some embodiments, the anti-Dectin-1 arm comprises a single-chain variable fragment (scFv) containing the VH and VL domains of the anti-Dectin-1 antigen-binding domain of this disclosure, as well as an antibody Fc region. In some embodiments, the anti-Dectin-1 arm comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAYGFPFTFPGGTKVDIKEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ IDNO:35) or QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLV TVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFT FGPGTKVDIKEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36). For example, in some embodiments, compositions comprising the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which comprise scFv-Fc fusion proteins containing the amino acid sequence of SEQ ID NO: 35, while other species comprise scFv-Fc fusion proteins containing the amino acid sequence of SEQ ID NO: 35.The scFv-Fc fusion protein has a 36-amino acid sequence. In some embodiments, the anti-Dectin-1 arm contains the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYY CQQAYGFPFTFPGGTKVDIKEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ IDNO:103) or QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTL VTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPF TFGPGTKVDIKEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:104). For example, in some embodiments, compositions comprising the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which comprise scFv-Fc fusion proteins containing the amino acid sequence of SEQ ID NO:103, while other species comprise scFv-Fc fusion proteins containing the amino acid sequence of SEQ ID NO:104.

[0088] In some embodiments, the anti-Dectin-1 arm comprises: an antibody heavy chain polypeptide comprising the VH domain of the anti-Dectin-1 antigen-binding domain of this disclosure; and an antibody light chain polypeptide comprising the VL domain of the anti-Dectin-1 antigen-binding domain of this disclosure. In some embodiments, the antibody heavy chain polypeptide further comprises an Fc region. In some embodiments, the anti-Dectin-1 arm comprises: an antibody heavy chain polypeptide,It comprises an amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 87) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88); and an antibody light chain polypeptide,It contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 87 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 89, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 88 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 89. In some implementations, the anti-Dectin-1 arm comprises: an antibody heavy chain polypeptide.It comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:32) or QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91); and an antibody light chain polypeptide,It contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES (SEQ ID NO: 33). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 32 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 33, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 91 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 33. In some implementations, the anti-Dectin-1 arm comprises: an antibody heavy chain polypeptide.It comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 28) or QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 90); and an antibody light chain polypeptide,It contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 28 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 29, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 90 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 29. In some implementations, the anti-Dectin-1 arm comprises: an antibody heavy chain polypeptide.It comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 111) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 112); and an antibody light chain polypeptide,It contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 111 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 89, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 112 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 89. In some implementations, the anti-Dectin-1 arm comprises: an antibody heavy chain polypeptide.It comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:101) or QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGCGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 102); and an antibody light chain polypeptide,It contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKCPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGES (SEQ ID NO: 33). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 101 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 33, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 102 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 33. In some implementations, the anti-Dectin-1 arm comprises: an antibody heavy chain polypeptide.It comprises the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:99) or QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGWINPNEGDTNYAQKFEGRITMTRDTSISTAYMELSRLRSDDTAVYYCARNTGAYSFGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 100); and an antibody light chain polypeptide,It contains the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYGASDLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYGFPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29). For example, in some embodiments, compositions comprising the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which comprise: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 99 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 29, while other species comprise: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 100 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 29.

[0089] In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein binds to human Dectin-1. In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein binds to human Dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, or granulocytes. In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein binds to human Dectin-1 isotype A and / or human Dectin-1 isotype B. In some embodiments, human Dectin-1 isotype A contains the amino acid sequence MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVKTTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO:84). In some embodiments, human Dectin-1 isotype B contains the amino acid sequence MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO:85).

[0090] In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein binds to human Dectin-1 expressed on the cell surface at EC50 values ​​of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM. In some embodiments, the anti-Dectin-1 antigen-binding domain, anti-Dectin-1 arm, or multispecific binding protein is capable of binding to both human Dectin-1 and monkey Dectin-1, such as cynomolgus monkey Dectin-1.

[0091] Anti-Trop-2 antigen-binding domain Various anti-Trop-2 antigen-binding domains are considered for use in the multispecific (e.g., bispecific) binding molecules of this disclosure. In some embodiments, the anti-Trop-2 antigen-binding domain is a human antigen-binding domain. In some embodiments, the anti-Trop-2 antigen-binding domain is a humanized antigen-binding domain.

[0092] In some embodiments, the anti-Trop-2 antigen-binding domain, the anti-Trop-2 arm, or the multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence SSNWWS (SEQ ID NO: 37); CDR-H2 containing the amino acid sequence EIYHDGSTDYFPSLKS (SEQ ID NO: 38); and CDR-H3 containing the amino acid sequence DNWGFDY (SEQ ID NO: 39); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence RASQSVSSSYL (SEQ ID NO: 40); CDR-L2 containing the amino acid sequence GASSRAT (SEQ ID NO: 41); and CDR-L3 containing the amino acid sequence QQYGSSHRT (SEQ ID NO: 42).

[0093] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GGSISSSNW (SEQ ID NO: 43); CDR-H2 containing the amino acid sequence IYHDGST (SEQ ID NO: 44); and CDR-H3 containing the amino acid sequence ARDNWGFDY (SEQ ID NO: 45); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence QSVSSSY (SEQ ID NO: 46); CDR-L2 containing the amino acid sequence GAS; and CDR-L3 containing the amino acid sequence QQYGSSHRT (SEQ ID NO: 42).

[0094] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GGSISSSN (SEQ ID NO: 48); CDR-H2 containing the amino acid sequence YHDGS (SEQ ID NO: 49); and CDR-H3 containing the amino acid sequence NWGFD (SEQ ID NO: 50); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence SQSVSSSY (SEQ ID NO: 51); CDR-L2 containing the amino acid sequence GAS; and CDR-L3 containing the amino acid sequence YGSSHR (SEQ ID NO: 52).

[0095] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence SYGMN (SEQ ID NO: 53); CDR-H2 containing the amino acid sequence WINTNTGNPTYAQGFTG (SEQ ID NO: 54); and CDR-H3 containing the amino acid sequence GYNWNDGDFDY (SEQ ID NO: 55); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence RASQGIRNDLG (SEQ ID NO: 56); CDR-L2 containing the amino acid sequence AASSLQS (SEQ ID NO: 57); and CDR-L3 containing the amino acid sequence LQNYNYPLT (SEQ ID NO: 58).

[0096] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GYTFTSYG (SEQ ID NO: 59); CDR-H2 containing the amino acid sequence INTNTGNP (SEQ ID NO: 60); and CDR-H3 containing the amino acid sequence ARGYNWNDGDFDY (SEQ ID NO: 61); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence QGIRND (SEQ ID NO: 62); CDR-L2 containing the amino acid sequence AAS; and CDR-L3 containing the amino acid sequence LQNYNYPLT (SEQ ID NO: 58).

[0097] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises: CDR-H1 containing the amino acid sequence GYTFTSY (SEQ ID NO: 64); CDR-H2 containing the amino acid sequence NTNTGN (SEQ ID NO: 65); and CDR-H3 containing the amino acid sequence YNWNDGDFD (SEQ ID NO: 66); and wherein the VL domain comprises: CDR-L1 containing the amino acid sequence SQGIRND (SEQ ID NO: 67); CDR-L2 containing the amino acid sequence AAS; and CDR-L3 containing the amino acid sequence NYNYPL (SEQ ID NO: 68).

[0098] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 69) and / or wherein the VL domain comprises the amino acid sequence DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 70). In some embodiments, the VH domain contains the amino acid sequence QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 69) and the VL domain contains the amino acid sequence DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 70).

[0099] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHDGSTDYFPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDNWGFDYWGQGTLVTVSS (SEQ ID NO: 74) and / or wherein the VL domain comprises the amino acid sequence DIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLISGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSHRTFGQGTKVEIK (SEQ ID NO: 75). In some embodiments, the VH domain contains the amino acid sequence QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHDGSTDYFPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDNWGFDYWGQGTLVTVSS (SEQ ID NO: 74) and the VL domain contains the amino acid sequence DIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLISGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSHRTFGQGTKVEIK (SEQ ID NO: 75).

