Anti-dectin-1 antibody and method of use thereof

Abstract

To provide an anti-dectin-1 antibody and a method for using the same. [Solution] The present invention relates to an anti-human dectin-1 antibody clone 2M24 (hIgG4) or 15E2, and a multispecificity binding molecule containing the same, as well as methods for producing and using the same. Disclosure relates to an antibody that binds to human dectin-1, a multispecificity (e.g., bispecificity) binding molecule, and related methods for using and producing the same. Disclosed herein is a targeted phagocytic method for removing disease-causing substances, including host cells / host cell products, microorganisms, or their products, by administering a multispecificity (e.g., bispecificity) binding molecule comprising a dectin-1 binding arm and a second arm that specifically binds to the substance.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 088,895, filed on October 7, 2020, and No. 63 / 174,439, filed on April 13, 2021, the disclosures of each of which are hereby incorporated by reference in their entirety.

[0002] Submission of Sequence Listing in ASCII Text File The content of the following submission in ASCII text file is hereby incorporated by reference in its entirety; a sequence listing in computer - readable format (CRF) (filename: 186542000440SEQLIST.TXT, recording date: October 6, 2021, size: 108,547 bytes).

[0003] This disclosure relates to antibodies that bind to human dectin - 1, multispecific (e.g., bispecific) binding molecules, and related methods of use and production methods.

Background Art

[0004] Phagocytosis is a major mechanism used to remove pathogens and cellular debris. Professional phagocytes such as monocytes, macrophages, dendritic cells, and granulocytes specifically recognize and engulf abnormal or disease - causing hosts or foreign substances. The engulfed substances are destroyed through the endolysosomal pathway of phagocytes. Furthermore, dendritic cells and macrophages can present antigens to cells of the adaptive immune system to further promote the elimination of disease - causing agents.

[0005] Dectin - 1 is a C - type lectin receptor that recognizes β - glucan and promotes antifungal phagocytic activity. Dectin - 1 is expressed on phagocytes and has been clearly shown to be sufficient for the activation of phagocytosis. Dectin - 1 can be utilized for antibody - targeted phagocytosis and the elimination of disease - causing agents.

[0006] Developing targeted removal and degradation of accumulated disease-causing substances without increasing overall phagocytosis can be beneficial. This disclosure provides a solution to this problem and describes other advantages.

[0007] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated herein by reference in whole, as if each individual reference were specifically and individually indicated so as to be incorporated by reference. [Overview of the project] [Means for solving the problem]

[0008] This disclosure relates to antibodies that bind to human dectin-1, multispecific (e.g., bispecific) conjugating molecules, and related methods of use and production. Disclosed herein is a method of targeted phagocytosis for removing disease-causing substances, including host cells / host cell products, microorganisms, or their products, by administering a multispecific (e.g., bispecific) conjugating molecule comprising a dectin-1 binding arm and a second arm that specifically binds to the substance. The multispecific (e.g., bispecific) conjugating molecule enables phagocytic cells to engage with the target substance, form synapses between them, and promote clustering of dectin-1 on the phagocytic cells. This stimulates phagocytosis of the target substance and simultaneously stimulates cytokine secretion by phagocytic cells via the dectin-1 / Syk / NfkB pathway. Furthermore, antigens of the taken-up substance are presented on the surface of dendritic cells / macrophages, enhancing the adaptive immune response to the disease-causing substance. In general, dectin-1 agonist multispecific (e.g., bispecific) binding molecules are thought to promote immune stimulation, targeted phagocytosis, and neoantigen presentation / activation of the adaptive immune system, thereby eliminating disease-causing substances.

[0009] Therefore, this disclosure describes, in particular, the preparation and functional characterization of agonist anti-human dectin-1 antibodies that exhibit high affinity binding to dectin-1 and can promote immune stimulation. Furthermore, the preparation of a bispecific antibody format, including an anti-human dectin-1 antibody and an antibody targeting an antigen on a disease-causing substance, is also described, along with data supporting target engagement, immune stimulation, phagocytosis, and antigen presentation.

[0010] In some embodiments, antibodies or antigen-binding fragments that bind to human dectin-1 are provided herein, wherein the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 containing the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 containing the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises CDR-L1 containing the sequence QGISSW (SEQ ID NO: 4), CDR-L2 containing the sequence GAS (SEQ ID NO: 5), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, an antibody or antigen-binding fragment that binds to human dectin-1 comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain sequence is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVS An antibody or fragment is provided herein that comprises CDR-H1, CDR-H2, and CDR-H3 from S (SEQ ID NO: 7), wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).In some embodiments, antibodies or antigen-binding fragments that bind to human dectin-1 are provided herein, wherein the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence DYYI (SEQ ID NO: 88), CDR-H2 containing the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 containing the sequence NSGSYSFGY (SEQ ID NO: 90), and the VL domain comprises CDR-L1 containing the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 containing the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the VH domain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7). The sequence contains; and / or the VL domain contains a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).In some embodiments, the VH domain comprises the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and / or the VL domain comprises the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, antibodies or antigen-binding fragments thereof that bind to human dectin-1 are provided herein, which bind to human dectin-1 expressed on the surface of a cell with an EC50 of less than 2 nM, less than 1 nM, or less than 0.5 nM. In some embodiments, antibodies or antigen-binding fragments thereof that bind to human dectin-1 and are capable of binding to human or cynomolgus monkey dectin-1 are provided herein. In some embodiments, antibodies or antigen-binding fragments thereof that bind to human dectin-1 and are not competitive with the natural ligand of dectin-1, for example, human dectin-1 (for example, with respect to binding to human dectin-1) are provided herein. In some embodiments, the antibody or fragment competes for binding to human dectin-1 with a reference antibody comprising: (a) a heavy chain variable (VH) domain comprising CDR-H1 comprising the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 comprising the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 comprising the sequence ARNSGSYSFGY (SEQ ID NO: 3), and a light chain variable (VL) domain comprising CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6); (b) a heavy chain variable (VH) domain comprising CDR-H1 comprising the sequence DYYI (SEQ ID NO: 88), CDR-H2 comprising the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 comprising the sequence NSGSYSFGY (SEQ ID NO: 90), and the sequence RASQGISSWL (c) A light chain variable (VL) domain comprising CDR-L1 containing A (SEQ ID NO: 91), CDR-L2 containing sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing sequence QQAYSFPFT (SEQ ID NO: 6); or (c) a heavy chain variable (VH) domain comprising sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and a light chain variable (VL) domain comprising sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).In some embodiments, the antibody or fragment binds to the same human dectin-1 epitope as a reference antibody comprising: (a) a heavy chain variable (VH) domain comprising CDR-H1 comprising the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 comprising the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 comprising the sequence ARNSGSYSFGY (SEQ ID NO: 3); and a light chain variable (VL) domain comprising CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6); (b) a heavy chain variable (VH) domain comprising CDR-H1 comprising the sequence DYYI (SEQ ID NO: 88), CDR-H2 comprising the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 comprising the sequence NSGSYSFGY (SEQ ID NO: 90); and the sequence RASQGISSWL A light chain variable (VL) domain comprising CDR-L1 containing sequence A (SEQ ID NO: 91), CDR-L2 containing sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing sequence QQAYSFPFT (SEQ ID NO: 6); or (c) a heavy chain variable (VH) domain comprising sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and a light chain variable (VL) domain comprising sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the antibody or fragment is a human, humanized, or chimeric antibody or fragment. In some embodiments, the antibody or fragment binds to human dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, and / or granulocytes. In some embodiments, the antigen-binding antibody fragment is Fab, Fab', F(ab')2, Fv, Fab'-SH, F(ab')2, single-chain antibody, nanobody, or scFv fragment. In some embodiments, the antibody further comprises an Fc region.In some embodiments, the antibody or fragment is a multispecific antibody or fragment. In some embodiments, the antibody or fragment is a bispecific antibody, fragment, or diabody comprising a first antigen-binding domain comprising a VH domain and a VL domain that bind to human dectin-1 and a second antigen-binding domain that binds to a target, or a first antigen-binding domain that binds to a target and a second antigen-binding domain comprising a VH domain and a VL domain that binds to human dectin-1. In some embodiments, the bispecific antibody comprises a single-stranded variable fragment (scFv) comprising a VH domain and a VL domain that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain of a second antigen-binding domain associated with an antibody light chain comprising a VL domain of a second antigen-binding domain, and a second antibody arm comprising a second Fc region attached to the VH domain of the second antigen-binding domain. In some embodiments, the second antigen-binding domain binds to CD20 and includes a VH domain containing the sequence of SEQ ID NO: 24 and a VL domain containing the sequence of SEQ ID NO: 25. In some embodiments, the second antigen-binding domain binds to Trop-2 and includes a VH domain containing the sequence of SEQ ID NO: 42 and a VL domain containing the sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding domain binds to light chain amyloid and includes a VH domain containing the sequence of SEQ ID NO: 44 and a VL domain containing the sequence of SEQ ID NO: 45. In some embodiments, the first Fc region includes one or more knob-forming mutations and the second Fc region includes one or more congeneral hole-forming mutations, or the second Fc region includes one or more knob-forming mutations and the first Fc region includes one or more congeneral hole-forming mutations. In some embodiments, the first Fc region includes a T366W substitution by EU numbering and the second Fc region includes T366S, L368A, and Y407V substitutions. In some embodiments, the first antibody arm includes a first linker between the VH domain and the VL domain, and a second linker between the VL domain and the first Fc region. In some embodiments, the first linker includes one or more repeats of the sequence GGGGS (SEQ ID NO: 26).In some embodiments, the first linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the second linker includes the sequence EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). In some embodiments, the bispecific antibody includes a first IgG antibody containing a first antigen-binding domain covalently linked to a second IgG antibody containing a second antigen-binding domain. In some embodiments, the bispecific antibody includes a first antibody arm containing a first antibody heavy chain containing a VH domain of the first antigen-binding domain and a first Fc region, and a second antibody arm containing a second antibody heavy chain containing a VH domain of the second antigen-binding domain and a second Fc region, wherein the first Fc region contains one or more knob-forming mutations, and the second Fc region contains one or more congeneral hole-forming mutations. In some embodiments, the first Fc region includes a T366W substitution according to EU numbering, and the second Fc region includes T366S, L368A, and Y407V substitutions. In some embodiments, the bispecific antibody includes a first antibody arm including a first antibody heavy chain and a first Fc region including a VH domain of a first antigen-binding domain, and a second antibody arm including a second antibody heavy chain and a second Fc region including a VH domain of a second antigen-binding domain, wherein the first Fc region includes one or more hole-forming mutations, and the second Fc region includes one or more congeneral knob-forming mutations. In some embodiments, the first Fc region includes a T366S, L368A, and Y407V substitution according to EU numbering, and the second Fc region includes a T366W substitution. In some embodiments, the bispecific antibody comprises a first IgG antibody containing a first antigen-binding domain linked to its biotin-binding derivative or avidin-binding derivative, and a second IgG antibody containing a second antigen-binding domain linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, wherein the biotin-binding derivative or avidin-binding derivative is conjugated to the avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative.In some embodiments, the bispecific antibody comprises a first IgG antibody containing a first antigen-binding domain linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and a second IgG antibody containing a second antigen-binding domain linked to its biotin-binding derivative or avidin-binding derivative, wherein the biotin-binding derivative or avidin-binding derivative is conjugated to the avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative. In some embodiments, the target is a disease causative agent. In some embodiments, the disease causative agent is a bacterial cell, a fungal cell, a virus, a senescent cell, a tumor cell, a protein aggregate (e.g., amyloid-beta, or lambda or kappa light chain amyloid), an LDL particle, a mast cell, an eosinophil, an ILC2 cell, or an inflammatory immune cell. In some embodiments, the target is an antigen expressed on the surface of bacterial cells, fungal cells, senescent cells, tumor cells, mast cells, eosinophils, ILC2 cells, or inflammatory immune cells. In some embodiments, the target is a viral surface antigen. In some embodiments, the target is an antigen expressed on the surface of cancer cells. In some embodiments, the target is CD70, HER2, DLL3, nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR.In some embodiments, the target is CD20; the second antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; the VH domain of the second antigen-binding domain comprises the sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24), and / or the VL domain of the second antigen-binding domain comprises the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25). In some embodiments, the antibody comprises two antibody heavy chains, each of which contains one or more amino acid substitutions at positions 234, 235, and 237 according to EU numbering. In some embodiments, each of the antibody heavy chains contains L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the antibody comprises two antibody heavy chains, with only one of the antibody heavy chains containing H435R and Y436F substitutions according to EU numbering. In some embodiments, the antibody comprises two arms, with only one of the antibody arms containing a heavy chain with F126C and C220V substitutions according to EU numbering and a light chain with S121C and C214V substitutions. In some embodiments, the bispecific antibody comprises a first antibody heavy chain and a second antibody heavy chain, 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 the VH domain of the second antibody heavy chain forms an antigen-binding domain with the VL domain of the second antibody light chain, and the first antibody heavy chain has an EU number. The first antibody light chain includes F126C, C220V, and T366W substitutions via rings, the second antibody heavy chain includes S121C and C214V substitutions, and the second antibody heavy chain includes T366S, L368A, Y407V, H435R, and Y436F substitutions. In some embodiments, the first and second antibody heavy chains further include L234A, L235E, and G237A substitutions via EU numbering. In some embodiments, the first and second antibody heavy chains include human IgG1 Fc domains. In some embodiments, the antibody includes a first antibody heavy chain and a second antibody heavy chain, at least one or two of the first and second antibody heavy chains are not fucosylated. In some embodiments, the antibody may be produced in cell lines having knockout of α1,6-fucosyltransferase (Fut8) or α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminetransferase (MGAT1). In some embodiments, the antibody may be produced in cell lines overexpressing β1,4-N-acetylglucosaminetransferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). In some embodiments, the antibody may be produced in cell lines treated with a mannosidase I inhibitor, such as kifunensin.