[0100] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; wherein the VH domain comprises the amino acid sequence QVQLVQSGSELKKPGASVKLSCKASGYTFTSYGMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYNWNDGDFDYWGQGTLVTVSS (SEQ ID NO: 79) and / or wherein the VL domain comprises the amino acid sequence AIQMTQSPSSLSPSVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCLQNYNYPLTFGGGTKVEIK (SEQ ID NO: 80). In some embodiments, the VH domain contains the amino acid sequence QVQLVQSGSELKKPGASVKLSCKASGYTFTSYGMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYNWNDGDFDYWGQGTLVTVSS (SEQ ID NO: 79) and the VL domain contains the amino acid sequence AIQMTQSPSSLSPSVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCLQNYNYPLTFGGGTKVEIK (SEQ ID NO: 80).

[0101] In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein of this disclosure comprises: a VH domain containing 1, 2, or 3 CDRs from a single VH domain listed in Table 7; and / or a VL domain containing 1, 2, or 3 CDRs from a single VL domain listed in Table 7. In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein of this disclosure comprises: a VH domain containing 1, 2, or 3 CDRs from a single VH domain listed in Table 8; and / or a VL domain containing 1, 2, or 3 CDRs from a single VL domain listed in Table 8. In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein of this disclosure comprises the VH and / or VL domains listed in Table 8. In some embodiments, any set of three VH CDRs shown in Table 7 may be combined with any set of three VL CDRs shown in Table 7 in the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein of this disclosure. In some embodiments, any set of three CDRs from the VH domain shown in Table 8 may be combined with any set of three CDRs from the VL domain shown in Table 8 in the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein of this disclosure. In some embodiments, any VH domain shown in Table 8 may be combined with any VL domain shown in Table 8 in the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein of this disclosure.

[0102] Table 7. CDR sequences of anti-Trop-2 antibodies

[0103] Table 8. Variable domain sequences of anti-Trop-2 antibodies

[0104] In some embodiments, the anti-Trop-2 arm comprises: an antibody heavy chain polypeptide comprising the VH domain of the anti-Trop-2 antigen-binding domain of this disclosure; and an antibody light chain polypeptide comprising the VL domain of the anti-Trop-2 antigen-binding domain of this disclosure. In some embodiments, the antibody heavy chain polypeptide further comprises an Fc region. In some embodiments, the anti-Trop-2 arm comprises: an antibody heavy chain polypeptide,It comprises the amino acid sequence QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 71) or QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72); and an antibody light chain polypeptide,It contains the amino acid sequence DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 73). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 71 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 73, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 72 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 73. In some implementations, the anti-Trop-2 arm comprises: an antibody heavy chain polypeptide,It comprises an amino acid sequence QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHDGSTDYFPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDNWGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 76) or QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHDGSTDYFPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDNWGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77); and an antibody light chain polypeptide,It contains the amino acid sequence DIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLISGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSHRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 78). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 76 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 78, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 77 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 78. In some implementations, the anti-Trop-2 arm comprises: an antibody heavy chain polypeptide,It comprises the amino acid sequence QVQLVQSGSELKKPGASVKLSCKASGYTFTSYGMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYNWNDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 81) or QVQLVQSGSELKKPGASVKLSCKASGYTFTSYGMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYNWNDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 82); and an antibody light chain polypeptide,It contains the amino acid sequence AIQMTQSPSSLSPSVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCLQNYNYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 81 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 83, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 82 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 83. In some implementations, the anti-Trop-2 arm comprises: an antibody heavy chain polypeptide,It comprises the amino acid sequence QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 105) or QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106); and an antibody light chain polypeptide,It contains the amino acid sequence DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 73). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 105 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 73, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 106 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 73. In some implementations, the anti-Trop-2 arm comprises: an antibody heavy chain polypeptide,It comprises the amino acid sequence QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHDGSTDYFPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDNWGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 107) or QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHDGSTDYFPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDNWGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 108); and an antibody light chain polypeptide,It contains the amino acid sequence DIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLISGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSHRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 78). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 107 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 78, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 108 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 78. In some implementations, the anti-Trop-2 arm comprises: an antibody heavy chain polypeptide,It comprises the amino acid sequence QVQLVQSGSELKKPGASVKLSCKASGYTFTSYGMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYNWNDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 109) or QVQLVQSGSELKKPGASVKLSCKASGYTFTSYGMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARGYNWNDGDFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 110); and an antibody light chain polypeptide,It contains the amino acid sequence AIQMTQSPSSLSPSVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIFAASSLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYYCLQNYNYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83). For example, in some embodiments, compositions containing the arms or multispecific binding molecules of this disclosure comprise a mixture of species, some of which contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 109 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 83, while other species contain: an antibody heavy chain polypeptide containing the amino acid sequence of SEQ ID NO: 110 and an antibody light chain polypeptide containing the amino acid sequence of SEQ ID NO: 83.

[0105] In some embodiments, an anti-Trop-2 antigen-binding domain, an anti-Trop-2 arm, or a multispecific binding protein binds to human Trop-2. In some embodiments, the anti-Trop-2 antigen-binding domain, anti-Trop-2 arm, or multispecific binding protein binds to human Trop-2 expressed on the surface of cells (e.g., cancer cells or tumor cells). In some embodiments, human Trop-2 comprises the amino acid sequence MARGPGLAPPPLRLPLLLLVLAAVTGHTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAPKNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGDLSLRCDELVR THHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPTIQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYYLDEIPPKFSMKRLTAGLIAVIVVVVVALVAGMAVLVITNRRKSGKYKKVEIKELGELRKEPSL (SEQ ID NO: 86).

[0106] Multispecific binding molecular format and other features In some embodiments, the antigen-binding arm of this disclosure (e.g., the anti-Dectin-1 or anti-Trop-2 arm of this disclosure) or multispecific binding molecule includes an Fc region. Antibodies can belong to any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD. The immunoglobulin Fc region of the molecule that induces targeted phagocytosis may play an important role in this process by binding to Fc receptors and inducing additional phagocytosis.

[0107] In some embodiments, the Fc region is a human IgG Fc region. In some embodiments, the Fc region is a human IgG1 or human IgG4 Fc region. In some embodiments, the Fc region is a human IgG1 Fc region substituted with S239D and I332E according to EU designations. In some embodiments, the Fc region is a human IgG1 Fc region substituted with S239D, A330L, and I332E according to EU designations. In some embodiments, the Fc region is a human IgG1 Fc region substituted with G236A, S239D, A330L, and I332E according to EU designations. In some embodiments, the Fc region is a human IgG4 Fc region substituted with S228P according to EU designations.

[0108] In some embodiments, the antigen-binding arm (e.g., the anti-Dectin-1 or anti-Trop-2 arm of this disclosure) or multispecific binding molecule includes an Fc region, wherein the carbohydrate structure linked to the Fc region has reduced or no fucose, for example, at least one or both heavy chains of the multispecific binding molecule are unfucosylated or contain reduced fucosylation. In some embodiments, a composition is provided herein comprising the multispecific binding molecule of this disclosure, the multispecific binding molecule including one or two Fc regions, wherein the carbohydrate structure linked to the Fc region has reduced or no fucose, for example, at least one or both heavy chains of the multispecific binding molecule are unfucosylated or contain reduced fucosylation. In some embodiments, less than 50% of the N-glycoside-linked carbohydrate chain in the composition contains fucose residues. In some embodiments, substantially no N-glycoside-linked carbohydrate chain contains fucose residues. In some embodiments, multispecific binding molecules with reduced or no fucose have improved ADCC function.

[0109] In some embodiments, the multispecific (e.g., bispecific) binding molecule of this disclosure also includes one or more mutations on only one antibody arm to reduce binding affinity to protein A. See, for example, Ollier, R. et al. (2019). MAbs11:1464-1478 and AU2018204314. In some embodiments, the multispecific (e.g., bispecific) binding molecule comprises two antibody light chains and two antibody heavy chains, wherein only one antibody heavy chain contains amino acid substitutions H435R and Y436F according to EU numbers.

[0110] In some embodiments, the multispecific (e.g., bispecific) binding molecule of this disclosure further includes one or more effector-reducing functions, such as mutations that reduce or eliminate the binding of the Fc region to the Fc receptor. In some embodiments, the multispecific (e.g., bispecific) binding molecule comprises two antibody Fc regions, wherein the antibody Fc regions contain amino acid substitutions at one or more of positions 234, 235, and 237 according to EU numbers. In some embodiments, the multispecific (e.g., bispecific) molecule comprises two antibody Fc regions, wherein the antibody Fc regions contain substitutions of L234A, L235E, and G237A according to EU numbers.

[0111] In other embodiments, the multispecific binding molecule of this disclosure (e.g., an IgG1 multispecific binding molecule) or a composition comprising the multispecific binding molecule of this disclosure (e.g., an IgG1 multispecific binding molecule) contains wild-type glycosylation of one or both Fc regions. In some embodiments, fucosylated multispecific binding molecules of this disclosure (e.g., IgG1 multispecific binding molecules) or compositions comprising the fucosylated multispecific binding molecules of this disclosure (e.g., IgG1 multispecific binding molecules) are provided herein.

[0112] Fucosylated or fucosylated antibodies refer to antibodies containing fucose residues within the oligosaccharides linked to the peptide backbone. Specifically, fucosylated antibodies contain α (1,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or both N-linked oligosaccharides linked to the antibody's Fc region, for example, at position Asn 297 (EU number of Fc region residues) in the human IgG1 Fc region. Due to minor sequence variations in immunoglobulins, Asn297 may also be located approximately +3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Non-fucosylated or fucose-deficient antibodies have reduced amounts of fucose compared to the same antibody produced in the cell line. Antibody fucosylation can be measured, for example, in antibody compositions treated with N-glycosidase F by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0113] In some embodiments, the Fc region contains one or more mutations that reduce or eliminate fucosylation, such as a substitution at Asn 297 (EU number of the Fc region residue) in the human IgG1 Fc region. Optionally, the Fc region also includes one or more amino acid substitutions therein that further improve ADCC, such as substitutions at positions 298, 333, and / or 334 in the Fc region (Eu number of the residue). Examples of publications involving "defucosylated" or "fucosylated" antibodies include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

[0114] Various techniques for assembling the two arms of the multispecific binding molecules disclosed herein are known in the art and are considered for use herein.

[0115] In some embodiments, the multispecific (e.g., bispecific) binding molecule of this disclosure comprises: a first antibody arm comprising an antibody heavy chain and an associated antibody light chain, wherein the antibody heavy chain comprises a first VH domain, the antibody light chain comprises a first VL domain, and a first Fc region attached to the first VH domain, wherein the first VH domain and the first VL domain form an antigen-binding domain binding to human Dectin-1; and a second antibody arm comprising an antibody heavy chain and an associated antibody light chain, wherein the antibody heavy chain comprises a second VH domain, the antibody light chain comprises a second VL domain, and a second Fc region attached to the second VH domain, wherein the second VH domain and the second VL domain form an antigen-binding domain binding to human Trop-2. That is, the first and second antibody arms may be in the form of conventional antibodies (see, for example...). Figure 1A and 1B ).

[0116] A well-established method for preparing bispecific antibodies is the "pestle-pot" or "protrusion-cavity" method. See, for example, U.S. Patent No. 5,731,168. Two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each contain an interface; the interface of one immunoglobulin polypeptide interacts with a corresponding or paired interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. In some embodiments, the interfaces can be engineered such that a "pestle" or "protrusion" on the interface of one immunoglobulin polypeptide corresponds to a paired "pot" or "cavity" on the interface of the other immunoglobulin polypeptide. In some embodiments, a pestle can be constructed by replacing a smaller amino acid side chain with a larger side chain. In some embodiments, a pothole can be constructed by replacing a larger amino acid side chain with a smaller side chain. The pestle or pothole may be present in the original interface, or they may be introduced synthetically. Polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding mutations forming a pestle or pothole can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, for example, U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; 8,679,785; 8,844,834; U.S. Publication No. 2013 / 0089553; Spiess et al., Nature Biotechnology 31: 753-758, 2013; and Ridgway and Carter (1996) Protein Eng. 9:617-621. Modified immunoglobulin peptides can be produced using prokaryotic host cells such as *Escherichia coli* or eukaryotic host cells such as mammalian cells (e.g., CHO cells) or yeast cells. The corresponding mortar and pestle-containing immunoglobulin peptides can be expressed in co-cultured host cells and purified together as heteropolymers, or they can be expressed in a single culture, purified individually, and assembled in vitro. Exemplary paired pestle and mortar mutations are provided below (according to EU index numbers). EU numbers used herein are known in the art; see, for example, IMGT resources www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html and www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html.

[0117] In some embodiments, this document provides a multispecific (e.g., bispecific) binding molecule comprising: a first antibody arm comprising a first antibody heavy chain and an associated first antibody light chain, wherein the first antibody heavy chain comprises a first VH domain, the first antibody light chain comprises a first VL domain, and a first Fc region connected to the first VH domain, wherein the first VH domain and the first VL domain form an antigen-binding domain binding to human Dectin-1; and a second antibody arm comprising a second antibody heavy chain and an associated second antibody light chain, wherein the second antibody heavy chain comprises a second VH domain, the second antibody light chain comprises a second VL domain, and a second Fc region connected to the second VH domain, wherein the second VH domain and the second VL domain form an antigen-binding domain binding to human Trop-2. In some embodiments, the first Fc region comprises one or more club-forming mutations, and the second Fc region comprises one or more paired acetabulum-forming mutations, or the second Fc region comprises one or more club-forming mutations, and the first Fc region comprises one or more paired acetabulum-forming mutations. Non-restrictive pestle and mortar formation mutant groups include: T366W substitution for pestle formation according to EU number, and T366S, L368A and Y407V substitution for mortar formation.

[0118] In some embodiments, the multispecific (e.g., bispecific) binding molecule of this disclosure comprises two antibody heavy chains and two antibody light chains, wherein the VH domain of the first antibody heavy chain forms an antigen-binding domain with the VL domain of the first antibody light chain, and wherein the VH domain of the second antibody heavy chain forms an antigen-binding domain with the VL domain of the second antibody light chain. According to EU designations, the first antibody heavy chain contains F126C, C220V, and T366W substitutions, the first antibody light chain contains S121C and C214V substitutions, and the second antibody heavy chain contains T366S, L368A, Y407V, H435R, and Y436F substitutions. In some embodiments, the first and second antibody heavy chains further contain L234A, L235E, and G237A substitutions according to EU designations. In some embodiments, the first and second antibody heavy chains contain a human IgG1 Fc domain.

[0119] In some implementations, multispecific (e.g., bispecific) antibodies also contain one or more mutations that improve heavy chain / light chain pairing on only one antibody arm. For example, amino acid substitutions can be used to replace native disulfide bonds at the CH1-CL interface of one antibody arm with engineered disulfide bonds (see example...). Figure 1B See, for example, Mazor, Y. et al. (2015). MAbs7:377-389 and EP3452089A2. In some embodiments, the multispecific or bispecific antibody comprises two antibody light chains and two antibody heavy chains, wherein, according to the EU designation, only one antibody heavy chain contains amino acid substitutions F126C and C220V, and only the corresponding or paired light chains contain amino acid substitutions S121C and C214V.

[0120] In some embodiments, the antibody heavy chain polypeptide of one arm of the multispecific (e.g., bispecific) binding molecule of this disclosure has a C→S amino acid substitution at position 5 according to IMGT hinge numbering, at position 220 according to EU indexing, or at position 233 according to Kabat numbering. Descriptions of the IMGT hinge, EU indexing, and Kabat numbering schemes are known in the art (see, for example, imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html), and therefore, those skilled in the art can determine the precise position of this residue using ordinary knowledge in the art. In some embodiments, the antibody light chain polypeptide of the corresponding arm of the multispecific (e.g., bispecific) binding molecule has a C→S substitution at the terminal residue of a light chain constant domain (e.g., CK domain). In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 30; the VL domain comprises the amino acid sequence of SEQ ID NO: 31; the antibody heavy chain of the antibody arm has a C→S amino acid substitution at position 5 according to the IMGT hinge number, position 220 according to the EU index, or position 233 according to the Kabat number; and the antibody light chain of the antibody arm has a C→S substitution at the terminal residue of the light chain constant domain (e.g., the CK domain). In some embodiments, the VH domain of the anti-Dectin-1 arm comprises the amino acid sequence of SEQ ID NO: 30, and the VL domain of the anti-Dectin-1 arm comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the VH and VL domains of the anti-Dectin-1 arm are part of an scFv comprising the amino acid sequence of SEQ ID NO: 34.

[0121] In some embodiments, the multispecific (e.g., bispecific) binding molecule of this disclosure comprises: a first antibody arm comprising a single-chain variable fragment (scFv) and a first Fc region, wherein the scFv comprises the VH domain and VL domain of this disclosure that bind to human Dectin-1; and a second antibody arm comprising an antibody heavy chain and an associated antibody light chain, wherein the antibody heavy chain comprises a VH domain, the antibody light chain comprises a VL domain, and a second Fc region connected to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to Trop-2. That is, the first arm may be in scFv format (e.g., an scFv-Fc fusion protein), and the second antibody arm may be in conventional antibody format (see, for example, ...). Figure 1C ).