[0011] In some embodiments, multispecific binding molecules are provided herein, comprising (a) a first antibody or antigen-binding fragment comprising a first antigen-binding domain that binds to human dectin-1, and (b) a second antibody or antigen-binding fragment comprising a second antigen-binding domain that binds to a target of interest. In some embodiments, the target of interest is a disease causative agent. In some embodiments, the disease causative agent is a bacterial cell, a fungal cell, a virus, a senescent cell, a tumor cell, a protein aggregate (e.g., amyloid-beta, or lambda or kappa light chain amyloid), an LDL particle, a mast cell, a eosinophil, an ILC2 cell, or an inflammatory immune cell. In some embodiments, the target of interest is an antigen expressed on the surface of a bacterial cell, a fungal cell, a senescent cell, a tumor cell, a mast cell, a eosinophil, an ILC2 cell, or an inflammatory immune cell. In some embodiments, the target of interest is a viral surface antigen. In some embodiments, the target is CD70, HER2, DLL3, Nectin-4, TROP-2, Mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR. In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 containing the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 containing the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises CDR-L1 containing the sequence QGISSW (SEQ ID NO: 4), CDR-L2 containing the sequence GAS (SEQ ID NO: 5), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6).In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 from the VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the VH domain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7). The sequence contains; and / or the VL domain contains a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).In some embodiments, the VH domain includes the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and / or the VL domain includes the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the first antigen-binding domain binds to human dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, or granulocytes; binds to human dectin-1 expressed on the cell surface with an EC50 of less than 2 nM; can bind to human or cynomolgus monkey dectin-1; and / or does not compete with the native ligand of human dectin-1. In some embodiments, the second antigen-binding domain binds to CD20 and includes a VH domain containing the sequence of SEQ ID NO: 24 and a VL domain containing the sequence of SEQ ID NO: 25. In some embodiments, the second antigen-binding domain binds to Trop-2 and includes a VH domain containing the sequence of SEQ ID NO: 42 and a VL domain containing the sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding domain binds to light chain amyloid and includes a VH domain containing the sequence of SEQ ID NO: 44 and a VL domain containing the sequence of SEQ ID NO: 45. In some embodiments, one or both of the first and second antibodies or fragments are human antibodies or fragments or humanized antibodies or fragments. In some embodiments, one or both of the first and second antibodies or fragments 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 antibodies or fragments further include an Fc domain. In some embodiments, the first antibody or fragment is a Fab fragment, and the second antibody or fragment is a full-length antibody, for example, comprising an antibody heavy chain and an antibody light chain. In some embodiments, both the first and second antibodies or fragments are full-length antibodies, for example, each comprising an antibody heavy chain and an antibody light chain. In some embodiments, the multispecificity binding molecule includes a single-stranded variable fragment (scFv) containing a VH domain and a VL domain that bind to human dectin-1, and a first antibody arm containing a first Fc region; an antibody heavy chain containing a VH domain of a second antigen-binding domain associated with an antibody light chain containing a VL domain of a second antigen-binding domain; and a second antibody arm containing a second Fc region attached to the VH domain of the second antigen-binding domain.In some embodiments, the first Fc region includes one or more knob-forming mutations and the second Fc region includes one or more congeneral hole-forming mutations, or the second Fc region includes one or more knob-forming mutations and the first Fc region includes one or more congeneral hole-forming mutations. In some embodiments, the first Fc region includes a T366W substitution by EU numbering and the second Fc region includes T366S, L368A, and Y407V substitutions. In some embodiments, the first antibody arm includes a first linker between the VH domain and the VL domain and a second linker between the VL domain and the first Fc region. In some embodiments, the first linker includes one or more repeats of the sequence GGGGS (SEQ ID NO: 26). In some embodiments, the first linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the second linker includes the sequence EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). In some embodiments, the first antibody or fragment is bound to its avidin-conjugated derivative, streptavidin-conjugated derivative, neutraavidin-conjugated derivative, or biotin-conjugated derivative, and the second antibody or fragment is linked to its biotin-conjugated derivative or avidin-conjugated derivative; or the second antibody or fragment is linked to its avidin-conjugated derivative, streptavidin-conjugated derivative, neutraavidin-conjugated derivative, or biotin-conjugated derivative, and the first antibody or fragment is linked to its biotin-conjugated derivative or avidin-conjugated derivative; and the first antibody or fragment is linked to the second antibody or fragment via interactions between its avidin-conjugated derivative, streptavidin-conjugated derivative, neutraavidin-conjugated derivative, or biotin-conjugated derivative and its biotin-conjugated derivative or avidin-conjugated derivative. In some embodiments, the first antibody or fragment is a Fab fragment linked to monomeric streptavidin (mSA), and the second antibody or fragment is a biotinylated antibody containing an antibody heavy chain and an antibody light chain.In some embodiments, the first antibody or fragment is a full-length antibody linked to monomeric streptavidin (mSA), and the second antibody or fragment is a biotinylated full-length antibody. In some embodiments, the multispecific binding molecule includes a first IgG antibody containing a first antigen-binding domain covalently linked to a second IgG antibody containing a second antigen-binding domain. In some embodiments, the multispecificity binding molecule comprises a first antibody arm comprising a first antibody heavy chain containing a VH domain of a first antigen-binding domain, and a first antibody light chain containing a first Fc region and a VL domain of a first antigen-binding domain; and a second antibody arm comprising a second antibody heavy chain containing a VH domain of a second antigen-binding domain, and a second antibody light chain containing a second Fc region and a VL domain of a second antigen-binding domain, wherein the first Fc region comprises one or more knob-forming mutations, and the second Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the first Fc region comprises a T366W substitution by EU numbering, and the second Fc region comprises T366S, L368A, and Y407V substitutions. In some embodiments, the multispecificity binding molecule comprises a first antibody arm comprising a VH domain of a first antigen-binding domain and a first Fc region, and a second antibody arm comprising a VH domain of a second antigen-binding domain and a second Fc region, wherein the first Fc region comprises one or more hole-forming mutations, and the second Fc region comprises one or more congeneral knob-forming mutations. In some embodiments, the first Fc region comprises T366S, L368A, and Y407V substitutions according to EU numbering, and the second Fc region comprises a T366W substitution. In some embodiments, the multispecificity binding molecule comprises two antibody Fc regions, each of which contains one or more amino acid substitutions at positions 234, 235, and 237 according to EU numbering. In some embodiments, each of which contains L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the multispecificity binding molecule comprises two antibody heavy chains, with only one of the antibody heavy chains containing H435R and Y436F substitutions according to EU numbering. In some embodiments, only one of the antibody arms comprises a heavy chain containing F126C and C220V substitutions according to EU numbering and a light chain containing S121C and C214V substitutions.In some embodiments, the multispecificity binding molecule comprises a first antibody heavy chain and a first antibody light chain, and a second antibody heavy chain and a second antibody light chain, wherein the VH domain of the first antibody heavy chain forms a first antigen-binding domain with the VL domain of the first antibody light chain, and the VH domain of the second antibody heavy chain forms a second antigen-binding domain with the VL domain of the second antibody light chain, and the first antibody heavy chain includes EU numbering substitutions F126C, C220V, and T366W, the first antibody light chain includes S121C and C214V, and the second antibody heavy chain includes T366S, L368A, Y407V, H435R, and Y436F. In some embodiments, the first and second antibody heavy chains further include EU numbering substitutions L234A, L235E, and G237A. In some embodiments, the first and second antibody heavy chains contain human IgG1 Fc domains. In some embodiments, at least one or two of the antibody heavy chains are not fucosylated. In some embodiments, the antibody may be produced in cell lines having α1,6-fucosyltransferase (Fut8) or α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminetransferase (MGAT1) knockout. In some embodiments, the antibody may be produced in cell lines overexpressing β1,4-N-acetylglucosaminetransferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). In some embodiments, the antibody may be produced in cell lines treated with a mannosidase I inhibitor, such as kifunensin.

[0012] In some embodiments, (a) a first antibody or antigen-binding fragment comprising a first antigen-binding domain that binds to a first target; (b) a second antibody or antigen-binding fragment comprising a second antigen-binding domain that binds to a second target; [wherein (i) the first antibody or fragment is linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the second antibody or fragment is linked to its biotin-binding derivative or avidin-binding derivative; or (ii) the second antibody or fragment is linked to its avidin-binding The following multispecific conjugation molecules are provided herein, comprising: a derivative, streptavidin-conjugated derivative, neutraavidin-conjugated derivative, or biotin-conjugated derivative; and the first antibody or fragment being linked to the biotin-conjugated derivative or avidin-conjugated derivative; wherein the first antibody or fragment is linked to the second antibody or fragment via an interaction between the avidin-conjugated derivative, streptavidin-conjugated derivative, neutraavidin-conjugated derivative, or biotin-conjugated derivative and the biotin-conjugated derivative or avidin-conjugated derivative. In some embodiments, the first target is human dectin-1. In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 containing the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 containing the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises CDR-L1 containing the sequence QGISSW (SEQ ID NO: 4), CDR-L2 containing the sequence GAS (SEQ ID NO: 5), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain,The VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (Sequence ID 7) contains CDR-H1, CDR-H2, and CDR-H3, and the VL domain contains CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (Sequence ID 8). In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence DYYI (SEQ ID NO: 88), CDR-H2 containing the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 containing the sequence NSGSYSFGY (SEQ ID NO: 90), and the VL domain comprises CDR-L1 containing the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 containing the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the VH domain comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7); and / or the VL domain comprises a sequence having at least 90%, at least 91%, identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8),The sequence contains at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity. In some embodiments, the VH domain contains the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and / or the VL domain contains the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the multispecific binding molecule is a sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFT A first polypeptide chain containing FGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31),Array QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV A multispecific binding molecule is provided herein, comprising a second polypeptide chain containing the sequence SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 32) and a third polypeptide chain containing the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33). In some embodiments, a multispecific binding molecule comprising a first arm containing a first antigen-binding domain and a second arm containing a second antigen-binding domain, wherein the first antigen-binding domain binds to human dectin-1 and the second antigen-binding domain binds to a target; the first arm,Array QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPC A multispecific binding molecule comprising a polypeptide chain containing PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31) is provided herein. In some embodiments, the first antigen-binding domain binds to human dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, or granulocytes. In some embodiments, one or both of the first and second antibodies or fragments are human antibodies or fragments or humanized antibodies or fragments. In some embodiments, one or both of the first and second antibodies or fragments 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 antibodies or fragments further include an Fc domain. In some embodiments, the first antibody or fragment is a Fab fragment, and the second antibody includes an antibody heavy chain and an antibody light chain. In some embodiments, the first antibody or fragment is a Fab fragment linked to monomeric streptavidin (mSA), and the second antibody is a biotinylated antibody including an antibody heavy chain and an antibody light chain. In some embodiments, the first antibody or fragment is a full-length antibody linked to monomeric streptavidin (mSA), and the second antibody is a biotinylated antibody containing an antibody heavy chain and an antibody light chain. In some embodiments, the second target is a disease causative agent. In some embodiments, the disease causative agent is a bacterial cell, fungal cell, virus, senescent cell, tumor cell, protein aggregate (e.g., amyloid-beta, or lambda or kappa light chain amyloid), LDL particle, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is an antigen expressed on the surface of a bacterial cell, fungal cell, senescent cell, tumor cell, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is a viral surface antigen. In some embodiments, the target is an antigen expressed on the surface of a cancer cell. In some embodiments, the target is CD70, HER2, DLL3, Nectin-4, TROP-2, Mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR.In some embodiments, the target is CD20; the second antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; the VH domain of the second antigen-binding domain comprises the sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24), and / or the VL domain of the second antigen-binding domain comprises the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25). In some embodiments, the second antigen-binding domain binds to Trop-2 and includes a VH domain containing the sequence of SEQ ID NO: 42 and a VL domain containing the sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding domain binds to light chain amyloid and includes a VH domain containing the sequence of SEQ ID NO: 44 and a VL domain containing the sequence of SEQ ID NO: 45.

[0013] In some embodiments, polynucleotides encoding any one of the antibodies or multispecificity conjugating molecules of the above embodiments are provided herein. In some embodiments, vectors (e.g., expression vectors) containing any one of the polynucleotides of the above embodiments are provided herein. In some embodiments, host cells (e.g., isolated host cells or cell lines) containing any one of the polynucleotides or vectors of the above embodiments are provided herein. In some embodiments, a method for producing an antibody or multispecificity conjugating molecule is provided herein, comprising culturing a host cell of any one of the above embodiments under conditions suitable for the production of the antibody or multispecificity conjugating molecule. In some embodiments, the method further includes recovering the antibody or multispecificity conjugating molecule. In some embodiments, a pharmaceutical composition comprising any one of the antibodies or multispecificity conjugating molecules of the above embodiments and a pharmaceutically acceptable carrier is provided herein.

[0014] In some embodiments, a method for producing a multispecific binding molecule, the method comprising: (a) providing a first antibody or antigen-binding fragment comprising a first antigen-binding domain that binds to a first target; and (b) providing a second antibody or antigen-binding fragment comprising a second antigen-binding domain that binds to a second target; [wherein (i) the first antibody or fragment is bound to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the second antibody or fragment is bound to its biotin-binding derivative or avidin-binding derivative; or (ii) the second antibody or fragment is bound to its avidin-binding (c) a first antibody or fragment is linked to a derivative, streptavidin-conjugated derivative, neutraavidin-conjugated derivative, or biotin-conjugated derivative, and the first antibody or fragment is linked to the biotin-conjugated derivative or avidin-conjugated derivative; (a) the first antibody or fragment is brought into contact with the second antibody or fragment under conditions suitable for the first antibody or fragment to bind to the second antibody or fragment via an interaction between the avidin-conjugated derivative, streptavidin-conjugated derivative, neutraavidin-conjugated derivative, or biotin-conjugated derivative and the biotin-conjugated derivative or avidin-conjugated derivative, thereby producing a multispecific binding molecule; a method is provided herein.In some embodiments, a method for identifying multispecific binding molecules that bind to a first target and a second target, the method comprising: (a) providing a first antibody or antigen-binding fragment comprising a first antigen-binding domain that binds to the first target; and (b) providing a second antibody or antigen-binding fragment comprising a second antigen-binding domain that binds to a second target; [wherein (i) the first antibody or fragment is bound to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the second antibody or fragment is bound to its biotin-binding derivative or avidin-binding derivative; or (ii) the second antibody or fragment is bound to its avidin-binding derivative, streptavidin-binding derivative (a) a first antibody or fragment is linked to a neutraavidin-binding derivative or a biotin-binding derivative, and the first antibody or fragment is linked to the biotin-binding derivative or avidin-binding derivative; (c) the first antibody or fragment is brought into contact with the second antibody or fragment under conditions suitable for the first antibody or fragment to bind to the second antibody or fragment via an interaction between the avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative and the biotin-binding derivative or avidin-binding derivative, thereby creating a multispecific binding molecule; (d) the binding between the multispecific binding molecule and at least one of the first target target and the second target; a method is provided herein that includes these steps. In some embodiments, the first target target is human dectin-1. In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 containing the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 containing the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises CDR-L1 containing the sequence QGISSW (SEQ ID NO: 4), CDR-L2 containing the sequence GAS (SEQ ID NO: 5), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6).In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 from the VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence DYYI (SEQ ID NO: 88), CDR-H2 containing the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 containing the sequence NSGSYSFGY (SEQ ID NO: 90), and the VL domain comprises CDR-L1 containing the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 containing the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6).In some embodiments, the VH domain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7). The sequence contains; and / or the VL domain contains a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the VH domain comprises the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7); and / or the VL domain comprises the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the first antigen-binding domain binds to human dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, or granulocytes. In some embodiments, one or both of the first and second antibodies or fragments are human antibodies or fragments or humanized antibodies or fragments.In some embodiments, one or both of the first and second antibodies or fragments 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 antibodies or fragments further include an Fc domain. In some embodiments, the first antibody or fragment is a Fab fragment, and the second antibody or fragment is a full-length antibody including, for example, an antibody heavy chain and an antibody light chain. In some embodiments, both the first and second antibodies or fragments are full-length antibodies each including, for example, an antibody heavy chain and an antibody light chain. In some embodiments, the first antibody or fragment is a Fab fragment linked to monomeric streptavidin (mSA), and the second antibody is a biotinylated antibody including an antibody heavy chain and an antibody light chain. In some embodiments, the first antibody or fragment is a full-length antibody linked to monomeric streptavidin (mSA), and the second antibody or fragment is a biotinylated full-length antibody. In some embodiments, the second target is a disease causative agent. In some embodiments, the disease causative agent is a bacterial cell, fungal cell, virus, senescent cell, tumor cell, protein aggregate (e.g., amyloid-beta, or lambda or kappa light chain amyloid), LDL particle, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is an antigen expressed on the surface of a bacterial cell, fungal cell, senescent cell, tumor cell, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is a viral surface antigen. In some embodiments, the target is an antigen expressed on the surface of a cancer cell. In some embodiments, the target is CD70, HER2, DLL3, nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR.In some embodiments, the target is CD20, and the second antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain of the second antigen-binding domain comprises the sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24), and / or the VL domain of the second antigen-binding domain comprises the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25). In some embodiments, the antibody comprises two antibody Fc regions, each of which contains one or more amino acid substitutions at positions 234, 235, and 237 according to EU numbering. In some embodiments, each of the antibody heavy chains contains L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the antibody comprises two antibody heavy chains, with only one of the antibody heavy chains containing H435R and Y436F substitutions according to EU numbering. In some embodiments, only one of the antibody arms comprises a heavy chain containing F126C and C220V substitutions according to EU numbering and a light chain containing S121C and C214V substitutions. In some embodiments, the bispecific antibody 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 the VH domain of the second antibody heavy chain forms an antigen-binding domain with the VL domain of the second antibody light chain, and the first antibody heavy chain includes substitutions F126C, C220V, and T366W according to EU numbering, the first antibody light chain includes substitutions S121C and C214V, and the second antibody heavy chain includes substitutions T366S, L368A, Y407V, H435R, and Y436F. In some embodiments, the first and second antibody heavy chains further include substitutions L234A, L235E, and G237A according to EU numbering.In some embodiments, the first and second antibody heavy chains contain human IgG1 Fc domains. In some embodiments, at least one or two of the antibody heavy chains are not fucosylated. In some embodiments, the antibody is α1,6-fucosyltransferase (Fut8) or α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminetransferase (2-β-N-acetylglucosaminetransferase). The antibody can be produced in cell lines having MGAT1 knockout. In some embodiments, the antibody can be produced in cell lines overexpressing β1,4-N-acetylglucosamine transferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). In some embodiments, the antibody can be produced in cell lines treated with a mannosidase I inhibitor, such as kifunensin.

[0015] In some embodiments, methods for treating a disease or disorder are provided herein, comprising administering an effective amount of any one of the antibodies, multispecific binding molecules, or compositions described in the above embodiments to an individual in need thereof. In some embodiments, the first target is human dectin-1, and the second target is a disease causative agent. In some embodiments, the disease causative agent is a bacterial cell, fungal cell, virus, senescent cell, tumor cell, protein aggregate (e.g., amyloid-beta, or lambda or kappa light chain amyloid), LDL particle, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is an antigen expressed on the surface of a bacterial cell, fungal cell, senescent cell, tumor cell, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is a viral surface antigen. In some embodiments, the disease or disorder is cancer, bacterial infection, fungal infection, viral infection, mast cell disease or disorder, systemic mastocytosis, amyloidosis (e.g., light chain amyloidosis or Alzheimer's disease), or age-related disease or disorder. In some embodiments, the target is CD70, HER2, DLL3, nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR. In some embodiments, the individual is human.