[0122] In some embodiments, this document provides a multispecific (e.g., bispecific) binding molecule comprising: a first antibody arm including a single-chain variable fragment (scFv) and a first Fc region, wherein the scFv includes the disclosed VH and VL domains binding to human Dectin-1; and a second antibody arm including an antibody heavy chain and an associated antibody light chain, wherein the antibody heavy chain includes a VH domain, the antibody light chain includes a VL domain, and a second Fc region linked to the VH domain, wherein the VH and VL domains of the second antibody arm form an antigen-binding domain binding to human Trop-2. In some embodiments, the first Fc region includes one or more club-forming mutations, and the second Fc region includes one or more paired mortise-forming mutations, or the second Fc region includes one or more club-forming mutations, and the first Fc region includes one or more paired mortise-forming mutations. In some embodiments, scFv includes a first connector of the present disclosure between the VH and VL domains and a second connector of the present disclosure between the VL domain and the first Fc region. Non-limiting pestle and mortar formation mutation groups include: T366W substitution for pestle formation according to EU designation, and T366S, L368A, and Y407V substitution for mortar formation.

[0123] Depending on the method, antibody variable domains with the desired binding specificity (antibody-antigen combination site) are fused with immunoglobulin constant domain sequences.

[0124] Multispecific (e.g., bispecific) binding molecules also include cross-linked or "heterologously conjugated" antibodies. Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, chemical linking can be used to prepare multispecific (e.g., bispecific) binding molecules. In some embodiments, the multispecific (e.g., bispecific) binding molecule comprises a first IgG antibody containing a first antigen-binding domain covalently linked to a second IgG antibody containing a second antigen-binding domain.

[0125] In some embodiments, the first arm is coupled to avidin, streptavidin, neutralizing avidin, or a biotin-binding derivative thereof, and the second arm is coupled to biotin or a biotin-binding derivative thereof. In some embodiments, the second arm is coupled to avidin, streptavidin, neutralizing avidin, or a biotin-binding derivative thereof, and the first arm is coupled to biotin or a biotin-binding derivative thereof. In some embodiments, the first arm binds to the second arm via an interaction between avidin, streptavidin, neutralizing avidin, or a biotin-binding derivative thereof and biotin or a biotin-binding derivative thereof. Exemplary avidin, streptavidin, neutralizing avidin, or a biotin-binding derivative thereof are known in the art. In some embodiments, streptavidin is monomeric streptavidin (mSA). Exemplary biotin-binding or avidin derivative thereof are known in the art. In some embodiments, the antigen-binding arm of this disclosure is biotinylated.

[0126] In some embodiments, one or both of the first and second arms contain a tag, for example, for affinity purification. In some embodiments, the tag is a polyhistidine tag.

[0127] In some embodiments, one or both of the first and second arms are Fab, Fab', F(ab')2, Fv, Fab'-SH, F(ab')2, single-chain antibody, nanobody, or scFv fragment. In some embodiments, one or both of the first and second arms also include an Fc domain. In some embodiments, the first arm is a Fab fragment, and the second arm is a full-length antibody. In some embodiments, both the first and second arms are full-length haptens.

[0128] In some embodiments, the multispecific (e.g., bispecific) binding molecule of this disclosure comprises: a first arm (e.g., an anti-Dectin-1 arm) comprising a first antibody heavy chain polypeptide and a first antibody light chain polypeptide; and a second arm (e.g., an anti-Trop-2 arm) comprising a second antibody heavy chain polypeptide and a second antibody light chain polypeptide. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73.In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0129] In some embodiments, the multispecific (e.g., bispecific) binding molecule of this disclosure comprises: a first arm (e.g., an anti-Dectin-1 arm) comprising a single-chain variable fragment (scFv) linked to an Fc region; and a second arm (e.g., an anti-Trop-2 arm) comprising an antibody heavy chain polypeptide and an antibody light chain polypeptide. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78. In some embodiments, the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83.

[0130] In some embodiments, this document provides a polynucleotide that encodes a multispecific binding molecule of any of the above embodiments. In some embodiments, this document provides a vector (e.g., an expression vector) that contains a polynucleotide of any of the above embodiments. In some embodiments, each arm of the multispecific (e.g., bispecific) binding molecule of this disclosure is encoded by a separate polynucleotide or vector. In some embodiments, a single polynucleotide or vector encodes both arms of the multispecific (e.g., bispecific) binding molecule of this disclosure.

[0131] In some embodiments, this document provides a host cell (e.g., an isolated host cell or cell line) containing a polynucleotide or vector from any of the above embodiments. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the host cell is an insect or mammalian host cell. Any of these can be used for the generation and / or treatment methods disclosed herein. Examples of useful mammalian host cell lines include: monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed with SV40; human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); and human cervical cancer cells (HELA, ATCC CCL 10). 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; CHOK1 cells, CHOK1SV cells or derivatives and human hepatocellular carcinoma cell line (HepG2).

[0132] In some embodiments, a non-fucosylated or non-fucosylated multispecific binding molecule is generated in a cell line having genetic modifications that lead to the generation of the non-fucosylated or non-fucosylated multispecific binding molecule. Examples of cell lines that produce fucosylation-free products include Lec13 CHO cells with protein fucosylation defects (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, such as CHO cells with α-1,6-fucosylation gene FUT8 knocked out (Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)), overexpression of β1,4-N-acetylglucosamine transferase III (GnT-III) and Golgi μ-mannosidase II. (ManII) cells, and mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1; see Byrne, G. et al., (2018)). PLoS Biol Cells knocked out (16:e2005817).

[0133] In some embodiments, non-fucosylated or non-fucosylated multispecific binding molecules are generated in cell lines treated with a sugar-processing enzyme inhibitor, such as a chif base, which is a mannosidase I inhibitor (see, for example, Elbein, AD et al. (1990)). J. Biol. Chem. 265:15599-15605). For example, cells can be centrifuged and resuspended in a growth medium containing chiffonine (e.g., 250 μg / mL), then cultured and used for production.

[0134] In some embodiments, this document provides a method for generating multispecific binding molecules, the method comprising culturing host cells from any of the above embodiments under conditions suitable for generating multispecific binding molecules. In some embodiments, the method further comprises recovering the multispecific binding molecules. Multispecific binding molecules can be generated using standard recombinant techniques as described herein and / or illustrated below.

[0135] Antibodies and antibody fragments can be generated using recombinant methods. For example, the nucleic acid encoding the antibody / fragment can be isolated and inserted into a reproducible vector for further cloning or expression. The DNA encoding the antibody / fragment can be readily isolated and sequenced using standard procedures, such as via oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody / fragment. Many vectors are known in the art; vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. Suitable host cells for cloning or expressing the DNA in the vectors described herein are prokaryotic cells, yeast cells, or higher eukaryotic cells. When using recombinant techniques, antibodies / fragments can be generated intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody / fragment is generated intracellularly, particulate debris, host cells, or lysed fragments are removed by, for example, centrifugation or ultrafiltration. When antibodies / fragments are secreted into the culture medium, the supernatant from such expression systems is typically first concentrated using a commercially available protein concentrater filter.

[0136] II. Instructions for Use In some aspects, this disclosure provides methods for treating cancer, including administering an effective amount of the multispecific (e.g., bispecific) binding molecule or composition of this disclosure to an individual in need. In some embodiments, the individual is a person. In some embodiments, the individual has or has been diagnosed with cancer. Any of the multispecific binding molecules of this disclosure (e.g., as described in Part I above) can be used in the treatments and methods of use disclosed herein, and in the compositions (e.g., pharmaceutical compositions) associated therewith.