[0016] In some embodiments, methods for treating cancer are provided herein, comprising administering an effective amount of a composition comprising a multispecific binding molecule to an individual in need thereof, wherein the multispecific binding molecule comprises (a) a first antibody or antigen-binding fragment comprising a first antigen-binding domain that binds to human dectin-1, and (b) a second antibody or antigen-binding fragment comprising a second antigen-binding domain that binds to CD70, HER2, DLL3, nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR. In some embodiments, the second antigen-binding domain binds to, for example, human CD70, human HER2, human DLL3, human nectin-4, human TROP-2, human mesothelin, human LIV-1, human C-MET, human FOLR1, human CD20, human CCR8, human CD33, or human EGFR expressed on the surface of cancer cells. In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 containing the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 containing the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises CDR-L1 containing the sequence QGISSW (SEQ ID NO: 4), CDR-L2 containing the sequence GAS (SEQ ID NO: 5), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6).In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 from the VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence DYYI (SEQ ID NO: 88), CDR-H2 containing the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 containing the sequence NSGSYSFGY (SEQ ID NO: 90), and the VL domain comprises CDR-L1 containing the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 containing the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6).In some embodiments, the VH domain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7). The sequence contains; and / or the VL domain contains a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the VH domain of the first antigen-binding domain comprises the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7); and / or the VL domain of the first antigen-binding domain comprises the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).In some embodiments, the second antigen-binding domain binds to CD20; the second antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; the VH domain of the second antigen-binding domain comprises the sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24), and / or the VL domain of the second antigen-binding domain comprises the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25). In some embodiments, the multispecificity binding molecule comprises a first antibody arm comprising a first antigen-binding domain and a first Fc region, and a second antibody arm comprising a second antigen-binding domain and a second Fc region, wherein the first Fc region comprises one or more knob-forming mutations, and the second Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the first Fc region comprises a T366W substitution by EU numbering, and the second Fc region comprises T366S, L368A, and Y407V substitutions. In some embodiments, the multispecificity binding molecule comprises a first antibody arm comprising a first antigen-binding domain and a first Fc region, and a second antibody arm comprising a second antigen-binding domain and a second Fc region, wherein the first Fc region comprises one or more hole-forming mutations, and the second Fc region comprises one or more congeneral knob-forming mutations. In some embodiments, the first Fc region includes the T366S, L368A, and Y407V substitutions according to EU numbering, and the second Fc region includes the T366W substitution. In some embodiments, the antibody includes two antibody Fc regions, each of which includes one or more amino acid substitutions at positions 234, 235, and 237 according to EU numbering. In some embodiments, each of the antibody heavy chains includes the L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the antibody includes two antibody heavy chains, with only one of the antibody heavy chains including the H435R and Y436F substitutions according to EU numbering. In some embodiments, only one of the antibody arms includes a heavy chain containing the F126C and C220V substitutions according to EU numbering, and a light chain containing the S121C and C214V substitution. In some embodiments, the bispecific antibody 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 the VH domain of the second antibody heavy chain forms an antigen-binding domain with the VL domain of the second antibody light chain, and the first antibody heavy chain comprises the substitutions F126C, C220V, and T366W according to EU numbering, the first antibody light chain comprises the substitutions S121C and C214V, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R,and Y436F substitution. In some embodiments, the first and second antibody heavy chains further include L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the first and second antibody heavy chains include human IgG1 Fc domains. In some embodiments (e.g., the second antigen-binding domain binds to CD20), the antibody comprises the following three polypeptide chains: sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQP A first polypeptide chain containing EDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31),Array QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT A second polypeptide chain comprising the sequence LPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 32), and a third polypeptide chain comprising the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33). In some embodiments, at least one or two of the antibody's heavy chains are not fucosylated. In some embodiments, the antibody may be produced in cell lines having α1,6-fucosyltransferase (Fut8) or α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminetransferase (MGAT1) knockout. In some embodiments, the antibody may be produced in cell lines overexpressing β1,4-N-acetylglucosaminetransferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). In some embodiments, the antibody may be produced in cell lines overexpressing α1,6-fucosyltransferase (Fut8) or α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminetransferase (MGAT1).It can be produced in cell lines treated with a mannosidase I inhibitor, such as kifunensin. In some embodiments, the organism is human.

[0017] In some embodiments, kits or products are provided herein that include an antibody, multispecificity conjugate molecule, or composition according to any one of the embodiments described above, and instructions for using the antibody, multispecificity conjugate molecule, or composition according to any one of the embodiments described above.

[0018] It will be understood that other embodiments of the Disclosure may be formed by combining one, some, or all of the characteristics of the various embodiments described herein. These and other embodiments of the Disclosure will be apparent to those skilled in the art. These and other embodiments of the Disclosure will be further described by embodiments for carrying out subsequent inventions. [Brief explanation of the drawing]