[0137] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is breast cancer, prostate cancer, urothelial carcinoma, cervical cancer, bladder cancer, pancreatic cancer, thyroid cancer, head and neck cancer, esophageal cancer, endometrial cancer, gastric cancer, liver cancer, colorectal cancer, skin cancer (e.g., melanoma), lung cancer, ovarian cancer, or uterine cancer. In some embodiments, the cancer cells express Trop-2. Exemplary cancer types demonstrating expression of Trop-2 and considered for treatment with the methods of this disclosure include, but are not limited to, cervical squamous cell carcinoma, bladder urothelial carcinoma, head and neck squamous cell carcinoma (HNSCC), esophageal squamous cell carcinoma, cervical endometrial adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, papillary thyroid carcinoma, mucinous carcinoma, endometrioid carcinoma, pancreatic adenocarcinoma, invasive lobular carcinoma of the breast, lung adenocarcinoma, uterine endometrioid carcinoma, serous ovarian cancer, and invasive breast carcinoma (N... OS), invasive ductal carcinoma of the breast, serous carcinoma of the uterus, metaplastic breast cancer, mixed endometrial carcinoma of the uterus, invasive mixed mucinous carcinoma of the breast, papillary cell carcinoma of the kidney, esophageal adenocarcinoma, gastric adenocarcinoma, invasive breast cancer, intrahepatic bile duct carcinoma, bile duct carcinoma, tubular gastric adenocarcinoma, intestinal-type gastric adenocarcinoma, mucinous gastric adenocarcinoma, papillary gastric adenocarcinoma, diffuse gastric adenocarcinoma, teratoma, gastric signet ring cell carcinoma, colorectal / rectal mucinous adenocarcinoma, uterine carcinosarcoma, rectal adenocarcinoma, thymoma, and perihilar bile duct carcinoma. In some embodiments, the sample obtained from the individual's cancer is positive for Trop-2 expression. In some embodiments, the method further includes obtaining a sample from the individual's cancer, wherein the sample contains cancer cells (e.g., cancer cells expressing Trop-2).

[0138] In some embodiments, the cancer responds to monotherapy (e.g., monotherapy using an immune checkpoint inhibitor), for example, before the administration of the multispecific (e.g., bispecific) binding molecule. In some embodiments, the cancer does not respond to monotherapy (e.g., monotherapy using an immune checkpoint inhibitor), for example, before the administration of the multispecific (e.g., bispecific) binding molecule. Advantageously, as demonstrated herein, the multispecific binding molecule of this disclosure has antitumor activity against tumor models with different tumor microenvironments, including antitumor activity against both "hot" (e.g., characterized by immune cell infiltration and / or response to monotherapy using immunotherapies such as immune checkpoint inhibitors) and "cold" (e.g., characterized by a lack of immune cell infiltration and / or reduced or minimal response to monotherapy using immunotherapies such as immune checkpoint inhibitors) tumor models.

[0139] III. Compositions and Kits In some respects, this disclosure provides pharmaceutical compositions comprising a multispecific (e.g., bispecific) binding molecule and a pharmaceutically acceptable carrier as described in any of the embodiments disclosed herein.

[0140] The pharmaceutical compositions and formulations described herein can be made by combining an active ingredient (such as a multispecific binding molecule) having the desired purity with one or more optional pharmaceutically acceptable carriers. Remington's Pharmaceutical Sciences 16th edition, Osol, A. (1980)) are mixed to prepare in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used and include, but are not limited to: buffers, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0141] Certain aspects of this disclosure relate to kits or articles of manufacture that contain any of the multispecific binding molecules disclosed herein. In some embodiments, the articles of manufacture include a container and a label or packaging insert on or accompanying the container. In some embodiments, the kits or articles of manufacture also include instructions for using the multispecific binding molecules, for example, to treat cancer, according to any method disclosed herein.

[0142] Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating the condition and may have a sterile dispensing port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody or a multispecific binding molecule as described herein. The label or packaging insert indicates that the composition is intended for the treatment of a specific condition. The label or packaging instructions will also include instructions for administering the multispecific binding molecule composition to a subject. Articles and kits containing the combination therapies described herein are also considered.

[0143] The following description is presented to enable those skilled in the art to make and use various embodiments. The description of specific apparatuses, techniques, and applications is provided only as examples. Various modifications to the embodiments described herein will be apparent to those skilled in the art, and the general principles defined herein can be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the embodiments described and shown herein, but are accorded the scope consistent with the claims.

[0144] Example Example 1: Characterization of binding to anti-Dectin-1 x Trop-2 bispecific antibody This embodiment characterizes the binding of the anti-Dectin-1 x anti-Trop-2 bispecific antibody to its target.

[0145] Materials and methods Bispecific antibodies for Figure 1A The Fab format shown indicates that the anti-Dectin-1 binding arm contains a variable domain, with the amino acid sequence of its VH domain being SEQ ID NO: 24 and the amino acid sequence of its VL domain being SEQ ID NO: 25. For Figure 1C The scFv format shown indicates that the anti-Dectin-1 binding arm contains an scFv with the amino acid sequence of SEQ ID NO: 34. For Figure 1B The Fab format shown has engineered disulfide bonds in the anti-Dectin-1 binding arm. The anti-Dectin-1 binding arm contains variable domains, with the amino acid sequence of the VH domain being SEQ ID NO: 30 and the amino acid sequence of the VL domain being SEQ ID NO: 31.

[0146] For the anti-Trop-2 binding arm in all formats, the amino acid sequences of the variable domains are as follows: Clone A has a VH domain of SEQ ID NO: 69 and a VL domain of SEQ ID NO: 70; Clone B has a VH domain of SEQ ID NO: 74 and a VL domain of SEQ ID NO: 75; Clone C has a VH domain of SEQ ID NO: 79 and a VL domain of SEQ ID NO: 80. All bispecific antibodies use the human IgG1 Fc region.

[0147] Flow cytometry Human cancer cell lines and CHO cells expressing human Trop-2 (CHO-K1-hTrop2 cells) were incubated with anti-Dectin-1 x anti-Trop-2 bispecific antibody, Trop-2 clones 15, 45, 76, 77, 63, 64, 65, and 123, or hIgG1 isotypes, followed by the addition of fluorescently labeled anti-human secondary antibody. Primary antibody was administered using a continuous dose-titration method at eight concentration points, starting at 300 nM or 100 nM with a dilution ratio of 1:3.

[0148] Biolayer Interferometry (BLI) Monovalent binding affinity was measured using a biosensor (BLI). Human Trop-2 ECD protein was captured on the biosensor via an anti-HIS tag. Binding and dissociation rates were measured against different concentrations of bispecific antibody (one arm of which binds to hTrop2). K was generated by fitting the binding and dissociation rate measurements. D value.

[0149] SEAP secretion HEK-BluehDectin-1a cells were engineered to express Dectin-1 isoform A and genes involved in the Dectin-1 / NF-κB / SEAP signaling pathway (and thus express secretory alkaline phosphatase (SEAP) in response to stimulation by Dectin-1 ligands). Human cancer cell lines expressing Trop-2 were treated with serial dilutions of anti-Dectin-1 x anti-Trop-2 bispecific antibodies in the presence of HEK cell lines expressing Dectin-1 reporter genes. After 24 hours of treatment, the supernatant was collected to test Dectin-1-induced NF-κB-driven SEAP activity.

[0150] result All three anti-Dectin-1 and anti-Trop-2 bispecific antibodies exhibited high affinity binding to cells expressing human Trop-2, including human cancer cell lines and CHO cells engineered to express human Trop-2. Figures 2A to 2D The EC50 binding values ​​of each antibody to each target are shown in Table 1. Without being bound by theory, it is assumed that the anti-Dectin-1 x anti-Trop-2 bispecific antibodies can effectively couple myeloid cells to Trop-2-expressing cancer cells, thereby promoting Dectin-1-mediated immunostimulation, tumor cell phagocytosis, and antigen presentation. Advantageously, anti-Trop-2 clones B and C are derived from humanized mice (unlike humanized antibodies such as clone A), and therefore may be more "human-like" and have a lower risk of immunogenicity.

[0151] Table 1. Testing the binding of anti-Dectin-1 x anti-Trop-2 bispecific antibodies to cell lines (EC50, nM).

[0152] The monovalent binding affinity of each anti-Dectin-1 x anti-Trop-2 bispecific antibody to human Trop-2 ECD was also measured using a biosensor (BLI). The measurements are provided in Table 2.

[0153] Table 2. Binding of anti-Dectin-1 x anti-Trop-2 bispecific antibody to hTrop-2 ECD by BLI.

[0154] The ability of the anti-Dectin-1 and anti-Trop-2 bispecific antibodies to activate the Dectin-1 pathway in the presence of hTrop-2-expressing cells was also measured. All three bispecific antibodies induced Dectin-1 pathway activation in the presence of Trop-2-expressing cell lines. Figures 3A to 3D The EC50 values ​​for each antibody in each cell line are shown in Table 3. SEAP secretion activity was found to correlate with binding data; the bispecific antibody with the clonal C anti-Trop-2 binding domain had the weakest EC50 and the lowest Ymax.