[0019] [Figure 1A] This paper presents the binding analysis of anti-human dectin-1 antibody (clone 2M24) to human and monkey monocytes derived from peripheral blood mononuclear cells (PBMCs) by flow cytometry. To identify monocytes, a single viable CD14+ cell was gated. Cells were incubated with either the 2M24 anti-dectin-1 primary antibody or an mIgG1 isotype control antibody, followed by incubation with a fluorescent anti-mouse secondary antibody. The primary antibody was used in continuous dose titration. The binding analysis of anti-human dectin-1 clone 2M24 in human monocytes is shown. [Figure 1B] This paper presents the binding analysis of anti-human dectin-1 antibody (clone 2M24) to human and monkey monocytes derived from peripheral blood mononuclear cells (PBMCs) by flow cytometry. To identify monocytes, a single viable CD14+ cell was gated. Cells were incubated with either the 2M24 anti-dectin-1 primary antibody or an mIgG1 isotype control antibody, followed by incubation with a fluorescent anti-mouse secondary antibody. The primary antibody was used in continuous dose titration. The binding analysis of anti-human dectin-1 clone 2M24 antibody in cynomolgus monkey monocytes is also shown. [Figure 1C] Binding analysis of anti-human dectin-1 antibody (clone 2M24) with human monocytes and cynomolgus monocytes derived from peripheral blood mononuclear cells (PBMC) by flow cytometry is shown. To identify monocytes, single viable CD14+ cells were gated. Cells were incubated with 2M24 anti-dectin-1 primary antibody or mIgG1 isotype control antibody and then with fluorescent anti-mouse secondary antibody. The primary antibody was used in serial dose titrations. Comparison of binding to human monocytes, HEK cells overexpressing human dectin-1, and cynomolgus monocytes among clone 2M24 and other dectin-1 antibodies identified from immunization of ATX-Gx Alloy transgenic mice and commercially available anti-dectin-1 antibodies is shown. Anti-human dectin-1 clone 2M24 antibody showed high affinity for human dectin-1 and cynomolgus dectin-1 expressed on monocytes and showed superior affinity compared to other anti-dectin-1 antibodies including commercially available antibodies. [Figure 2A] Phagocytosis of pHrodo-labeled polystyrene anti-mouse Fc IgG beads conjugated with anti-dectin-1 antibody 2M24 or isotype control antibody by HEK-Blue h dectin-1a cells and human monocytes is shown. Polystyrene anti-mouse Fc IgG beads (approx. 3.4 μm) were labeled with a pH-sensitive fluorescent dye (pHrodo Red) and conjugated with dectin-1 antibody 2M24 or isotype control. Next, the beads were incubated with cultured HEK-Blue h dectin-1a cells or human monocytes at a ratio of 1:2 (cells:beads). HEK-Blue h dectin-1a cells were labeled with the cell-permeable dye calcein AM. Phagocytosis of the beads was monitored by IncuCyte live cell imaging. Phagocytosis was quantified using IncuCyte analysis software and represented as the overlap of the number of red objects (pHrodo) with calcein-positive cells. Phagocytosis of the beads over 2.5 hours in HEK-Blue h dectin-1a cells (top) and a representative image of pHrodo-positive cells after 2.5 hours of phagocytosis (bottom) are shown. [Figure 2B]This study demonstrates the phagocytic activity of pHrodo-labeled polystyrene anti-mouse Fc IgG beads conjugated with anti-dectin-1 antibody 2M24 or isotype control antibodies by HEK-Blue h Dectin-1a cells and human monocytes. Polystyrene anti-mouse Fc IgG beads (approximately 3.4 μm) were labeled with a pH-sensitive fluorescent dye (pHrodo Red) and conjugated with Dectin-1 antibody 2M24 or an isotype control. The beads were then incubated with cultured HEK-Blue h Dectin-1a cells or human monocytes in a 1:2 (cell:bead) ratio. HEK-Blue h Dectin-1a cells were labeled with the cell-permeable dye calcein AM. Phagocytosis of the beads was monitored by IncuCyte live-cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the overlap of red object counts (pHrodo) relative to calcein-positive cells. The images show the phagocytic activity of beads in human monocytes over 4 hours (top) and representative images of pHrodo-positive cells 2.5 hours after phagocytosis (bottom). In the representative images, the ingested beads fluoresce brightly within the phagosome. [Figure 3] This shows the binding of fully human 2M24 anti-dectin-1 antibody (hIgG4) or isotype control antibody to HEK-Blue h Dectin-1a cells and primary human monocytes. A shows the binding analysis of fully human 2M24 anti-dectin-1 antibody to HEK cells. B shows the binding to primary human monocytes. The primary antibody was used in sequential dose titration, followed by the use of a fluorescent secondary antibody against the primary antibody. The fully human 2M24 anti-dectin-1 hIgG4 antibody bound to dectin-1 expressing cells with high affinity. [Figure 4]Targeted phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated with full-length human 2M24 anti-dectin-1 antibody (hIgG4) or isotype control antibody by dectin-1-expressing cells is shown. Polystyrene biotin beads were labeled with pHrodo Red and conjugated to anti-dectin-1 antibody 2M24 or isotype control via streptavidin. The conjugated beads were mixed with cells at a ratio of 1:3, and phagocytosis of the beads was monitored by IncuCyte live cell imaging. Phagocytosis of phrodo-biotin beads conjugated to streptavidin 2M24 anti-dectin-1 hIgG4 antibody is shown for HEK-Blue h dectin-1a cells (upper left), human monocytes (upper right), and human macrophages (lower). The full-length human 2M24 anti-dectin-1 antibody (hIgG4) promoted phagocytosis of dectin-1-expressing cells. [Figure 5A] Results of a secreted alkaline phosphatase reporter assay of dectin-1 in HEK-Blue h dectin-1a cells are shown. Results of a secreted alkaline phosphatase assay performed using immobilized full-length human 2M24 anti-dectin-1 antibody are shown. Full-length human 2M24 (hIgG4) anti-dectin-1 antibody or isotype control antibody was immobilized overnight in a U-bottom polypropylene microtiter plate at a quantity of 0.1–10 μg per well, then HEK-BLUE h dectin-1a cells were cultured for 22 hours, and alkaline phosphatase secretion was evaluated by OD 630 nm in the supernatant. [Figure 5B]The results of a secreted alkaline phosphatase reporter assay of dectin-1 in HEK-Blue h dectin-1a cells are shown. The results of the secreted alkaline phosphatase assay performed using a bead-conjugated fully human 2M24 anti-dectin-1 antibody are presented. Biotin beads of sizes 3, 10, and 16.5 μm were conjugated with streptavidin 2M24(hIgG4) anti-dectin-1 antibody. The antibody-conjugated beads were mixed with HEK-Blue h dectin-1a cells for 22 hours, and the supernatant was evaluated for alkaline phosphatase secretion at OD 630 nm. Bars represent mean ± standard deviation; n=2 replicates. 2M24(hIgG4) anti-dectin-1 antibody induced alkaline phosphatase secretion in HEK-Blue h dectin-1a cells in both immobilized and bead-conjugated forms. [Figure 6A] This graph shows the cytokine secretion levels of primary human macrophages stimulated with anti-dectin-1 (15E2) antibody in solution. Primary human macrophages and primary monocytes were stimulated with 10 μg / ml of 15E2 anti-dectin-1 antibody or isotype antibody in solution for 24 hours, and TNFα and IL6 secretion levels were evaluated by ELISA analysis of the supernatant. Zymosan was used as a positive control for cytokine secretion. Bars represent the mean ± standard deviation. n=2 replicates. A shows the results for primary human monocytes stimulated with soluble 15E2 anti-dectin-1 antibody, and B shows the results for stimulated primary human macrophages. Soluble 15E2 anti-dectin-1 antibody did not induce cytokine secretion in primary human monocytes or macrophages. [Figure 6B]This graph shows the cytokine secretion levels of primary human macrophages stimulated with anti-dectin-1 (15E2) antibody in solution. Primary human macrophages and primary monocytes were stimulated with 10 μg / ml of 15E2 anti-dectin-1 antibody or isotype antibody in solution for 24 hours, and TNFα and IL6 secretion levels were evaluated by ELISA analysis of the supernatant. Zymosan was used as a positive control for cytokine secretion. Bars represent the mean ± standard deviation. n=2 replicates. A shows the results for primary human monocytes stimulated with soluble 15E2 anti-dectin-1 antibody, and B shows the results for stimulated primary human macrophages. Soluble 15E2 anti-dectin-1 antibody did not induce cytokine secretion in primary human monocytes or macrophages. [Figure 7A] This graph shows cytokine secretion levels from human primary monocytes and PBMCs stimulated with immobilized 2M24 or 15E2 anti-dectin-1 antibodies. Anti-dectin-1 antibodies or isotype control antibodies were immobilized overnight at 10 μg per well in U-bottom polypropylene microtiter plates, and human monocytes or human PBMCs were cultured for 24 hours. TNFα, IL6, and IFNg secretion levels were evaluated by ELISA analysis of the supernatant. A shows cytokine secretion levels from human monocytes after stimulation with immobilized anti-dectin-1 antibodies. B shows cytokine secretion levels from cultured human PBMCs after stimulation. Bars represent mean ± standard deviation; n=2 replicates. The 2M24 anti-dectin-1 antibody induced cytokine secretion in both primary human monocytes and PBMCs, showing superior immunostimulation compared to the 15E2 dectin-1 agonist antibody. [Figure 7B]This graph shows cytokine secretion levels from human primary monocytes and PBMCs stimulated with immobilized 2M24 or 15E2 anti-dectin-1 antibodies. Anti-dectin-1 antibodies or isotype control antibodies were immobilized overnight at 10 μg per well in U-bottom polypropylene microtiter plates, and human monocytes or human PBMCs were cultured for 24 hours. TNFα, IL6, and IFNg secretion levels were evaluated by ELISA analysis of the supernatant. A shows cytokine secretion levels from human monocytes after stimulation with immobilized anti-dectin-1 antibodies. B shows cytokine secretion levels from cultured human PBMCs after stimulation. Bars represent mean ± standard deviation; n=2 replicates. The 2M24 anti-dectin-1 antibody induced cytokine secretion in both primary human monocytes and PBMCs, showing superior immunostimulation compared to the 15E2 dectin-1 agonist antibody. [Figure 8] The results of competitive assays using the 12M4 anti-dectin-1 antibody clone and the native ligand for dectin-1 are presented. HEK-Blue h-dectin-1a cells were incubated on ice at 300 nM in the presence of 8 ug / ml biotin-laminarin with either the 2M24(hIgG4) anti-dectin-1 antibody or the 15E2, 259931, and GE2 anti-dectin-1 commercial antibodies in 1 / 3 serial dose titration. Binding of laminarin to dectin-1 was evaluated by flow cytometry using Streptavidin-Alexa fluor 647. The 2M24(hIgG4) anti-dectin-1 antibody did not compete with the native ligand for binding to dectin-1. [Figure 9] A summary of the functional characterization of 2M24 and 15E2 anti-dectin-1 antibodies is presented. [Figure 10] This diagram shows a schematic representation of the production of bispecific antibodies by click chemistry. A shows the differential labeling of antibodies with MTA or FOL reagents. B shows the covalent crosslinking of antibodies via specific MTA-FOL interactions. [Figure 11A]It exhibits potential modes of activity deployed by anti-dectin-1 agonist bispecific antibodies to eliminate target cancer cells. These include immunostimulation, phagocytosis, neoantigen presentation, and activation of T and B lymphocytes of the adaptive immune system. [Figure 11B] Here is a list of potential targets for depleting cancer cells. [Figure 12A] This document describes the characterization of click chemistry conjugate bispecific antibodies containing anti-dectin-1 (clone 2M24) and anti-hCD70 arms. It also presents SDS-PAGE analysis of covalent antibody pairs (2M24 / anti-hCD20, 2M24 / anti-hCD70, and isotype controls) under non-reducing and reducing conditions. [Figure 12B] This document describes the characterization of click chemistry conjugate-type bispecific compounds containing anti-dectin-1 (clone 2M24) and anti-hCD70 arms. Flow cytometry-based characterization of bispecific compound binding (2M24 / anti-hCD70 or isotype control) to dectin-1 expressing HEK293 cells (top left) and two renal cancer cell lines -A498 (top right) and 786-0 (bottom left) is shown. EC50 concentrations (nM) based on nonlinear regression fitting are also shown (bottom right). The anti-dectin-1 / anti-hCD70 bispecific compounds bind to dectin-1 or CD70 expressing cells with affinities of 1.8 nM and 12.34 nM, respectively. [Figure 13]This shows the ligation of Dectin-1-expressing HEK293 cell lines with A498 kidney cancer cell lines induced by the 2M24 / anti-hCD70 bispecific antibody. The left image shows flow cytometry analysis of co-cultures of HEK293 cells (labeled with calcein green) and A498 cells (labeled with calcein red) in the presence of the 2M24 / anti-hCD70 bispecific antibody or isotype control. Ligation between HEK293 and A498 cells is indicated by a double-positive signal (green + red +, square box). The right image also shows quantified ligation efficiency as the percentage of all target cells (A498) that form a doublet with HEK293 cells. Bars represent mean ± standard deviation; n=3 replicates. The 2M24 / anti-hCD70 bispecific antibody induced ligation between Dectin-1-expressing HEK293 cell lines and A498 kidney cancer cell lines. [Figure 14A] This shows the linkage between Dectin-1-expressing cells and B cells induced by an anti-Dectin-1 / anti-hCD20 bispecific antibody. It also shows the linkage between Dectin-1-expressing HEK293 cells and B cells induced by an anti-Dectin-1 / anti-hCD20 bispecific antibody. The left image shows flow cytometry analysis of co-cultures of HEK293 cells (labeled with calcein green) and Raji cells (labeled with calcein red) in the presence of a 2M24 / anti-hCD70 bispecific agent or isotype control. Linkage between HEK293 and Raji cells is indicated by a double-positive signal (green + red +; square box). The right image also shows the linkage efficiency quantified as the percentage of all target cells (Raji) forming a doublet with HEK293 cells. Bars represent mean ± standard deviation; n=2 replicates. [Figure 14B] This shows the syncretism between Dectin-1-expressing cells and B cells induced by an anti-Dectin-1 / anti-hCD20 bispecific antibody. The results of a similar experiment conducted to evaluate the syncretism between human M0 macrophages and Raji cells induced by an anti-Dectin-1 / anti-hCD20 bispecific antibody are also shown. Bars represent mean ± standard deviation; n=2 replicates. The 2M24 / anti-hCD20 bispecific antibody induced syncretism between Dectin-1-expressing cells and CDC20-positive B cells (Raji cells). [Figure 15]This report presents the results of a Dectin-1-mediated secreted alkaline phosphatase reporter assay in HEK-Blue h Dectin-1a cells using an anti-Dectin-1 / anti-CD20 bispecific antibody in the presence of Raji cells. Raji cells were incubated with 2M24(hIgG4) / anti-CD20 bispecific antibody, followed by two washes to remove unbound bispecific antibody. Raji cells were then mixed with HEK-Blue h Dectin-1a cells in a ratio of 200,000 Raji cells to 100,000 HEK cells for 22 hours. Secreted alkaline phosphatase was evaluated in the supernatant at OD 630 nm. Bars represent mean ± standard deviation; n=2 replicates. Raji cells coated with the anti-Dectin-1 / anti-CD20 bispecific antibody induced alkaline phosphatase secretion in HEK-Blue h Dectin-1a cells. [Figure 16] This image shows the induction of phagocytosis of Raji cells by Dectin-1-expressing HEK293 cells using an anti-dectin-1 / anti-hCD20 bispecific antibody. Representative incucyte images showing phagocytosis of Raji cells by HEK cells at 16 hours and 0 hours (arrows) are shown (left). Co-localization is indicated by yellow fluorescence. The decrease in calcein red signal of Raji cells at 16 hours indicates cell death due to phagocytosis. The image on the right shows quantification of overlap or co-localization of HEK (calcein green) and Raji (calcein red) in different treatment groups. Pre-incubation of HEK cells with the ADCP inhibitor latrunclin A inhibits phagocytosis mediated by the 15E2 / anti-hCD20 bispecific antibody (n=2 replicates). [Figure 17]This shows the linkage between Dectin-1-expressing cells and HER2-expressing cells induced by an anti-Dectin-1 / anti-hHER2 bispecific antibody. Flow cytometry analysis of co-cultures of Dectin-1-expressing HEK293 cells (labeled with calcein green) and HER2-expressing SKBR3 cells (labeled with pHrodo red) in the presence of a 15E2 / anti-hHER2 bispecific antibody or isotype control is shown (left). Linkage between HEK293 and SKBR3 cells is indicated by a double-positive signal (green + red +; square box). Linkage efficiency, quantified as the percentage of all target cells (SKBR3) that form a doublet with Dectin-1-expressing cells, is also shown (right). Bars represent mean ± standard deviation; n=2 replicates. The anti-Dectin-1 / anti-hHER2 bispecific antibody induces linkage between Dectin-1 and HER2-positive cancer cells. [Figure 18] This shows the ligation of Dectin-1-expressing HEK293 cells and CD94-expressing BaF3 cells induced by the anti-Dectin-1 / anti-hCD94 bispecificity. Flow cytometry analysis of co-cultures of HEK293 cells (labeled with calcein green) and BaF3 cells (labeled with pHrodo red) in the presence of 2M24 / anti-hCD94 bispecificity or isotype control is shown (left). Ligation between HEK293 and BaF3 cells is indicated by a double-positive signal (green + red +; square box). Ligation efficiency, quantified as the percentage of all target cells (BaF3) forming a doublet with HEK293 cells, is also shown (right). Bars represent mean ± standard deviation; n=2 replicates. The anti-Dectin-1 / anti-hCD94 bispecificity induced ligation between Dectin-1-expressing cells and CD94-expressing cells. [Figure 19]A schematic diagram of Fab 2M24-mSA or full-length 2M24-mSA bound to a biotinylated target antibody is shown. A shows a chimeric fusion of monomeric streptavidin (mSA) and Fab 2M24 or full-length 2M24. The mSA is fused to either Fab 2M24 or full-length 2M24. B shows the ligation of Fab 2M24-mSA or 2M24-mSA to a biotinylated target antibody. The chimeric fusion was incubated with a biotinylated target antibody to create a bispecific compound containing a dectin-1 binding arm and a second arm that binds to the target receptor or the protein of interest. [Figure 20] This document describes the biochemical and functional characterization of the Fab 2M24-mSA fusion protein. A shows the HPLC characterization of recombinant Fab 2M24-mSA. B shows the SDS-PAGE analysis of purified Fab 2M24-mSA under reducing conditions. C shows the flow cytometry characterization of Fab 2M24-mSA (EC50 = 1.45 nM) bound to HEK293 cells stably overexpressing human dectin-1. The Fab 2M24 fusion to monomeric streptavidin bound to dectin-1 expressing cells with an affinity of 1.45 nM. [Figure 21A] This study demonstrates the phagocytic activity of pHrodo-labeled polystyrene biotin beads conjugated with monomeric streptavidin-tagged Fab-2M24 anti-dectin-1 antibody (Fab-2M24-mSA). Flow cytometry evaluation shows duplet formation between HEK-Blue h dectin-1a cells and Fab-2M24-mSA conjugated to biotin beads, as well as the phagocytic activity of the beads. [Figure 21B]This image demonstrates the phagocytic activity of pHrodo-labeled polystyrene biotin beads conjugated with monomeric streptavidin-tagged Fab-2M24 anti-dectin-1 antibody (Fab-2M24-mSA). The image shows the phagocytic activity of pHrodo-labeled biotin beads (approximately 3 μm) conjugated to Fab-2M24-mSA (top) and a representative image of pHrodo-positive cells (incorporated beads emitting bright red fluorescence in phagosomes) versus a control without beads, evaluated by IncuCyte live imaging (bottom). The Fab 2M24-mSA fusion induced bead binding and phagocytosis by dectin-1-expressing HEK293 cells. [Figure 22] Panels A-D show bispecific complexes containing Fab 2M24-mSA and targeted biotinylated antibodies. HPLC analyses of Fab 2M24-mSA complexed with biotinylated anti-hCD20 (A), biotinylated anti-hCD19 (B), biotinylated anti-hCD70 (C), or biotinylated anti-amyloid β1-42 (D) are shown. The panel includes Fab 2M24-mSA alone, the targeted biotinylated antibody alone, and an overlay of A280 traces containing Fab 2M24-mSA complexed with the biotinylated target antibody. [Figure 23] This shows the ligation of Dectin-1-expressing HEK293 cells and CD20-expressing Raji cells induced by the Fab 2M24-mSA / biotin anti-hCD20 bispecific antibody. Flow cytometry analysis of co-cultures of HEK293 cells (labeled with calcein green) and Raji cells (labeled with calcein red) in the presence of the Fab 2M24-mSA / biotin anti-hCD20 bispecific agent or isotype bispecific control is shown (left). Co-cultures were incubated at 4°C or 37°C. Ligation between HEK293 and Raji cells is indicated by a bipositive signal (green + red + dotted square). Ligation efficiency, quantified as the percentage of all target cells (Raji) forming a doublet, is also shown (right). Bars represent mean ± standard deviation; n=4 replicates. Fab 2M24-mSA / biotin anti-hCD20 bispecificity induced ligation between dectin-1 expressing HEK293 cells and Raji cells. [Figure 24] This is a schematic diagram illustrating the targeted phagocytic activity of amyloid deposits in amyloidosis using a dectin-1 agonist bispecific antibody. [Figure 25] This paper presents a strategy for targeted depletion of mast cells using a dectin-1 agonist bispecific antibody. A is a schematic diagram of mast cell depletion by a dectin-1 agonist bispecific antibody. B shows a list of possible targets for mast cell depletion. [Figure 26] This image demonstrates the phagocytic activity of large (approximately 16.2 μm) Phrodo-labeled beads by human dendritic cells. The left image shows quantification of bead phagocytosis over 12 hours, and the right image shows representative images of Phrodo-positive cells 3 hours after phagocytosis (the ingested beads fluoresce bright red in phagosomes). Dectin-1 antibody enhanced the targeted phagocytosis of beads in cultured monocyte-derived dendritic cells. [Figure 27] This is a schematic diagram of targeted depletion of microorganisms using dectin-1 agonist bispecific antibodies. Bispecific antibodies with a dectin-1 binding arm and a microbial agent binding arm were constructed to target bacteria, viruses, or fungi (top). The dectin-1 bispecific antibodies deployed dectin-1 expressing phagocytic cells and eliminated the bacterial, viral, or fungal pathogens (bottom). [Figure 28A] This paper demonstrates the binding of a bispecific antibody consisting of a dectin-1 antibody (15E2 clone) conjugated to an anti-H3N2 hemagglutinin antibody (12CA5 clone) to H3N2 influenza virus and to dectin-1 expressing cells. The binding analysis of the anti-dectin-1 / anti-hemagglutinin bispecific antibody to H3N2 influenza virus, evaluated by ELISA, is also shown. 96-well microtiter plates were coated with H3N2 influenza virus particles, and then single antibodies, bispecific antibodies, and isotype controls were incubated. After thorough washing, the primary antibody was detected using a secondary anti-mouse FcgR HRP antibody. [Figure 28B]This paper demonstrates the binding of a bispecific antibody consisting of a dectin-1 antibody (15E2 clone) conjugated to an anti-H3N2 hemagglutinin antibody (12CA5 clone) to both the H3N2 influenza virus and dectin-1 expressing cells. Flow cytometry analysis of the binding of the anti-dectin-1 / anti-hemagglutinin bispecific antibody to dectin-1 expressing HEK cells is also shown. After incubation of HEK cells with the primary antibody, the anti-dectin-1 antibody (anti-mIgG2a APC) or hemagglutinin antibody (anti-mIgG2b PB) was detected using a secondary fluorescent antibody. The anti-dectin-1 / anti-hemagglutinin bispecific antibody efficiently bound to both the H3N2 influenza virus and dectin-1 expressing HEK cells. [Figure 29A] A schematic diagram shows the use of anti-dectin-1 antibodies to deliver antigens for vaccine production, illustrating two scenarios: (A) using anti-dectin-1 antibodies fused to the target antigen for delivery to the APC, and (B) using anti-dectin-1 bispecific antibodies to target and deliver disease-causing substances (e.g., cells, microorganisms, proteins, etc.) to the APC. [Figure 29B] A schematic diagram shows the use of anti-dectin-1 antibodies to deliver antigens for vaccine production, illustrating two scenarios: (A) using anti-dectin-1 antibodies fused to the target antigen for delivery to the APC, and (B) using anti-dectin-1 bispecific antibodies to target and deliver disease-causing substances (e.g., cells, microorganisms, proteins, etc.) to the APC. [Figure 30]This study demonstrates the phagocytic activity of human dendritic cells towards pHrodo-labeled polystyrene anti-mouse Fc IgG beads (approximately 3.4 μm) conjugated with Dectin-1 antibody (15E2) or an isotype control antibody. Polystyrene anti-mouse Fc IgG beads were labeled with a pH-sensitive fluorescent dye (pHrodo Red) and conjugated with Dectin-1 antibody or an isotype control. The beads were then incubated with cultured monocyte-derived dendritic cells in a 1:3 (cell:bead) ratio. Phagocytosis of the beads was monitored using IncuCyte live-cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the total cumulative intensity (sum fluorescence intensity) of red objects (pHrodo) in the image. Figure 30 shows the quantification of bead phagocytosis over 9 hours (top) and a representative image of pHrodo-positive cells 3 hours after phagocytosis (incorporated beads emitting bright red fluorescence in phagosomes; bottom). [Figure 31A] This shows the phagocytic activity of Dectin-1-expressing HEK293 cells towards SARS-CoV-2 spike protein-coated beads. The diagram is a schematic of the experiment. Beads coated with SARS-CoV-2-derived spike protein were ligated to Dectin-1-expressing HEK293 cells using an anti-Dectin-1 bispecific antibody containing Dectin-1 protein-binding arms and spike protein-binding arms. [Figure 31B] This study demonstrates the phagocytic activity of dectin-1-expressing HEK293 cells towards SARS-CoV-2 spike protein-coated beads. It also shows flow cytometry characterization of the engagement between the effector (HEK293 cells) and target (spike-coated beads) using bispecific and isotype controls (Panel A), and quantification of coupling efficiency based on the doublet population (Panel B). [Figure 31C]This shows the phagocytic activity of SARS-CoV-2 spike protein-coated beads by Dectin-1-expressing HEK293 cells. It also demonstrates the phagocytic activity of SARS-CoV-2 spike protein-coated beads by HEK 293 cells in a co-culture experiment. The phagocytic activity of pHrodo-labeled beads was monitored by changes in pHrodo fluorescence resulting from the acidic pH within the phagosome. The quantification of phagocytosis, expressed as the number of red overlapping objects (pHrodo) relative to calcein-positive cells (left), and a representative image of pHrodo-positive cells 2 hours after phagocytosis (right) are shown (incorporated beads emitting bright red fluorescence within the phagosome). [Figure 32A] This document describes the design of a bispecific antibody for human dectin-1 and a disease target or antigen (e.g., a human IgG1 bispecific antibody). A schematic diagram of the design is provided. One arm (2M24A.X) having VH domain A and VL domain B targets human dectin-1, while the other arm (2M24B.X) having VH domain C and VL domain D targets the disease target or antigen. [Figure 32B] This paper presents the design of bispecific antibodies (e.g., human IgG1 bispecific antibodies) targeting dectin-1 and disease targets or antigens. It provides a schematic diagram of the exemplary mechanism of action of anti-dectin-1 bispecific antibodies that target h-dectin-1 on bone marrow cells (via the first arm), antigens on target cells / disease causative agents (via the second arm), and Fc receptors on bone marrow cells and NK cells, and that have an active Fc domain that induces robust immune stimulation and phagocytosis. [Figure 33A]This shows that a bispecific antibody, with one arm targeting hdectin-1 and the other arm targeting hCD20 (using the variable domain of rituximab), binds to cells expressing human dectin-1 or human CD20. The upper panel shows the binding of a bispecific antibody targeting both hdectin-1 and hCD20 (2M24 / CD20) or a bispecific antibody targeting both hdectin-1 and RSV (2M24 / RSV) to HEK293 cells stably expressing human dectin-1, as evaluated by flow cytometry. The lower panel shows the binding of the bispecific antibody 2M24 / RSV hIgG1-FITC conjugate and 2M24 bivalent hIgG1-FITC conjugate to PBMCs, as evaluated by flow cytometry. [Figure 33B] This study demonstrates that a bispecific antibody, with one arm targeting hdectin-1 and the other arm targeting hCD20 (using the variable domain of rituximab), binds to cells expressing either human dectin-1 or human CD20. It also shows the binding of rituximab (human IgG1), 2M24 / CD20 with active human IgG1 Fc, 2M24 / CD20 with inactive human IgG1 Fc, 2M24 / RSV with active human IgG1 Fc, or 2M24 / RSV with inactive human IgG1 Fc to the CD20-expressing B-cell lymphoma Raji cell line. [Figure 34A] This study demonstrates that bispecific antibodies targeting hDectin-1 and hCD20 (2M24 / CD20) induce ligation between Dectin-1 and CD20-expressing cells. To evaluate the ligation between Dectin-1-expressing HEK293 cells (effector) and CD20-expressing Raji cells (target), cells were differentially labeled with either calcein green (effector) or calcein red (target). Labeled cells were co-cultured and treated with bispecific antibodies against inactive hIgG1 2M24 / CD20 or 2M24 / RSV (control) to induce effector:target ligation. Successful ligation of effector:target cells was indicated by double positive staining (calcein green+, calcein red+, square box). [Figure 34B]This study demonstrates that bispecific antibodies targeting h-dectin-1 and hCD20 (2M24 / CD20) induce ligation between dectin-1 and CD20-expressing cells. Effector cells are shown as dose titration of the bispecific antibodies in co-cultures of target cells. Ligation efficiency was quantified as the percentage of all target cells that bind to or ligate to effector cells. [Figure 35A] This study demonstrates that bispecific antibodies targeting hdectin-1 and hCD20 (2M24 / CD20) with active hIgG1 Fc do not induce monocyte depletion via antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP). PBMCs from two healthy donors, donor 76(A) and donor 77(B), were treated for 24 hours with increasing concentrations of 2M24 / CD20 bispecific antibody (active or inactive hIgG1 isotype) and rituximab, followed by flow cytometry analysis to quantify the level of remaining viable CD14+ monocytes (as a percentage relative to isotype control). [Figure 35B] This study demonstrates that bispecific antibodies targeting hdectin-1 and hCD20 (2M24 / CD20) with active hIgG1 Fc do not induce monocyte depletion via antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP). PBMCs from two healthy donors, donor 76(A) and donor 77(B), were treated for 24 hours with increasing concentrations of 2M24 / CD20 bispecific antibody (active or inactive hIgG1 isotype) and rituximab, followed by flow cytometry analysis to quantify the level of remaining viable CD14+ monocytes (as a percentage relative to isotype control). [Figure 36A] This study demonstrates that a bispecific antibody possessing active hIgG1 Fc, targeting hdectin-1 and hCD20 (2M24 / CD20), induces superior B cell depletion compared to rituximab. PBMCs from two healthy donors, donor 83(A) and donor 84(B), were treated with the indicated antibody at increasing concentrations for 24 hours, followed by flow cytometry analysis to quantify the levels of remaining viable CD19+ B cells (reported as a percentage of B cells in PBMCs treated with isotype control). [Figure 36B] This study demonstrates that a bispecific antibody possessing active hIgG1 Fc, targeting hdectin-1 and hCD20 (2M24 / CD20), induces superior B cell depletion compared to rituximab. PBMCs from two healthy donors, donor 83(A) and donor 84(B), were treated with the indicated antibody at increasing concentrations for 24 hours, followed by flow cytometry analysis to quantify the levels of remaining viable CD19+ B cells (reported as a percentage of B cells in PBMCs treated with isotype control). [Figure 37A] This study demonstrates that rituximab induces higher B cell shaving (CD19 downregulation) compared to a 2M24 / CD20 active IgG1 bispecific antibody. CD19+ expression on B cells from two healthy donors, donor 83(A) and donor 84(B), was quantified by flow cytometry after 24-hour incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab, or isotype control. The effects of 2M24 / CD20 bispecificity and rituximab on CD19 expression on B cells were evaluated using mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugate). EC50 values ​​were calculated based on nonlinear regression analysis. [Figure 37B] This study demonstrates that rituximab induces higher B cell shaving (CD19 downregulation) compared to a 2M24 / CD20 active IgG1 bispecific antibody. CD19+ expression on B cells from two healthy donors, donor 83(A) and donor 84(B), was quantified by flow cytometry after 24-hour incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab, or isotype control. The effects of 2M24 / CD20 bispecificity and rituximab on CD19 expression on B cells were evaluated using mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugate). EC50 values ​​were calculated based on nonlinear regression analysis. [Figure 38] This study shows the difference in cytokine release induced by a 2M24 / CD20 active hIgG1 bispecific antibody compared to rituximab. ELISA-based (mesoscale discovery) quantification of cytokines was performed on the supernatants isolated from healthy donor PBMCs treated with 2M24 / CD20 active hIgG1 bispecific, rituximab, or isotype control. PBMCs were stimulated with the antibody overnight, and the supernatant was subsequently analyzed in MSD. The cytokines tested were IFNγ, IL-12p70, IL-6, TNFα, IL-1β, IL-4, IL-13, IL-10, and IL-8. Each plot shows cytokine secretion (pg / mL) as a function of the antibody used for treatment (from left to right: 2M24 / CD20 hIgG1 bispecific, 2M24 / RSV hIgG1 bispecific, rituximab hIgG1, and isotype control hIgG1). [Figure 39A] This study demonstrates that a bispecific antibody against 2M24 / CD20 hIgG1 (active isotype) induces superior B cell depletion and lower CD19 shaving compared to rituximab in co-cultures of human macrophages and GFP-expressing Raji B cells. Flow cytometry analysis of co-cultures of human macrophages and Raji-GFP cells (3:1 ratio) was performed in the presence of 2M24 / CD20 hIgG1 (active isotype) bispecificity, 2M24 / RSV control, fucosylated rituximab, or isotype hIgG1 control. Co-cultures were incubated at 37°C for 24 hours, then stained with PE anti-CD206 antibody to label macrophages and BV-605 anti-CD19 antibody to label Raji cells. The number of remaining viable / Raji-GFP+ cells was assessed at the end of the experiment. Primary antibodies were used in continuous dose titration. [Figure 39B]This study demonstrates that a 2M24 / CD20 hIgG1 (active isotype) bispecific antibody induces superior B cell depletion and lower CD19 shaving compared to rituximab in co-cultures of human macrophages and GFP-expressing Raji B cells. CD19 levels in Raji-GFP cells were assessed after 24 hours. B cell receptor shaving is indicated by a decrease in CD19 MFI in the presence of the anti-dectin-1 / anti-hCD20 bispecific antibody or rituximab. [Figure 40A] This study demonstrates that a 2M24 / CD20 active IgG1 bispecific antibody significantly induces tissue B cell depletion compared to rituximab in single-cell suspensions from renal cancer specimens. Single-cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of a 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, a 2M24 / RSV hIgG1 control, fucosylated rituximab, and their respective isotype controls. Renal cancer tissue specimens were dissociated into single-cell suspensions and treated with primary antibody (2 μg / ml) at 37°C for 24 hours. The immune cell population was analyzed by flow cytometry. Cells were first gated for viable cells, then separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells), and then CD19+ (B cells) and CD3+ (T cells) cells were identified within the CD45+ population (A and B). The number of remaining B cells was evaluated using an anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 40B]This study demonstrates that a 2M24 / CD20 active IgG1 bispecific antibody significantly induces tissue B cell depletion compared to rituximab in single-cell suspensions from renal cancer specimens. Single-cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of a 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, a 2M24 / RSV hIgG1 control, fucosylated rituximab, and their respective isotype controls. Renal cancer tissue specimens were dissociated into single-cell suspensions and treated with primary antibody (2 μg / ml) at 37°C for 24 hours. The immune cell population was analyzed by flow cytometry. Cells were first gated for viable cells, then separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells), and then CD19+ (B cells) and CD3+ (T cells) cells were identified within the CD45+ population (A and B). The number of remaining B cells was evaluated using an anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 40C] This study demonstrates that a 2M24 / CD20 active IgG1 bispecific antibody significantly induces tissue B cell depletion compared to rituximab in single-cell suspensions from renal cancer specimens. Single-cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of a 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, a 2M24 / RSV hIgG1 control, fucosylated rituximab, and their respective isotype controls. Renal cancer tissue specimens were dissociated into single-cell suspensions and treated with primary antibody (2 μg / ml) at 37°C for 24 hours. The immune cell population was analyzed by flow cytometry. Cells were first gated for viable cells, then separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells), and then CD19+ (B cells) and CD3+ (T cells) cells were identified within the CD45+ population (A and B). The number of remaining B cells was evaluated using an anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 41]Figures A-C demonstrate that the anti-dectin-1 antibody (clone 2M24) induces dectin-1 clustering and TNFα secretion from human macrophages. Cytokine secretion was tested using cultured macrophages stimulated with immobilized anti-dectin-1 antibody (clone 2M24) or 2M24 / CD20 bispecific antibody, as well as single-cell suspensions from renal cancer specimens. Anti-dectin-1 antibody (clone 2M24), isotype control, or 2M24 / CD20 bispecific antibody was immobilized overnight at 10 μg per well in U-bottom polypropylene microtiter plates, followed by the cultivation of human monocyte-derived macrophages (A and B) or single-cell suspensions from renal cancer specimens (C). Cells were cultured for 24 hours, and TNFα secretion in the supernatant was evaluated by ELISA. As a positive control, cells were stimulated with zymosan. [Figure 42] This study demonstrates that immobilized anti-dectin-1 antibody (clone 2M24) promotes immunostimulation in single-cell suspensions from kidney cancer specimens. Single-cell suspensions from kidney cancer specimens were treated with either immobilized anti-dectin-1 antibody (clone 2M24) or isotype-controlled hIgG4 antibody for 24 hours. The supernatant was analyzed by ELISA for the release of various cytokines, including IFNγ, IL-6, TNFα, IL-23, IL-12p70, IL-10, and IL-13. Each plot shows the amount of cytokine (pg / mL) as a function of antibody treatment. Results are shown for treatment with anti-dectin-1 antibody (clone 2M24) or isotype-controlled hIgG4 antibody using kidney cancer donor 3 (left) or donor 4 (right). [Figure 43] This study demonstrates the effect of a 2M24 / CD20 bispecific antibody on CD16 expression in human NK cells compared to rituximab or an isotype control (RSV). The results show that CD16 antigen levels on NK cells are better maintained in PBMCs treated with the 2M24 / CD20 bispecific antibody compared to rituximab. [Figure 44]This study demonstrates the effect of a 2M24 / CD20 bispecific antibody on CD19 expression in human B cells compared to rituximab or an isotype control (2M24 / RSV bispecific antibody). The results show that CD19 antigen levels are better maintained in B cells treated with the 2M24 / CD20 bispecific antibody compared to rituximab. [Figure 45] This study demonstrates human B cell depletion by either a rituximab-derived or obinutuzumab-derived 2M24 / CD20 bispecific antibody. The results indicate that the 2M24 / CD20 bispecific antibody derived from the rituximab arm is superior to the bispecific antibody derived from obinutuzumab in depleting B cells. [Figure 46] This paper describes the design of an exploratory study on the safety and efficacy of a 2M24 / CD20 bispecific antibody in non-human primates. [Figure 47] This image shows the depletion of circulating B cells in cynomolgus monkeys using a 2M24 / CD20 hIgG1 bispecific antibody produced from cells treated with kifunensin (KIF). It also shows B cell depletion in monkeys treated with 5 mg / kg of 2M24 / CD20 hIgG1 KIF (top) or inactive 2M24 / CD20 hIgG1 (bottom). [Figure 48] This shows the depletion of circulating B cells in cynomolgus monkeys using a 2M24 / CD20 hIgG1 bispecific antibody produced from cells treated with kifunensin (KIF). This shows the depletion of B cells in monkeys treated with 5 mg / kg rituximab hIgG1 KIF. [Figure 49A] This shows the depletion of tissue-resident B cells in cynomolgus monkeys using a 2M24 / CD20 hIgG1 bispecific antibody produced from cells treated with kifunensin (KIF). This shows the depletion of B cells in the bone marrow of monkeys treated with 5 mg / kg of 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF. [Figure 49B]This shows the depletion of tissue-resident B cells in cynomolgus monkeys using a 2M24 / CD20 hIgG1 bispecific antibody produced from cells treated with kifunensin (KIF). This shows the depletion of B cells in the lymph nodes of monkeys treated with 5 mg / kg of 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF. [Figure 50] This demonstrates B cell depletion from cynomolgus monkey PBMCs in ex vivo conditions. [Figure 51] Using knob-into-hole technology, we present the format of a bispecific molecule (2M24 scFv / CD20) paired with a conventional anti-CD20 half-antibody and an anti-dectin-1 single-strand variable fragment (scFv) Fc fusion arm. H represents the 2M24 VH domain, and L represents the 2M24 VL domain. [Figure 52A] This document describes the purification and functional characterization of the 2M24 / CD20 bispecific antibody. It also shows the molecular purification by size exclusion chromatography (SEC). [Figure 52B] This report describes the purification and functional characterization of the 2M24 / CD20 bispecific antibody. It demonstrates that the purified bispecific antibody promoted targeted immunostimulation, as evaluated by the NFκB reporter assay. [Figure 52C] This document describes the purification and functional characterization of the 2M24 / CD20 bispecific antibody. It also demonstrates the depletion of human B cells by the 2M24 scFv / CD20 bispecific antibody. [Figure 53A] This document describes the preparation and characterization of anti-dectin-1 (2M24) / anti-Trop-2 bispecific antibodies. The purification of the 2M24 / Trop-2 bispecific antibody by SEC is shown (left). The purified antibody was analyzed by SDS-PAGE under non-reducing (NR) or reducing (R) conditions (right). [Figure 53B] This report describes the production and characterization of a bispecific anti-dectin-1 (2M24) / anti-Trop-2 antibody. It demonstrates high affinity binding of the antibody to dectin-1-expressing HEK cells. [Figure 53C] This report describes the production and characterization of a bispecific anti-dectin-1 (2M24) / anti-Trop-2 antibody. It exhibits moderate affinity binding to the Trop-2 expressing A431 cancer cell line. [Figure 54] This shows the expression level of Trop-2 in cancer cells. [Figure 55A] This shows the binding of the 2M24 / Trop-2 bispecific antibody to the Trop-2 expressing cell line HeLa. The binding EC50 for each cell line is shown, calculated using 4-parameter logistic (4PL) nonlinear regression. [Figure 55B] This shows the binding of the 2M24 / Trop-2 bispecific antibody to BxPC-3. The binding EC50 for each cell line is calculated using 4-parameter logistic (4PL) nonlinear regression. [Figure 55C] This shows the binding of the 2M24 / Trop-2 bispecific antibody to SiHa. The binding EC50 for each cell line is calculated using 4-parameter logistic (4PL) nonlinear regression. [Figure 55D] This shows the binding of the 2M24 / Trop-2 bispecific antibody to Capan-2. The binding EC50, calculated for each cell line using 4-parameter logistic (4PL) nonlinear regression, is also shown. [Figure 56A] This shows the depletion of Trop-2 expressing cell lines (SKBR3 cells) using a 2M24 / Trop-2 bispecific antibody. [Figure 56B] This shows the depletion of the Trop-2 expressing cell line (A431 cells) using a 2M24 / Trop-2 bispecific antibody. [Figure 57A] This shows the expression of Trop-2 and Dectin-1 in lung cancer specimens. [Figure 57B] This shows the expression of Trop-2 and Dectin-1 in lung cancer specimens. [Figure 58] This shows the depletion of Trop-2-positive cancer cells in lung cancer samples. [Figure 59A] This demonstrates the activity of the 2M24 / Trop-2 bispecific antibody in the NFκB reporter assay. [Figure 59B] This study demonstrates that the 2M24 / Trop-2 bispecific antibody promotes antigen presentation and T cell activation. A schematic diagram of the assay setup is provided. [Figure 59C]This study demonstrates that a 2M24 / Trop-2 bispecific antibody promotes antigen presentation and T cell activation. Macrophages and SKBR3 breast cancer cells were co-incubated in the presence of either a 2M24 / Trop-2 hIgG1 or control 2M24 / RSV hIgG1 bispecific antibody. Phagocytosis or depletion of SKBR3 cells was evaluated by flow cytometry by staining EPCAM expression on SKBR3 cells. Data were reported in comparison to the control bispecific 2M24 / RSV. [Figure 59D] This study demonstrates that the 2M24 / Trop-2 bispecific antibody promotes antigen presentation and T cell activation. IFNγ levels in the supernatant were quantified using the BD OptiEIA kit. [Figure 59E] This study demonstrates that the 2M24 / Trop-2 bispecific antibody promotes antigen presentation and T cell activation. The expression of CD69, an early activation marker on T cells, was evaluated by flow cytometry. The data are reported in comparison to those of all CD3+ T cells. [Figure 60A] This document describes the design and production of a 2M24 / nectin-4 bispecific antibody. A diagram of the bispecific molecule is also shown. [Figure 60B] This document describes the design and production of a 2M24 / nectin-4 bispecific antibody. It also shows the purification of the bispecific antibody using protein A chromatography. [Figure 61A] This shows nectin-4 expression in cancer cell lines. [Figure 61B] This shows nectin-4 expression in cancer cells from primary tumor samples. [Figure 62] This shows the binding of the 2M24 / nectin-4 bispecific antibody to dectin-1 expressing HEK cells (top) or nectin-4 expressing A431 cells (bottom). [Figure 63] This shows the stimulation of Dectin-1 in an NFκB reporter assay using a 2M24 / Nectin-4 bispecific antibody. The upper panel shows the assay diagram. The lower panel shows the quantified results based on SEAP levels in the culture medium. [Figure 64A]Figure 64A shows the depletion of nectin-4 expressing cancer cells by a 2M24 / nectin-4 bispecific antibody. Flow cytometry detection of phagocytosis / depletion is also shown. [Figure 64B] Figure 64B shows the depletion of nectin-4 expressing cancer cells by a 2M24 / nectin-4 bispecific antibody. Figure 64B shows the depletion compared to the RSV control. [Figure 65A] This shows that the 2M24 / 11-1F4 bispecific antibody binds to light chain amyloid. The purification of the parental anti-amyloid antibody 11-1F4 (top) and the 2M24 / 11-1F4 bispecific antibody (bottom) by SEC is also shown. [Figure 65B] This shows that the 2M24 / 11-1F4 bispecific antibody binds to light chain amyloid. Octet demonstrates the binding of either the 11-1F4 parent antibody (B) or the 2M24 / 11-1F4 bispecific antibody (C) to recombinant light chain amyloid derived from different patients (AL30, AL47, AL48, and AL55). [Figure 65C] This shows that the 2M24 / 11-1F4 bispecific antibody binds to light chain amyloid. Octet demonstrates the binding of either the 11-1F4 parent antibody (B) or the 2M24 / 11-1F4 bispecific antibody (C) to recombinant light chain amyloid derived from different patients (AL30, AL47, AL48, and AL55). [Figure 66] It shows phagocytic activity of light chain amyloid fibers by monocytes. [Modes for carrying out the invention]