[0155] Table 3. Activation of the Dectin-1 pathway in the presence of the indicated hTrop-2-expressing cell lines (EC50, μg / mL).

[0156] The anti-Dectin-1 x anti-Trop-2 bispecific antibody exhibited dose-dependent NF-κB activation in the presence of human cancer cell lines and CHO-K1-hTrop2 cells. Trop-2 expression levels in the cell lines used ranged from 50,000 to 400,000 copies, indicating that the anti-Dectin-1 x anti-Trop-2 bispecific antibody can induce Dectin-1 aggregation and activate the downstream NF-κB pathway in the presence of high or low Trop-2 expression levels.

[0157] Anti-Dectin-1 x anti-Trop-2 bispecific antibody also Figure 1C The format shown was tested. An anti-Dectin-1 x anti-Trop-2 bispecific antibody against the anti-Dectin-1 arm was constructed using the scFv format, targeting hDectin-1 expression (…). Figure 4A ) or hTrop-2 ( Figure 4B Cells showed binding specificity comparable to the Fab format. The use of the anti-Dectin-1x anti-Trop-2 bispecific antibody in the scFv format also induced Dectin-1 activation, as measured by SEAP secretion (…). Figure 4CUsing the scFv-based Dectin-1 arm (as opposed to traditional antibody Fab-based arms) is considered advantageous in manufacturing because these bispecific antibodies exhibit reduced dimer formation during purification and increased long-term stability. This specific anti-Dectin-1 antigen-binding domain also demonstrates improved affinity compared to the parental 2M24 antigen-binding domain and may have a lower liability risk due to the removal of certain amino acids.

[0158] Anti-Dectin-1 × anti-Trop-2 bispecific antibodies also Figure 1B The format shown was tested, which has engineered disulfide bonds in the anti-Dectin-1 Fab arm. Using the Fab format of the anti-Dectin-1 x anti-Trop-2 bispecific antibody with engineered disulfide bonds in the anti-Dectin-1 arm, the expression of hDectin-1 ( Figure 5A ) or hTrop-2 ( Figure 5B Cells showed binding specificity comparable to the Fab format. The use of the Fab format anti-Dectin-1 x anti-Trop-2 bispecific antibody with engineered disulfide bonds in the anti-Dectin-1 arm also induced Dectin-1 activation, as measured by SEAP secretion (…). Figure 5C Advantageously, the use of an anti-Dectin-1 binding arm with engineered disulfide bonds is thought to facilitate proper pairing of the heavy and light chains during co-expression of the bispecific antibody arm, thereby optimizing the manufacturing process. This specific anti-Dectin-1 antigen-binding domain also exhibits improved affinity compared to the parental 2M24 antigen-binding domain and may have a lower liability risk due to the removal of certain amino acids.

[0159] In summary, all three formats provide anti-Dectin-1 x anti-Trop-2 bispecific antibodies that bind to their respective targets with high affinity and induce activation of the Dectin-1 pathway.

[0160] Example 2: Functional characterization of anti-Dectin-1 x Trop-2 bispecific antibody This embodiment characterizes the functional activity of the anti-Dectin-1 x anti-Trop-2 bispecific antibody.

[0161] Materials and methods Phagocytosis Flow cytometry analysis was performed on co-cultures of human macrophages and calcein AM-labeled human cancer cells (effectant:target ratio 3:1) in the presence of anti-Dectin-1x anti-Trop-2 bispecific antibody (containing human IgG1 Fc) or an allotype control. The co-cultures were incubated at 37°C for 24 hours, followed by staining with PE-labeled anti-CD206 antibody to label macrophages. Phagocytosis was quantified as the percentage of FITC+ cancer cells phagocytosed by macrophages in a single phylum. Primary antibodies were administered using a continuous dose-titration method. Bispecific antibodies were used... Figure 1A and Figure 1B The Fab format is shown. The anti-Dectin-1 binding arm contains variable domains, with the amino acid sequence of the VH domain being SEQ ID NO: 24 and the amino acid sequence of the VL domain being SEQ ID NO: 25. For the anti-Trop-2 binding arm, the amino acid sequences of the variable domains are as follows: Clone A: VH domain is SEQ ID NO: 69, VL domain is SEQ ID NO: 70; Clone B: VH domain is SEQ ID NO: 74, VL domain is SEQ ID NO: 75; Clone C: VH domain is SEQ ID NO: 79, VL domain is SEQ ID NO: 80. All bispecific antibodies use the human IgG1 Fc region.

[0162] Cytokine release PBMCs and CHO-K1-hTrop2 cells were co-cultured at a 3:1 ratio in the presence of anti-Dectin-1 x anti-Trop-2 bispecific antibody or an allotype control. The co-cultures were incubated at 37°C for 24 hours, and the supernatant was collected for cytokine analysis using MSD (Meso Scale Discovery). The bispecific antibody was used... Figure 1A The Fab format is shown. The anti-Dectin-1 binding arm contains variable domains, with the amino acid sequence of its VH domain being SEQ ID NO: 24 and the amino acid sequence of its VL domain being SEQ ID NO: 25. For the anti-Trop-2 binding arm, the amino acid sequences of the variable domains are as follows: clone A: VH domain is SEQ ID NO: 69, VL domain is SEQ ID NO: 70; clone B: VH domain is SEQ ID NO: 74, VL domain is SEQ ID NO: 75; clone C: VH domain is SEQ ID NO: 79, VL domain is SEQ ID NO: 80. All bispecific antibodies use the human IgG1 Fc region.

[0163] Homogeneous tumor model Eight-week-old female Balb / c or C57BL6 mice were subcutaneously inoculated with 2 million human Trop2-overexpressing mouse colon cancer cells (hTrop2-CT26) or 500,000 human Trop2-overexpressing mouse melanoma cancer cells (hTrop2-B16F10). When the tumor reached 80 mm... 3 Mice were randomly divided into four groups of 12 each and received six doses of an anti-Dectin-1 x anti-Trop-2 bispecific antibody containing anti-mDectin-1Fab and anti-Trop-2 clones A, B, or C, as well as mIgG2a Fc (or an isotype control), administered intraperitoneally at a dose of 10 mg / kg twice weekly. Tumor volume and body weight were measured 2-3 times weekly. The bispecific antibody was used... Figure 1A The Fab format is shown. The anti-Dectin-1 binding arm uses the anti-mouse Dectin-1 variable domain. For the anti-Trop-2 binding arm, the amino acid sequences of the variable domains are as follows: VH domain of clone A is SEQ ID NO: 69, and VL domain is SEQ ID NO: 70; VH domain of clone B is SEQ ID NO: 74, and VL domain is SEQ ID NO: 75; VH domain of clone C is SEQ ID NO: 79, and VL domain is SEQ ID NO: 80. All bispecific antibodies use the mouse IgG2a Fc region.

[0164] To directly test the 2M24.119 anti-Dectin-1 binding arm, 500,000 human Trop2-overexpressing mouse melanoma cancer cells (hTrop2-B16F10) were subcutaneously injected into eight-week-old mixed-sex C57BL6 mice expressing human Dectin1. When the tumor reached 80 mm... 3 Mice were randomly divided into groups of 12 each and received 6 doses of an anti-Dectin-1 x anti-Trop-2 bispecific antibody containing mIgG2a Fc (or an isotype control) via intraperitoneal injection at a dose of 10 mg / kg, twice weekly. Tumor volume and body weight were measured 2-3 times weekly. The bispecific antibody was used... Figure 1BThe Fab format is shown. The VH domain of the anti-Dectin-1 binding arm uses the amino acid sequence of SEQ ID NO: 30, and the VL domain uses the amino acid sequence of SEQ ID NO: 31. For the anti-Trop-2 binding arm, the amino acid sequences of the variable domains are as follows: VH domain of clone A is SEQ ID NO: 69, and VL domain is SEQ ID NO: 70; VH domain of clone B is SEQ ID NO: 74, and VL domain is SEQ ID NO: 75; VH domain of clone C is SEQ ID NO: 79, and VL domain is SEQ ID NO: 80. All bispecific antibodies use the mouse IgG2a Fc region.

[0165] Cancer cell depletion in human tissue biopsy Human cancer biopsy tissue was enzymatically dissociated to generate a single-cell suspension. 300,000 to 500,000 cells were treated with a bispecific antibody containing the human IgG1 Fc region (anti-Dectin-1 x anti-Trop-2) at 5 μg / ml for 24 hours. Cancer cells were stained with APC-EPCAM antibody. Remaining cancer cells were assessed as single / viable / CD45- / EPCAM+ cells and expressed as the percentage of viable cells.