[0020] Several embodiments are described below with reference to illustrative applications for illustrative purposes. It should be understood that many specific details, relationships, and methods are described herein in order to provide a complete understanding of the features described herein. However, those skilled in the art will readily recognize that the features described herein can be carried out by other methods without one or more specific details. Since some actions can occur in different orders and / or simultaneously with other actions or events, the features described herein are not limited to the order of the illustrated actions or events. Furthermore, not all illustrated actions or events are required to carry out a method according to the features described herein.

[0021] As used herein, the singular forms "a," "an," and "the" are intended to include the plural form unless otherwise clearly indicated by the context. Furthermore, to the extent that the terms "including," "includes," "having," "has," and "with," or their variations thereof, are used in any form for carrying out the invention and / or in the claims, such terms are intended to be comprehensive in the same manner as the term "comprising." As used herein, the term "comprising" is synonymous with "including" or "containing," and is comprehensive or non-restrictive.

[0022] In this specification, references to “or” are intended to include “and / or” unless otherwise specified. Where used herein, the term “approximately” with respect to a number means a number that is +10% or -10% of that number. With respect to a range, the term “approximately” means the range minus 10% of its minimum value plus 10% of its maximum value.

[0023] I. Antibodies and multispecificity-binding proteins In certain embodiments, the disclosure provides antigen-binding domains, antibodies, and antibody fragments that bind to human dectin-1, as well as multispecific (e.g., bispecific) binding molecules containing the same.

[0024] In some embodiments, antibodies and immunoglobulins are used interchangeably and, in the broadest sense herein, encompass 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 (described in more detail herein), as long as they exhibit the desired antigen-binding activity.

[0025] In some embodiments, an antibody (immunoglobulin) refers to a protein having a structure substantially similar to that of a naturally occurring antibody, or a protein having heavy chains and light chain variable regions having a structure substantially similar to that of a naturally occurring heavy chain and light chain variable region. Natural antibodies refer to naturally occurring immunoglobulin molecules with diverse structures. For example, the IgG class of natural immunoglobulins is a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy 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 domain or light chain variable domain, followed by a constant light (CL) domain, also called a light chain constant region. The subunit structures and three-dimensional arrangements of different classes of immunoglobulins are well known, for example, Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 1. This is generally described in 91 (2007). Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies (immunoglobulins) are assigned to different classes. Antibodies have five main classes: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further classified into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an immunoglobulin can be assigned one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. An immunoglobulin basically consists of two Fab molecules and one Fc domain linked via the hinge region of the immunoglobulin.

[0026] In some embodiments, Fc, Fc region, or Fc domain refers to the C-terminal region of an antibody heavy chain, including at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. Fc can refer to all or part of the flexible hinges located on the N-terminal side of these domains, as well as the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. In the case of IgA and IgM, Fc may include the J chain. The IgG Fc region includes the IgG CH2 and IgG CH3 domains, and optionally the hinges. Unless otherwise specified herein, the numbering of amino acid residues within the Fc region or constant region follows the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md, 1991. Human IgG Fc domains are particularly useful in this disclosure and may be Fc domains derived from human IgG1, IgG2, or IgG4.

[0027] Several definitions of CDR sequences for antibody variable domains are known in the art. See, for example, Kabat (Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3) and Chothia. Unless otherwise specified, CDR sequences are described herein according to the IMGT definition. See, for example, www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html.

[0028] In some embodiments, the antigen-binding domain, antibody, or antibody fragment that binds to human dectin-1 comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1 containing the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 containing the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 containing the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises CDR-L1 containing the sequence QGISSW (SEQ ID NO: 4), CDR-L2 containing the sequence GAS (SEQ ID NO: 5), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the antigen-binding domain, antibody, or antibody fragment that binds to human dectin-1 comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 from the VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).

[0029] As described above, Kabat definitions of CDR sequences can also be used. In some embodiments, an antigen-binding domain, antibody, or antibody fragment that binds to human dectin-1 includes a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain includes CDR-H1 containing the sequence DYYI (SEQ ID NO: 88), CDR-H2 containing the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 containing the sequence NSGSYSFGY (SEQ ID NO: 90), and the VL domain includes CDR-L1 containing the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 containing the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6).

[0030] In some embodiments, the antigen-binding domain, antibody, or antibody fragment that binds to human dectin-1 is an amino acid sequence A VH domain containing a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (Sequence ID 7), and / or comprises a VL domain containing a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8). In some embodiments, the antigen-binding domain, antibody, or antibody fragment that binds to human dectin-1 includes a VH domain containing the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and / or a VL domain containing the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).In some embodiments, the antigen-binding domain, antibody, or antibody fragment that binds to human dectin-1 comprises a VH domain containing the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7) and a VL domain containing the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).

[0031] In some embodiments, the antigen-binding domain, antibody, or antibody fragment is humanized.

[0032] In some embodiments, the antibody that binds to human dectin-1 is sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT The heavy chain comprises ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11), and / or the light chain comprises the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).In some embodiments, the antibody that binds to human dectin-1 is sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE It comprises a heavy chain containing the sequence KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11) and a light chain containing the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).

[0033] In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to human dectin-1. In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to human dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, or granulocytes. In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to human dectin-1 isoform A and / or human dectin-1 isoform. In some embodiments, star dectin-1 isoform A comprises the amino acid sequence MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVKTTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO: 9). In some embodiments, the human dectin-1 isoform B contains the amino acid sequence MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO: 10). In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to human dectin-1 expressed on the cell surface at an EC50 of less than 5 nM, less than 2 nM, less than 1 nM, or less than 0.5 nM. In some embodiments, the antigen-binding domain, antibody, or antibody fragment can bind to human dectin-1 and monkey dectin-1, such as cynomolgus monkey dectin-1.

[0034] In some embodiments, the antigen-binding domain, antibody, or antibody fragment competes for binding to human dectin-1 with a reference antibody comprising: a heavy chain variable (VH) domain comprising CDR-H1 comprising the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 comprising the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 comprising the sequence ARNSGSYSFGY (SEQ ID NO: 3); and a light chain variable (VL) domain comprising CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the antigen-binding domain, antibody, or antibody fragment competes for binding to human dectin-1 with a reference antibody comprising: a heavy chain variable (VH) domain comprising CDR-H1 comprising sequence DYYI (SEQ ID NO: 88), CDR-H2 comprising sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 comprising sequence NSGSYSFGY (SEQ ID NO: 90); and a light chain variable (VL) domain comprising CDR-L1 comprising sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the antigen-binding domain, antibody, or antibody fragment competes for binding to human dectin-1 with a reference antibody comprising: APGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7) and a heavy-chain variable (VH) domain comprising the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).

[0035] In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to the same human dectin-1 epitope as a reference antibody, which includes: a heavy chain variable (VH) domain comprising CDR-H1 containing the sequence GYTFTDYY (SEQ ID NO: 1), CDR-H2 containing the sequence INPNSGDT (SEQ ID NO: 2), and CDR-H3 containing the sequence ARNSGSYSFGY (SEQ ID NO: 3); and a light chain variable (VL) domain comprising CDR-L1 containing the sequence QGISSW (SEQ ID NO: 4), CDR-L2 containing the sequence GAS (SEQ ID NO: 5), and CDR-L3 containing the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to the same human dectin-1 epitope as a reference antibody, which includes: a heavy chain variable (VH) domain comprising CDR-H1 containing sequence DYYI (SEQ ID NO: 88), CDR-H2 containing sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and CDR-H3 containing sequence NSGSYSFGY (SEQ ID NO: 90); and a light chain variable (VL) domain comprising CDR-L1 containing sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 containing sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 containing sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to the same human dectin-1 epitope as the reference antibody, which includes: a heavy chain variable (VH) domain containing the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable (VL) domain containing the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).

[0036] Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibodies, nanobodies, scFv fragments, VH, and multispecific (e.g., bispecific) antibodies / fragments formed from antibody fragments.

[0037] "Fab" (fragment antigen binding) is the part of the antibody that binds to the antigen, and includes a variable region of the heavy chain and CH1 linked to the light chain via interchain disulfide bonds.

[0038] In some embodiments, the antibodies of this disclosure include an Fc region. The antibodies may be of 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 a significant role in the process by binding to the Fc receptor and inducing further phagocytosis. In some embodiments, the molecule has a modified Fc region with reduced ADCC activity compared to wild-type human IgG1 (e.g., including one or more mutations that reduce the effector function described herein).

[0039] In some embodiments, the antibodies of the Disclosure include an Fc region, and the carbohydrate structure bound to the Fc region is fucose-depleted or fucose-deficient, for example, at least one or two of the antibody's heavy chains are not fucosylated. In some embodiments, compositions comprising the antibodies of the Disclosure including an Fc region are provided herein, and the carbohydrate structure bound to the Fc region is fucose-depleted or fucose-deficient, for example, at least one or two of the antibody's heavy chains are not fucosylated. In some embodiments, less than 50% of the N-glycosidic carbohydrate chains in the composition contain fucose residues. In some embodiments, none of the N-glycosidic carbohydrate chains substantially contain fucose residues. In some embodiments, the fucose-depleted or fucose-deficient antibodies have improved ADCC function.

[0040] In other embodiments, the antibody of the Disclosure (e.g., IgG1 antibody), or a composition comprising the antibody of the Disclosure (e.g., IgG1 antibody), comprises wild-type glycosylation of the Fc region. In some embodiments, compositions comprising the fucosylated antibody of the Disclosure (e.g., IgG1 antibody), or the fucosylated antibody of the Disclosure (e.g., IgG1 antibody) are provided herein.

[0041] Fucosylation, or fucosylated antibodies, can refer to the presence of a fucose residue within an oligosaccharide bound to the peptide backbone of an antibody. Specifically, fucosylated antibodies contain α(l,6)-bound fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or both N-linked oligosaccharides bound to the Fc region of the antibody, for example, at position Asn297 (EU numbering of Fc region residues) of the human IgG1 Fc region. Asn297 can also be located approximately +3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to slight variations in immunoglobulin sequences. Non-fucosylated antibodies, or fucose-deficient antibodies, have reduced fucose levels compared to the amount of fucose in the same antibody produced in a cell line. Fucosylation of an antibody can be measured in antibody compositions treated with N-glycosidase F, for example, by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI TOF MS).

[0042] In some embodiments, the Fc region includes one or more mutations that reduce or eliminate fucosylation, e.g., a substitution at Asn297 (EU numbering of the Fc region residue) in the human IgG1 Fc region. Optionally, the Fc region further includes one or more amino acid substitutions that further improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of the residue) in the Fc region. Examples of publications related to "defucosylated" or "fucose-deficient" antibodies include US2003 / 0157108;WO2000 / 61739;WO2001 / 29246;US2003 / 0115614;US2002 / 0164328;US2004 / 0093621;US2004 / 0132140;US2004 / 0110704;US2004 / 0110282;US2004 / 0109865;WO2003 / 085119;WO2003 / 084570;WO2005 / 035586;WO2005 / 035778;WO2005 / 053742;Okazaki et al. References include al. J. Mol. Biol. 336:1239-1249 (2004) and Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0043] In some embodiments, defucosylated or nonfucosylated antibodies are produced in cell lines having gene modifications that result in defucosylated or nonfucosylated antibodies. An example of a cell line that produces defucosylated antibodies is Lec13, which lacks protein fucosylation. CHO cells (Ripka et al. Examples include cells in which the α-1,6-fucosyltransferase gene, FUT8, knockout cell lines such as knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)), cells overexpressing β1,4-N-acetylglucosaminetransferase III (GnT-III) and Golgi μ-mannosidase II (ManII), and cells in which mannosyl glycoprotein 2-β-N-acetylglucosaminetransferase (MGAT1; see Byrne, G. et al. (2018) PLoS Biol. 16:e2005817) has been knocked out.

[0044] In some embodiments, defucosylated or non-fucosylated antibodies are produced in cell lines treated with inhibitors of sugar-processing enzymes, such as kifunensin, which is an inhibitor of mannosidase I (see, e.g., Elbein, AD et al. (1990) J. Biol. Chem. 265:15599-15605). For example, cells can be centrifuged, resuspended in growth medium containing kifunensin (e.g., at 250 μg / mL), and cultured for antibody production.

[0045] In certain embodiments, the Disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments comprising a first antigen-binding domain that binds to a first target and a second antigen-binding domain that binds to a second target. In some embodiments, the Disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments comprising a first antigen-binding domain that binds to human dectin-1 and a second antigen-binding domain that binds to a target.

[0046] In some embodiments, multispecific (e.g., bispecific) antibodies and antibody fragments comprise a first antibody or antigen-binding fragment containing a first antigen-binding domain and a second antibody or antigen-binding fragment containing a second antigen-binding domain. In some embodiments, the first antibody or fragment is linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the second antibody or fragment is linked to its biotin-binding derivative or avidin-binding derivative. In some embodiments, the second antibody or fragment is linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the first antibody or fragment is linked to its biotin-binding derivative or avidin-binding derivative. In some embodiments, the first antibody or fragment conjugates to the second antibody or fragment via an interaction between its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative and its biotin-binding derivative or avidin-binding derivative.

[0047] Exemplary avidin-conjugated derivatives, streptavidin-conjugated derivatives, neutraavidin-conjugated derivatives, or biotin-conjugated derivatives are known in the art. In some embodiments, streptavidin is monomeric streptavidin (mSA). Exemplary biotin-conjugated derivatives or avidin-conjugated derivatives are known in the art. In some embodiments, the antibody or its antigen-conjugated fragment of the Disclosure is biotinylated. Kits for biotinylation of the antibody of interest are known in the art and are commercially available. In some embodiments, the mSA comprises the sequence EFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGS (SEQ ID NO: 14).

[0048] In some embodiments, the antibody that binds to human dectin-1 and links to mSA via a linker includes the sequence (SEQ ID NO: 15).In some embodiments, the antibody fragment that binds to human dectin-1 and is linked to mSA via a linker is sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVK Includes PSAASGS (sequence number 16) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH (sequence number 17).

[0049] In some embodiments, one or both of the first and second antigen-binding domains, the antibody, or the fragment are humanized.

[0050] In some embodiments, one or both of the first and second antigen-binding domains, the antibody, or the fragment include, for example, a tag for affinity purification. In some embodiments, the tag is a polyhistidine tag.

[0051] In some embodiments, one or both of the first and second antibodies or fragments 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 antibodies or fragments further include an Fc domain. In some embodiments, the first antibody or fragment is a Fab fragment and the second antibody or fragment is a full-length antibody. In some embodiments, both the first and second antibodies or fragments are full-length antibodies. In some embodiments, the first antibody or fragment is a Fab fragment linked to monomeric streptavidin (mSA) and the second antibody or fragment is a biotinylated full-length antibody. In some embodiments, the first antibody or fragment is a full-length antibody linked to monomeric streptavidin (mSA) and the second antibody or fragment is a biotinylated full-length antibody.