[0166] result The ability of anti-Dectin-1 x anti-Trop-2 bispecific antibodies to induce phagocytosis of cancer cell lines by human macrophages was tested in vitro. All three anti-Dectin-1 x anti-Trop-2 bispecific antibodies were found to induce phagocytosis of A-431, NCI-N87, and SKBR3 cells. Figures 6A to 6C The EC50 values ​​for each antibody against each cell type are shown in Table 4.

[0167] Table 4. Phagocytosis induced in vitro by the specified anti-Dectin-1 x anti-Trop-2 bispecific antibodies against the specified cell lines (EC50, nM).

[0168] In summary, the anti-Dectin-1 x anti-Trop-2 bispecific antibody exhausted cancer cells in vitro via phagocytosis. This exhaustion was mediated by the Fcγ receptor. These data support the ability of the anti-Dectin-1 x anti-Trop-2 bispecific antibody to promote the binding and elimination of Trop-2-expressing human cancer cells by human macrophages.

[0169] Cytokine secretion was also measured in a co-culture of human PBMCs and hTrop-2-expressing CHO-K1 cells treated with anti-Dectin-1 and anti-Trop-2 bispecific antibodies. Figures 7A to 7D As shown, the anti-Dectin-1 x anti-Trop-2 bispecific antibodies did not significantly affect cytokine secretion.

[0170] In a syngeneic mouse tumor model, the anti-Dectin-1 x anti-Trop-2 bispecific antibody was tested using an alternative anti-mDectin-1 binding arm and mIgG2a Fc. The anti-Dectin-1 x anti-Trop-2 bispecific antibody was found to be effective in colon cancer (…). Figure 8A ) and melanoma ( Figure 8B Both models inhibited tumor growth and demonstrated significant efficacy in both preclinical models. Advantageously, the bispecific antibody with anti-Trop-2 clone B exhibited the strongest antitumor activity. These models are considered to have fundamental differences in their tumor microenvironments. CT26 tumors are considered "hot" tumors with more infiltrating immune cells and generally respond to immunomodulatory agents. In contrast, B16F10 is considered a "cold" tumor with less immune cell infiltration and is unresponsive to monotherapy. The efficacy generated in both models supports the versatility of anti-Dectin-1 x anti-Trop-2 bispecific antibodies, which act in both "hot" tumors and more difficult-to-treat "cold" tumors, stemming from their ability to modulate the tumor microenvironment.

[0171] In mice expressing human Dectin-1, an anti-Dectin-1 x anti-Trop-2 bispecific antibody was tested in a syngeneic mouse B16F10 tumor model using a disulfide-engineered anti-Dectin-1 variable domain. The anti-Dectin-1 x anti-Trop-2 bispecific antibody demonstrated significant antitumor efficacy in this melanoma model. Figure 9 This study directly demonstrates the ability of anti-Dectin-1 x anti-Trop-2 bispecific antibodies to conjugate anti-tumor effects by binding to human Dectin-1, thus supporting the application of anti-Dectin-1 x anti-Trop-2 bispecific antibodies in oncology.

[0172] The ability of the anti-Dectin-1 x anti-Trop-2 bispecific antibody to deplete single-cell suspensions derived from tumor biopsy tissue was also tested. It was found that the anti-Dectin-1 x anti-Trop-2 bispecific antibody induced depletion of single-cell suspensions from human lung cancer (…). Figure 10A ovarian cancer Figure 10B ) and uterine cancer ( Figure 10CThe depletion of tumor cells in biopsy tissues. Anti-Dectin-1 x anti-Trop-2 bispecific antibodies reduced Trop-2-expressing cancer cells by 20% to 50% in various human cancer biopsy tissues. Advantageously, bispecific antibodies with anti-Trop-2 clone B showed improved activity.

[0173] These data indicate that bispecific antibodies can induce phagocytosis and elimination of cancer cells by tumor-associated macrophages in solid cancer biopsy tissues, demonstrating their activity in the endogenous cancer tissue environment. As determined by mRNA expression levels, Trop-2 is widely expressed in human cancers (…). Figure 10D Therefore, this strategy can be widely applied to solid tumors.

[0174] Although this disclosure has been described in detail by way of illustration and examples for purposes of clarity, the description and examples should not be construed as limiting the scope of this disclosure. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

Claims

1. A bispecific binding molecule comprising: (a) A first arm, the first arm comprising a first antigen-binding domain that binds to human Dectin-1; and (b) A second arm, the second arm comprising a second antigen-binding domain that binds to human Trop-2; The second antigen-binding domain comprises a second heavy chain variable (VH) domain and a second light chain variable (VL) domain; and in: (a) The second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; or (b) The second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 53, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 54, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 55, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 56, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 57, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

58.

2. A bispecific binding molecule comprising: (a) A first arm, the first arm comprising a first antigen-binding domain that binds to human Dectin-1; and (b) A second arm, the second arm comprising a second antigen-binding domain that binds to human Trop-2; The second antigen-binding domain comprises a second heavy chain variable (VH) domain and a second light chain variable (VL) domain; and in: (a) The second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 48, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 49, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 50, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 51, CDR-L2 comprising the amino acid sequence of GAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 52; or (b) The second VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 64, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 65, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 66, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 67, CDR-L2 comprising the amino acid sequence of AAS, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

68.

3. The bispecific binding molecule as described in claim 1 or claim 2, wherein: (a) The second VH domain contains the amino acid sequence of SEQ ID NO: 74, and the second VL domain contains the amino acid sequence of SEQ ID NO: 75; or (b) The second VH domain contains the amino acid sequence of SEQ ID NO: 79, and the second VL domain contains the amino acid sequence of SEQ ID NO:

80.

4. The bispecific binding molecule of any one of claims 1 to 3, wherein the second arm comprises: an antibody heavy chain polypeptide comprising the second VH domain and an antibody light chain polypeptide comprising the second VL domain.

5. The bispecific binding molecule of claim 4, wherein the antibody heavy chain polypeptide in the second arm comprises an Fc region.

6. The bispecific binding molecule of claim 5, wherein the Fc region of the second arm is the human IgG Fc region.

7. The bispecific binding molecule of claim 6, wherein the Fc region of the second arm is the human IgG1 Fc region.

8. The bispecific binding molecule of claim 6, wherein the Fc region of the second arm is the human IgG4 Fc region.

9. The bispecific binding molecule of any one of claims 5 to 8, wherein the first arm comprises an Fc region; wherein the Fc region of the first arm comprises one or more mutations forming a club, and the Fc region of the second arm comprises one or more paired mutations forming a mortise.

10. The bispecific binding molecule of claim 9, wherein, according to the EU designation, the Fc region of the first arm comprises a T366W substitution, and wherein the Fc region of the second arm comprises T366S, L368A, and Y407V substitutions.

11. The bispecific binding molecule of any one of claims 5 to 8, wherein the first arm comprises an Fc region; wherein the Fc region of the second arm comprises one or more mutations forming a club, and the Fc region of the first arm comprises one or more paired mutations forming a mortise.

12. The bispecific binding molecule of claim 11, wherein, according to the EU designation, the Fc region of the second arm comprises a T366W substitution, and wherein the Fc region of the first arm comprises T366S, L368A, and Y407V substitutions.

13. The bispecific binding molecule of claim 4, wherein: (a) The second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 76 or 77 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 78; (b) The second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 81 or 82 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 83; (c) The second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 107 or 108 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 78; or (d) The second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 109 or 110 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO:

83.

14. A bispecific binding molecule comprising: (a) A first arm, the first arm comprising a first antigen-binding domain that binds to human Dectin-1; and (b) A second arm, the second arm comprising a second antigen-binding domain that binds to human Trop-2; The second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 71 or 72 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 73, or wherein the second arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 105 or 106 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO:

73.

15. The bispecific binding molecule of any one of claims 1 to 14, wherein the first antigen-binding domain comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain; and wherein the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

16. The bispecific binding molecule of any one of claims 1 to 14, wherein the first antigen-binding domain comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain; and wherein the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

6.

17. The bispecific binding molecule of claim 15 or claim 16, wherein the first VH domain comprises the amino acid sequence of SEQ ID NO: 24, and the first VL domain comprises the amino acid sequence of SEQ ID NO:

25.

18. The bispecific binding molecule of any one of claims 1 to 14, wherein the first antigen-binding domain comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain; and wherein the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

20.