[0052] In some embodiments, an antibody or fragment is linked via a linker to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, or to its biotin-binding derivative or avidin-binding derivative. Linkers for constructing antibody fusion proteins are known in the art. In some embodiments, the linker comprises, or essentially comprises, glycine and / or serine residues. In some embodiments, the linker is 15 to 20 amino acids long. In some embodiments, the linker comprises the sequence GGGSGGGSGGGS (SEQ ID NO: 13). In some embodiments, the linker comprises one or more repeats of the sequence GGGGS (SEQ ID NO: 26). In some embodiments, the linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28). Further linker sequences are described in Chen, X. et al. (2013) Adv. Drug Deliv. Rev. 65:1357-1369. In some embodiments (for example, in the scFv of this disclosure), the scFv includes a type of linker between the VH domain and the VL domain, and another type of linker connecting the VL domain to the rest of the semi-antibody, e.g., the Fc region. For example, in some embodiments, the linker between the VH domain and the VL domain includes glycine and / or serine residues such as GGGSGGGSGGGS (SEQ ID NO: 13), GGGGSGGGGSGGGGS (SEQ ID NO: 27), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28), or one or more repeats of the sequence GGGGS (SEQ ID NO: 26); and / or the linker connecting the VL domain to the Fc region includes EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). In some embodiments, the linker between the VH domain and the VL domain comprises glycine and / or serine residues and is 15 to 20 amino acids long.

[0053] In some embodiments, the first target is human dectin-1 (e.g., isoform(s) A and / or B). In some embodiments, the second target is a disease-causing agent. In some embodiments, the second target is human dectin-1 (e.g., isoform(s) A and / or B). In some embodiments, the first target is a disease-causing agent.

[0054] In certain embodiments, the Disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments comprising at least one antigen-binding domain that binds to human dectin-1. Any of the human dectin-1-binding antigen-binding domains of the Disclosure can be used in multispecific (e.g., bispecific) binding molecules, antibodies, or antibody fragments. In some embodiments, the multispecific (e.g., bispecific) binding molecules, antibodies, or antibody fragments further comprise at least one antigen-binding domain that binds to a target (e.g., those described herein). In some embodiments, the target is a disease-causing substance.

[0055] In some embodiments, a multispecific (e.g., bispecific) binding molecule comprises a first antibody arm comprising a single-stranded variable fragment (scFv) comprising the VH domain and VL domain of the Disclosure that binds to human dectin-1, and a first Fc region; an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain; and a second antibody arm comprising a second Fc region attached to the VH domain. In some embodiments, the scFv arm binds to dectin-1, and the conventional antibody arm having the VH domain and VL domain on a separate polypeptide binds to a target (e.g., as described herein), such as a disease causative agent. In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the first antibody arm includes a first linker (e.g., 15-20 amino acid length) between the VH domain and the VL domain, and a second linker between the VL domain and the first Fc region. In some embodiments, the first linker includes one or more repeats of the sequence GGGGS (SEQ ID NO: 26), e.g., the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the second linker includes the sequence EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). Further linker sequences are described in Chen, X. et al. (2013) Adv. Drug Deliv. Rev. 65:1357-1369. A non-limiting example of this format is shown in Figure 51.

[0056] In some embodiments, the disease causative agent is a bacterial cell, fungal cell, virus, senescent cell, tumor cell, protein aggregate, LDL particle, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is an antigen expressed on the surface of a bacterial cell, fungal cell, senescent cell, tumor cell, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is a viral surface antigen. In some embodiments, the target is a protein aggregate or its monomer, e.g., amyloid-beta (e.g., in Alzheimer's disease), or lambda or kappa light chain amyloid (e.g., in light chain amyloidosis). In some embodiments, for example, in the case of applications in the field of oncology, the second target is, for example, CD70, HER2, DLL3, nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR expressed on the surface of cancer cells.

[0057] In some embodiments, the target is CD20, for example, human CD20. In some embodiments, the antigen-binding domain that binds to CD20 includes the CDR-H1, CDR-H2, and CDR-H3 sequences from the VH domain sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24), and / or the CDR-L1, CDR-L2, and CDR-L3 sequences from the VL domain sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25). In some embodiments, the antigen-binding domain that binds to CD20 includes a VH domain containing the sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24), and / or a VL domain containing the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25). In some embodiments, the antigen-binding domain that binds to CD20 includes VH and VL domain sequences derived from rituximab. In some embodiments, the antigen-binding domain that binds to CD20 includes VH and VL domain sequences derived from obinituzumab. In some embodiments, the antigen-binding domain that binds to CD20 includes a VH domain containing the sequence of SEQ ID NO: 46 and / or a VL domain containing the sequence of SEQ ID NO: 47.

[0058] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to a target (e.g., a disease causative agent of the Disclosure). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).

[0059] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to CD20 (e.g., human CD20). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence of SEQ ID NO: 24 and a VL domain containing the sequence of SEQ ID NO: 25. In some embodiments, the second antibody arm is QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE The molecule comprises a second polypeptide containing the sequence PQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 32), and a third polypeptide containing the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0060] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to HER2 (e.g., human HER2). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 34) and a VL domain containing the sequence DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 35). In some embodiments, the antigen-binding domain that binds to HER2 includes the trastuzumab-derived VH and VL domain sequences.

[0061] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the present disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached 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 (e.g., human Trop-2). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 42) and a VL domain containing the sequence DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 43). In some embodiments, the second antibody arm includes a VH domain containing the sequence QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTKTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKKEDTATYFCGRGGYGSSYWYFDVWGAGTTVTVSS (SEQ ID NO: 56) and a VL domain containing the sequence DIVMTQSHKFMSTSVGDRVSITCKASQDVSIAVAWYQQKPGQSPKVLIYSASYRYTGVPDRFTGSGSGTDFTFTISRVQAEDLAVYYCQQHYITPLTFGAGTKLELK (SEQ ID NO: 57). In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLQQSGPELVRPGTSVRISCKASGYTFTIYWLGWVKQRPGHGLEWIGNIFPGSAYINYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREGSNSGYWGQGTTLTVSS (SEQ ID NO: 58) and a VL domain containing the sequence DIVMTQSPSSLSVSAGEKVTMTCKSSQSLLNSGNQQNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQSDHIYPYTFGGGTKLEIK (SEQ ID NO: 59).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISSYGVHWIRQPPGKGLEWIGVIWTGGSTDYNSALMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGDYDRYTMDYWGQGTLVTVSS (SEQ ID NO: 66) and a VL domain containing the sequence DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPYTFGQGTKLEIK (SEQ ID NO: 67). In some embodiments, the antigen-binding domain that binds to Trop-2 includes VH and VL domain sequences derived from sacituzumab, AR47A6.4.2, h7E6, or Pr1E11.

[0062] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a first antibody arm comprising a single-stranded variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human dectin-1, and a first Fc region; an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain; and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to light chain amyloid (e.g., human light chain amyloid, e.g., human kappa light chain amyloid, human lambda light chain amyloid, or both human kappa and lambda light chain amyloid). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region.In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31). In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLKESGPGLVAPSQSLSITCTVSGFSLSSYGVSWVRQPPGKGLEWLGVIWGDGSTNYKPNLMSRLSISKDISKSQVLFKLNSLQTDDTATYYCVTLDYWGQGTSVTVSS (SEQ ID NO: 44) and a VL domain containing the sequence DVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCFQTTYVPNTFGGGTKLEIK (SEQ ID NO: 45).In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGRLVQPKGSLKLSCAASGFTFNTYAMYWIRQAPGKGLEWVARIRSKSNNYAIYYADSVKDRFTIFRDDSQSMLYLQMNNLKTEDTAMYYCVRPYSDSFAYWGQGTLVTVSA (SEQ ID NO: 52) and a VL domain containing the sequence DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSTGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHVPFTFGGGTKLEIK (SEQ ID NO: 53). In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGRLVQPKGSLKLSCAASGFTFNTYAMYWIRQAPGKGLEWVARIRSKSNNYAIYYADSVKDRFTIFRDDSQSMLYLQMNNLKTEDTAMYYCVRPYSDSFAYWGQGTLVTVSA (SEQ ID NO: 54) and a VL domain containing the sequence DVVMTQTPLSLPVSLGDQASISCRSSLSLVHSTGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHVPFTFGGGTKLEIK (SEQ ID NO: 55). In some embodiments, the antigen-binding domain that binds to light chain amyloid includes VH domain and VL domain sequences derived from antibody 11-1F4, 2A4, or 7D8.

[0063] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the present disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to amyloid-beta (e.g., human amyloid-beta). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO: 48) and a VL domain containing the sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK (SEQ ID NO: 49). In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGGLVQPGGSLRLSCSASGFTFSSFGMHWVRQAPGKGLEWVAYISSGSSTIYYGDTVKGRFTISRDNAKNSLFLQMSSLRAEDTAVYYCAREGGYYYGRSYYTMDYWGQGTTVTVSS (SEQ ID NO: 50) and a VL domain containing the sequence DVVMTQSPLSLPVTPGAPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCFQGSHVPPTFGPGTKLEIK (SEQ ID NO: 51). In some embodiments, the antigen-binding domain that binds to amyloid-beta includes VH domain and VL domain sequences derived from aducanumab or lecanemab.

[0064] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to CD70 (e.g., human CD70). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS (SEQ ID NO: 38) and a VL domain containing the sequence QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVL (SEQ ID NO: 39). In some embodiments, the antigen-binding domain that binds to CD70 includes the VH domain and VL domain sequences derived from 4ID12.

[0065] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the present disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to nectin-4 (e.g., human nectin-4). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSS (SEQ ID NO: 40) and a VL domain containing the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIK (SEQ ID NO: 41). In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFISRLSISKDTSKSQVFFKMNSLQADDTAIYYCARELIHAMDNWGQGTSVTVSS (SEQ ID NO: 60) and a VL domain containing the sequence DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGNSPQLLVFAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPTFGGGTKLEIK (SEQ ID NO: 61). In some embodiments, the antigen-binding domain that binds to nectin-4 includes VH and VL domain sequences derived from N41 or Ha22-2.

[0066] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to EGFR (e.g., human EGFR). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 62) and a VL domain containing the sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 63). In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 64) and a VL domain containing the sequence EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIK (SEQ ID NO: 65). In some embodiments, the antigen-binding domain that binds to EGFR includes VH domain and VL domain sequences derived from cetuximab or nesitumumab.

[0067] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to DLL3 (e.g., human DLL3). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARIGDSSPSDYWGQGTLVTVSS (SEQ ID NO: 68) and a VL domain containing the sequence EIVMTQSPATLSVSPGERATLSCKASQSVSNDVVWYQQKPGQAPRLLIYYASNRYTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDYTSPWTFGQGTKLEIK (SEQ ID NO: 69). In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVYYSGTTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSS (SEQ ID NO: 70) and a VL domain containing the sequence EIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK (SEQ ID NO: 71). In some embodiments, the antigen-binding domain that binds to DLL3 includes VH domain and VL domain sequences derived from lovalpituzumab or DLL3-4.

[0068] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the present disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to mesothelin (e.g., human mesothelin). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 72) and a VL domain containing the sequence DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 73). In some embodiments, the antigen-binding domain that binds to mesothelin includes the VH domain and VL domain sequences derived from amatsuximab.

[0069] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to CD33 (e.g., human CD33). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS (SEQ ID NO: 74) and a VL domain containing the sequence DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK (SEQ ID NO: 75). In some embodiments, the antigen-binding domain that binds to CD33 includes gemtuzumab-derived VH and VL domain sequences.

[0070] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) comprising a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to CCR8 (e.g., human CCR8). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGRLVQPKGSLKLSCAASGFAFNTYALYWIRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRARFYYSDYGYAMDYWGQGTSVTVSS (SEQ ID NO: 76) and a VL domain containing the sequence DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK (SEQ ID NO: 77). In some embodiments, the antigen-binding domain that binds to CCR8 includes the VH domain and VL domain sequences derived from 10A11.

[0071] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) comprising a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to CTLA4 (e.g., human CTLA4). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 78) and a VL domain containing the sequence EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 79). In some embodiments, the antigen-binding domain that binds to CTLA4 includes the ipilimumab-derived VH and VL domain sequences.

[0072] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the Disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to cMET (e.g., human cMET). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv comprises a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 80) and a VL domain containing the sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIK (SEQ ID NO: 81). In some embodiments, the antigen-binding domain that binds to cMET includes the VH domain and VL domain sequences derived from onartuzumab.

[0073] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the present disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to LIV-1 (e.g., human LIV-1). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLVQSGAEVKKPGASVKVSCKASGYTIEDYYMHWVRQAPGQGLEWMGWIDPENGDTEYAPTFQGRVTMTRDTSINTAYMELSRLRSDDTAVYYCARHDAHYGTWFAYWGQGTLVTVSS (SEQ ID NO: 82) and a VL domain containing the sequence DVVMTQSPLSLPVTLGQPASISCRSSQSIIRNDGNTYLEWYQQRPGQSPRRLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGGGTKVEIK (SEQ ID NO: 83). In some embodiments, the antigen-binding domain that binds to LIV-1 includes the VH domain and VL domain sequences derived from hLIV14.

[0074] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the present disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to ROR-1 (e.g., human ROR-1). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLQESGPGLVKPSQTLSLTCTVSGYAFTAYNIHWVRQAPGQGLEWMGSFDPYDGGSSYNQKFKDRLTISKDTSKNQVVLTMTNMDPVDTATYYCARGWYYFDYWGHGTLVTVSS (SEQ ID NO: 84) and a VL domain containing the sequence DIVMTQTPLSLPVTPGEPASISCRASKSISKYLAWYQQKPGQAPRLLIYSGSTLQSGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQHDESPYTFGEGTKVEIK (SEQ ID NO: 85). In some embodiments, the antigen-binding domain that binds to ROR-1 includes the VH domain and VL domain sequences derived from Ab1.

[0075] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising a single-stranded variable fragment (scFv) containing a VH domain and a VL domain of the present disclosure that binds to human dectin-1, a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region attached to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to serum amyloid-P (SAP), e.g., human SAP. In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more congeneral hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more congeneral hole-forming mutations. In some embodiments, the scFv includes a first linker of the Disclosure between the VH domain and the VL domain, and a second linker of the Disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF Contains the amino acid sequence GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31).In some embodiments, the second antibody arm includes a VH domain containing the sequence QVQLVQSGAEVKKPGSSVKVSCKASGFTFATYNMHWVRQAPGQGLEWMGYIYPGDGNANYNQQFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGDFDYDGGYYFDSWGQGTLVTVSS (SEQ ID NO: 86) and a VL domain containing the sequence DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIHNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGAPLTFGQGTKLEIK (SEQ ID NO: 87). In some embodiments, the antigen-binding domain that binds to SAP includes the VH domain and VL domain sequences derived from dezamizumab.

[0076] Multispecific antibodies have binding specificity to at least two different epitopes (usually derived from different antigens). Multispecific or bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0077] To enable targeted removal of disease-causing substances by phagocytosis, the antigen-binding domain of this disclosure may be selected from IgG, intrabody, peptidebody, nanobody, single-domain antibody, SMTP, and multispecific antibody (e.g., bispecific antibody, diabody, triabody, tetrabody, tandem di-scFV, tandem tri-scFv, ADAPTIR).

[0078] Methods for producing bispecific antibodies are known in the art. Established methods for producing bispecific antibodies include the "knobs-into-holes" or "protuberance-into-cavity" approach. 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 congeneral interface on the other immunoglobulin polypeptide, thereby enabling the association of the two immunoglobulin polypeptides. In some embodiments, the interfaces can be manipulated so that a "knob" or "protuberance" located at the interface of one immunoglobulin polypeptide corresponds to a congeneral "hole" or "cavity" located at the interface of the other immunoglobulin polypeptide. In some embodiments, knobs can be constructed by replacing a smaller amino acid side chain with a larger side chain. In some embodiments, holes can be constructed by replacing a larger amino acid side chain with a smaller side chain. The knobs or holes may be present within the original interface or introduced by synthesis. Polynucleotides encoding modified immunoglobulin polypeptides having one or more corresponding knob or hole-forming mutations can be expressed and purified using standard recombination techniques and cell lines known in the art. See, for example, U.S. Patents 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. Patent Application Publication 2013 / 0089553; Spiess et al., Nature Biotechnology 31:753-758, 2013; and Ridgway and Carter (1996) Protein Eng. 9:617-621. Modified immunoglobulin polypeptides can be produced using prokaryotic host cells such as E. coli, or eukaryotic host cells such as mammalian cells (e.g., CHO cells) or yeast cells.Immunoglobulin polypeptides with corresponding knobs and holes can be expressed in a co-culture in host cells and purified together as a heteromultimer, or expressed in a single culture, purified separately, and constructed in vitro. Exemplary homologous knob and hole mutations are shown below (numbering follows EU index). The EU numbering used herein is known in the art. See, for example, the IMGT resources at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html and www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html. As used herein, “antibody arm” may refer to a pair of antibody heavy chain and antibody light chain, where the variable domains of the heavy and light chains form an antigen-binding site that binds to a target antigen. [Table 1]

[0079] According to a different approach, an antibody variable domain (antibody-antigen binding site) with desired binding specificity fuses with the immunoglobulin constant domain sequence.