19. The bispecific binding molecule of any one of claims 1 to 14, wherein the first antigen-binding domain comprises a first heavy chain variable (VH) domain and a first light chain variable (VL) domain; and wherein the first VH domain comprises: CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 23 and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18, and the second VL domain comprises: CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 19 and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

20.

20. The bispecific binding molecule of claim 18 or claim 19, wherein the first VH domain comprises the amino acid sequence of SEQ ID NO: 26, and the first VL domain comprises the amino acid sequence of SEQ ID NO:

27.

21. The bispecific binding molecule of claim 18 or claim 19, wherein the first VH domain comprises the amino acid sequence of SEQ ID NO: 30, and the first VL domain comprises the amino acid sequence of SEQ ID NO:

31.

22. The bispecific binding molecule of any one of claims 1 to 21, wherein the first arm comprises: an antibody heavy chain polypeptide comprising the first VH domain and an antibody light chain polypeptide comprising the first VL domain.

23. The bispecific binding molecule of claim 22, wherein the antibody heavy chain polypeptide of the first arm comprises an Fc region.

24. The bispecific binding molecule of any one of claims 1 to 21, wherein the first arm comprises a single-stranded variable fragment (scFv) comprising the first VH domain and the first VL domain.

25. The bispecific binding molecule of claim 24, wherein the scFv includes a linker between the first VH domain and the first VL domain.

26. The bispecific binding molecule of claim 24 or claim 25, wherein the scFv comprises the amino acid sequence of SEQ ID NO:

34.

27. The bispecific binding molecule of any one of claims 24 to 26, wherein the first arm further comprises an Fc region connected to the scFv.

28. The bispecific binding molecule of claim 23 or claim 27, wherein the Fc region of the first arm is the human IgG Fc region.

29. The bispecific binding molecule of claim 28, wherein the Fc region of the first arm is the human IgG1 Fc region.

30. The bispecific binding molecule of claim 28, wherein the Fc region of the first arm is the human IgG4 Fc region.

31. The bispecific binding molecule of any one of claims 28 to 30, wherein the second arm comprises an Fc region; wherein the Fc region of the first arm comprises one or more mutations forming a club, and the Fc region of the second arm comprises one or more paired mutations forming a mortise.

32. The bispecific binding molecule of claim 31, wherein, according to the EU designation, the Fc region of the first arm comprises a T366W substitution, and wherein the Fc region of the second arm comprises T366S, L368A, and Y407V substitutions.

33. The bispecific binding molecule of any one of claims 28 to 30, wherein the second arm comprises an Fc region; wherein the Fc region of the second arm comprises one or more mutations forming a club, and the Fc region of the first arm comprises one or more paired mutations forming a mortise.

34. The bispecific binding molecule of claim 33, wherein, according to the EU designation, the Fc region of the second arm comprises a T366W substitution, and wherein the Fc region of the first arm comprises T366S, L368A, and Y407V substitutions.

35. The bispecific binding molecule of claim 22, wherein the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 87 or 88 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 89, or wherein the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 111 or 112 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO:

89.

36. The bispecific binding molecule of claim 22, wherein the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 28 or 90 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 29, or wherein the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 99 or 100 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO:

29.

37. The bispecific binding molecule of claim 22, wherein the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 32 or 91 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO: 33, or wherein the first arm comprises: an antibody heavy chain polypeptide comprising the amino acid sequence of SEQ ID NO: 101 or 102 and an antibody light chain polypeptide comprising the amino acid sequence of SEQ ID NO:

33.

38. The bispecific binding molecule of claim 24, wherein the first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, or wherein the first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104.

39. The bispecific binding molecule of any one of claims 1 to 38, wherein at least one or both of the first arm and the second arm are unfucosylated or contain reduced fucosylation.

40. The bispecific binding molecule of any one of claims 1 to 39, wherein the human Dectin-1 comprises the amino acid sequence of SEQ ID NO: 84 or 85.

41. The bispecific binding molecule of any one of claims 1 to 40, wherein the human Trop-2 comprises the amino acid sequence of SEQ ID NO:

86.

42. A bispecific binding molecule comprising: (a) A first arm, the first arm comprising a first antigen-binding domain that binds to human Dectin-1; and (b) A second arm, the second arm comprising a second antigen-binding domain that binds to human Trop-2; The first arm comprises a first antibody heavy chain polypeptide and a first antibody light chain polypeptide, and the second arm comprises a second antibody heavy chain polypeptide and a second antibody light chain polypeptide; and wherein: (a) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73; (b) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78; (c) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 87 or 88, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

83. (d) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73; (e) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

78. (f) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 28 or 90, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

83. (g) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73; (h) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

78. (i) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 32 or 91, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 83; (j) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

73. (k) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

78. (l) The first antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 111 or 112, the first antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO: 89, the second antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO: 83; (m) The first antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO: 73; (n) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

78. (o) The first antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 99 or 100, the first antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO: 29, the second antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO:

83. (p) The first antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 105 or 106, and the second antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO:

73. (q) The first antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the second antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78; or (r) The first antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 101 or 102, the first antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO: 33, the second antibody heavy chain polypeptide contains the amino acid sequence of SEQ ID NO: 109 or 110, and the second antibody light chain polypeptide contains the amino acid sequence of SEQ ID NO:

83.

43. A bispecific binding molecule comprising: (a) A first arm, the first arm comprising a first antigen-binding domain that binds to human Dectin-1; and (b) A second arm, the second arm comprising a second antigen-binding domain that binds to human Trop-2; The first arm comprises a single-chain variable fragment (scFv) linked to the Fc region, and the second arm comprises an antibody heavy chain polypeptide and an antibody light chain polypeptide; and wherein: (a) The first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 71 or 72, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 73; (b) The first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 76 or 77, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78; (c) The first arm comprises the amino acid sequence of SEQ ID NO: 35 or 36, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 81 or 82, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

83. (d) The first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 105 or 106, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

73. (e) The first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 107 or 108, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO: 78; or (f) The first arm comprises the amino acid sequence of SEQ ID NO: 103 or 104, the antibody heavy chain polypeptide comprises the amino acid sequence of SEQ ID NO: 109 or 110, and the antibody light chain polypeptide comprises the amino acid sequence of SEQ ID NO:

83.

44. A polynucleotide encoding a bispecific binding molecule as described in any one of claims 1 to 43.

45. A vector comprising the polynucleotide of claim 44.

46. ​​The carrier of claim 45, wherein the carrier is an expression carrier.

47. An isolated host cell comprising the polynucleotide of claim 44, or the vector of claim 45 or claim 46.

48. The isolated host cell of claim 47, wherein the host cell is a yeast cell, insect cell, plant cell, or prokaryotic cell.

49. The isolated host cell of claim 47, wherein the host cell is a mammalian cell.

50. The isolated host cell of claim 49, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

51. The isolated host cell of claim 49 or claim 50, wherein the host cell comprises α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) knockout.

52. The isolated host cell as claimed in claim 49 or claim 50, wherein the host cell overexpresses β1,4-N-acetylglucosamine transferase III (GnT-III).

53. The isolated host cell of claim 52, wherein the host cell further overexpresses Golgi μ-mannosidase II (ManII).

54. A method for generating a bispecific binding molecule, comprising culturing a host cell according to any one of claims 47 to 53 under conditions suitable for generating said bispecific binding molecule.

55. The method of claim 54, further comprising recovering the bispecific binding molecule from the host cell.

56. The method of claim 54 or claim 55, wherein, The host cells are treated with chiffon base prior to the generation of the bispecific binding molecule.

57. A bispecific binding molecule, which is produced by the method of any one of claims 54 to 56.

58. A pharmaceutical composition comprising the bispecific binding molecule of any one of claims 1 to 43 and 57 and a pharmaceutically acceptable carrier.

59. A method of treating cancer, comprising administering to an individual in need an effective amount of any one of claims 1 to 43 and 57 of the bispecific binding molecule or the pharmaceutical composition of claim 58.

60. The method of claim 59, wherein the cancer is a solid cancer.

61. The method of claim 59 or claim 60, wherein the cancer is breast cancer, prostate cancer, urothelial carcinoma, cervical cancer, bladder cancer, pancreatic cancer, thyroid cancer, head and neck cancer, esophageal cancer, endometrial cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, lung cancer, ovarian cancer, or uterine cancer.

62. The method of any one of claims 59 to 61, wherein the cancer cells express Trop-2.

63. The method of any one of claims 59 to 62, wherein the individual is a person.

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