[0080] In some embodiments, multispecific (e.g., bispecific) antibodies further include one or more mutations in only one of the antibody arms to improve heavy / light chain pairing. For example, amino acid substitutions can be used to replace the native disulfide bond at the CH1-CL interface of one antibody arm with an engineered disulfide bond. See, for example, Mazor, Y. et al. (2015) MAbs 7:377-389 and EP3452089A2. In some embodiments, multispecific or bispecific antibodies comprise two antibody light chains and two antibody heavy chains, where only one of the antibody heavy chains contains the amino acid substitutions F126C and C220V by EU numbering, and only the corresponding or homogeneous light chain contains the amino acid substitutions S121C and C214V.

[0081] Examples of multispecific (e.g., bispecific) antibodies include cross-linked or "heteroconjugated" antibodies. Techniques for producing bispecific antibodies from antibody fragments are also described in this literature. For example, bispecific antibodies can be prepared using chemical bonding. In some embodiments, the bispecific antibody comprises a first IgG antibody containing a first antigen-binding domain covalently linked to a second IgG antibody containing a second antigen-binding domain.

[0082] In some embodiments, multispecific (e.g., bispecific) antibodies further include one or more mutations in only one of the antibody arms to reduce binding affinity to protein A. See, for example, Ollier, R. et al. (2019) MAbs 11:1464-1478 and AU2018204314. In some embodiments, multispecific or bispecific antibodies comprise two antibody light chains and two antibody heavy chains, with only one of the antibody heavy chains containing the amino acid substitutions H435R and Y436F according to EU numbering.

[0083] In some embodiments, monospecific or multispecific (e.g., bispecific) antibodies further include one or more mutations to reduce effector function, for example, to reduce or eliminate binding of the Fc region to the Fc receptor. In some embodiments, the antibody comprises two antibody Fc regions, each antibody Fc region containing one or more amino acid substitutions at positions 234, 235, and 237 according to EU numbering. In some embodiments, the antibody comprises two antibody Fc regions, each antibody Fc region containing L234A, L235E, and G237A substitutions according to EU numbering.

[0084] In some embodiments, a monospecific or multispecific (e.g., bispecific) antibody 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 the VH domain of the second antibody heavy chain forms an antigen-binding domain with the VL domain of the second antibody light chain, and the first antibody heavy chain includes substitutions F126C, C220V, and T366W according to EU numbering, the first antibody light chain includes substitutions S121C and C214V, and the second antibody heavy chain includes substitutions T366S, L368A, Y407V, H435R, and Y436F. In some embodiments, the first and second antibody heavy chains further include substitutions L234A, L235E, and G237A according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain include human IgG1 Fc domains.

[0085] In some embodiments, polynucleotides encoding any one of the antibodies or multispecificity conjugating molecules of the embodiments described above are provided herein. In some embodiments, vectors (e.g., expression vectors) containing any one of the polynucleotides of the embodiments described above are provided herein. In some embodiments, host cells (e.g., isolated host cells or cell lines) containing any one of the polynucleotides or vectors of the embodiments described above are provided herein. In some embodiments, pharmaceutical compositions comprising any one of the antibodies or multispecificity conjugating molecules of the embodiments described above and a pharmaceutically acceptable carrier are provided herein. Any of these can be used in the production and / or processing methods disclosed herein.

[0086] In some embodiments, methods for producing antibodies or multispecificity conjugate molecules are provided herein, comprising culturing host cells of any one of the above embodiments under conditions suitable for the production of said antibodies or multispecificity conjugate molecules. In some embodiments, the method further comprises recovering the antibodies or multispecificity conjugate molecules. The antibodies or multispecificity conjugate molecules can be produced using standard recombinant techniques described herein and / or illustrated below.

[0087] Antibodies and antibody fragments can be produced using recombinant methods. For example, nucleic acids encoding antibodies / fragments can be isolated and inserted into a replicable vector for further cloning or expression. The DNA encoding the antibody / fragment can be readily isolated and sequenced using conventional procedures (e.g., via oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody / fragment). Many vectors are known in the art. Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Suitable host cells for cloning or expressing DNA in vectors as described herein are prokaryotes, yeasts, or higher eukaryotes. When recombinant techniques are used, antibodies / fragments can be produced intracellularly, in the perimembrane space, or directly secreted into the culture medium. If antibodies / fragments are produced intracellularly, particulate residue (either host cells or lysed fragments) can be removed, for example, by centrifugation or ultrafiltration. When antibodies / fragments are secreted into the culture medium, the supernatant of such an expression system is typically first concentrated using a commercially available protein concentration filter.

[0088] In some embodiments, the antibody or multispecific binding molecule of this disclosure is part of a pharmaceutical composition comprising, for example, an antibody and one or more pharmaceutically acceptable carriers. The pharmaceutical compositions and formulations described herein may be prepared in the form of lyophilized formulations or aqueous solutions by mixing an active ingredient (e.g., a fusion protein) of a desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmacochemically acceptable carriers are generally nontoxic to the recipient at the dosage and concentration used, and include, but are not limited to, buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG).

[0089] Certain aspects of this disclosure relate to kits or products comprising either an antibody or a multispecificity conjugate molecule disclosed herein. In some embodiments, the product includes a container and a label or accompanying documentation on or attached to the container. In some embodiments, the kit or product further includes instructions for using the antibody or multispecificity conjugate molecule in accordance with any of the methods disclosed herein to treat a disease or disorder, such as cancer.

[0090] Suitable containers include, for example, bottles, vials, and syringes. Containers may be formed from various materials such as glass or plastic. The container may hold a composition effective in treating a medical condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper puncturable by a subcutaneous needle). At least one activator in the composition is an antibody or multispecific conjugating molecule as described herein. The label or accompanying information indicates that the composition is used to treat a specific medical condition. The label or accompanying information further includes instructions for administering the antibody or multispecific conjugating molecule composition. Products and kits containing the concomitant therapeutic agents described herein are also intended.

[0091] II. Production Methods and Identification Methods In certain embodiments, the Disclosure provides methods for producing or creating multispecific (e.g., bispecific) antibodies and antibody fragments. In some embodiments, the Method includes: providing a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to a first target; providing a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to a second target; and contacting the first antibody or fragment with the second antibody or fragment under conditions suitable for binding of the first antibody or fragment to the second antibody or fragment via interactions between its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative and its biotin-binding derivative or avidin-binding derivative, thereby creating a multispecific binding molecule. In some embodiments, the first antibody or fragment is linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the second antibody or fragment is linked to its biotin-binding derivative or avidin-binding derivative. In some embodiments, the second antibody or fragment is linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the first antibody or fragment is linked to its biotin-binding derivative or avidin-binding derivative. Any antigen-binding domain, antibody, and antibody fragment of this disclosure (e.g., those described above in Section I) can be produced or produced using the methods for producing or constructing multispecific (e.g., bispecific) antibodies and antibody fragments disclosed herein. Advantageously, this platform provides a modular form for constructing a variety of multispecific (e.g., bispecific) binding molecules in which different antigen-binding domains, antibodies, and / or antibody fragments are linked together via high-affinity avidin:biotin interactions.

[0092] In certain embodiments, the Disclosure provides a method for identifying multispecific (e.g., bispecific) binding molecules that bind to a first target and a second target. In some embodiments, the method includes: providing a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to a first target; providing a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to a second target; contacting the first antibody or fragment with the second antibody or fragment under conditions suitable for binding of the first antibody or fragment to the second antibody or fragment via an interaction between the avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative and the biotin-binding derivative or avidin-binding derivative, thereby creating a multispecific binding molecule; and measuring the binding of the multispecific binding molecule to at least one of the first target and the second target. In some embodiments, the first antibody or fragment is linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the second antibody or fragment is linked to its biotin-binding derivative or avidin-binding derivative. In some embodiments, the second antibody or fragment is linked to its avidin-binding derivative, streptavidin-binding derivative, neutraavidin-binding derivative, or biotin-binding derivative, and the first antibody or fragment is linked to its biotin-binding derivative or avidin-binding derivative. Advantageously, this platform allows for screening of various antigen-binding domains for binding to target organisms in a multispecific (e.g., bispecific) manner.

[0093] In some embodiments, the first target is human dectin-1 (e.g., isoform(s) A and / or B). In some embodiments, the second target is a disease-causing agent. In some embodiments, the second target is human dectin-1 (e.g., isoform(s) A and / or B). In some embodiments, the first target is a disease-causing agent.

[0094] Any antigen-binding domain, antibody, and antibody fragment of this disclosure (e.g., those described above in Section I) can be used in methods for identifying multispecific (e.g., bispecific) antibodies and antibody fragments disclosed herein. In some embodiments, the antigen-binding domain, antibody, and antibody fragment bind to human dectin-1. For example, in some embodiments, the antibody or fragment binds to human dectin-1 and is linked to an mSA via a linker. In some embodiments, the antibody or fragment contains a sequence selected from the group consisting of SEQ ID NOs: 15-17.

[0095] Assays for measuring the binding of a multispecific binding molecule to at least one of a first target and a second target are known in the art. In some embodiments, the binding of a multispecific binding molecule to a purified antigen is measured, for example, in an ELISA or SPR binding assay. In some embodiments, the binding of a multispecific binding molecule to a cell expressing an antigen on its surface is measured, for example, in a flow cytometry-based binding assay. In some embodiments, the binding of a multispecific binding molecule to an antigen-coated bead or other solid substrate is measured. In some embodiments, a functional assay is used to detect interactions between two or more cells (each expressing a surface antigen bound by the antigen-binding domain of the multispecific binding molecule) brought together by the binding of the multispecific binding molecule, for example, by measuring cytokine production, cell death / phagocytosis, etc.

[0096] III.How to use In certain embodiments, the Disclosure provides a method for treating a disease or disorder, comprising administering an effective amount of an antibody, an antibody fragment, a multispecific (e.g., bispecific) conjugating molecule, or a composition of the Disclosure to an individual in need thereof. In some embodiments, the individual is a human.

[0097] Any antigen-binding domain, antibody, and antibody fragment of this disclosure (e.g., those described above in Section I) can be used in the therapeutic methods and methods of use disclosed herein, as well as in related compositions (e.g., pharmaceutical compositions). For example, in some embodiments, the method involves using a multispecific (e.g., bispecific) binding molecule of this disclosure having a first antigen-binding domain that binds to human dectin-1 and a second antigen-binding domain that binds to a disease-causing substance. In some embodiments, the disease-causing substance is a bacterial cell, fungal cell, virus, senescent cell, tumor cell, protein aggregate (e.g., amyloid-beta, or lambda or kappa light chain amyloid), LDL particle, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is an antigen expressed on the surface of a bacterial cell, fungal cell, senescent cell, tumor cell, mast cell, eosinophil, ILC2 cell, or inflammatory immune cell. In some embodiments, the target is a viral surface antigen. In some embodiments, the target is CD70, HER2, DLL3, Nectin-4, TROP-2, Mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR. Binding of molecules mediating the targeted removal of disease-causing substances by phagocytosis is possible with or without avidity, i.e., with or without induction of dimerization of the target antigen present on the phagocytic receptor such as Dectin-1 or on the disease-causing substance.

[0098] In some embodiments, the disease or disorder is cancer, bacterial infection, fungal infection, viral infection, mast cell disease or disorder, systemic mastocytosis, amyloidosis, or age-related disease or disorder. Various abnormal host cells accumulate and are not removed, including tumors, lymphomas, dead cells, necrotic cells, apoptotic cells, dying cells, infected cells, and damaged cells associated with the disease. In addition, various cell products such as aggregated proteins (β-amyloid plaques, tau aggregates, or antibody-lambda or kappa light chain amyloids) and lipoprotein particles can cause disease when their accumulation increases. Disease-causing cells may have glycoproteins, surface proteins, or glycolipids typical of abnormal cells associated with the disease, disorder, or other undesirable pathological conditions. Besides host-produced substances, various exogenous pathogens, such as infectious microorganisms (e.g., viruses, fungi, and bacteria) and products and residues produced by these microorganisms (e.g., viral particle envelopes, endotoxins), may not be adequately removed from the patient. In some embodiments, the virus is the influenza virus. In some embodiments, the virus is SARS-CoV-2.

[0099] The abnormalities described above can lead to diseases such as cancer, Alzheimer's disease, fibrosis, Parkinson's disease, Huntington's disease, HIV, hepatitis A, B, or C, and sepsis. Many of these disorders or diseases are characterized by the accumulation of disease-causing substances in different organs of the human subject. In addition to the beneficial removal of disease-causing substances through phagocytosis, this molecule can induce the production of inflammatory mediators, thereby altering the disease microenvironment of tumors, cancers, and lymphomas. While we do not wish to be bound by theory, it is thought that molecules that perform targeted phagocytosis may show clear benefits to patients with, for example, Alzheimer's disease, Parkinson's disease, cancer, infections (viral, bacterial, fungal, protozoan infections), inflammation, or immune diseases (e.g., autoimmune diseases, inflammatory bowel disease, multiple sclerosis), degenerative diseases (e.g., joints and cartilage), rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD, etc. In addition, targeted phagocytic antibody therapy may have greater activity in depleting cells within tissues than ADCC, which relies on NK cells. This therapy may have more selective activity in removing specific disease-causing substances than therapies that target bone marrow cells and improve phagocytosis in general. For example, potential targets for cancer treatment include, but are not limited to, CD70, HER2, DLL3, Nectin-4, TROP-2, Mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, and EGFR.

[0100] The following description is provided to enable those skilled in the art to fabricate and use various embodiments. Specific descriptions of devices, techniques, and applications are provided solely as examples. Various modifications to the embodiments described herein will be readily 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. Accordingly, the various embodiments are not intended to be limited to the embodiments described and shown herein, but should be given a scope consistent with the claims. [Examples]

[0101] Example 1: Functional characterization of the 2M24 anti-dectin-1 antibody This example describes the production of a monoclonal antibody specific to human dectin-1. This example also describes the characterization of a novel anti-human dectin-1 antibody.

[0102] Materials and methods Production of anti-dectin-1 antibodies Four-week-old ATX-Gx transgenic mice were subcutaneously inoculated with recombinant human dectin-1 isoform B for five weeks, and bolus antigens were administered weekly. Antibody titers in mouse serum were evaluated before and after bolus administration via ELISA and flow cytometry. Mice with the highest serum antibody titers were selected and supplied with B cells for hybridoma generation.

[0103] Prior to cell fusion, mice were given a single additional inoculation with recombinant human dectin-1 isoform B. The mice were sacrificed and their spleens were harvested. Splenocytes and SP2 / 0-Ag14 myeloma cells were mixed and incubated at 37°C, and fusion was induced in the presence of polyethylene glycol (PEG) or by electroporation. The cells were then harvested and plated in 96-well plates at limiting dilutions to one cell per well. Subsequently, the cells were treated with hypoxanthine, aminopterin, and thymidine (HAT) medium and cultured for at least two weeks before selection.

[0104] To identify dectin-1-specific candidates, hybridoma supernatants were screened by flow cytometry on cells overexpressing dectin-1 and on human primary monocytes. Cross-reactivity of cynomolgus monkey dectin-1 was evaluated using flow cytometry with antibodies that bind to cynomolgus monkey primary monocytes.

[0105] Healthy donor sample Fresh, healthy donor buffy coat was obtained from Stanford Blood Center. Peripheral blood mononuclear cells were isolated by ficoll paque (GE Healthcare, Chicago, IL) and cryopreserved in Bambanker cell freezing medium (Bulldog Bio, Portsmouth, NH). Briefly, buffy coat was diluted with phosphate-buffered saline (1:1 ratio), then the diluted buffy coat was overlaid on ficoll and centrifuged at 760g. The PBMC layer was isolated and washed with PBS before downstream analysis. Peripheral blood leukocytes were isolated by erythrolysis. Cryopreserved cynomolgus monkey PBMCs were obtained from Human Cells Biosciences.

[0106] Primary cells and cell cultures Human monocytes were isolated from healthy donor PBMCs according to the manufacturer's instructions for the panmonocyte isolation kit (Miltenyi Biotec, Inc., Auburn, CA). For differentiation into macrophages and dendritic cells, monocytes were cultured for 6 days in RPMI containing 10 human serum (Millipore Sigma) in the presence of 50 ng / ml MCSF (Peprotech, Rocky Hill, NJ) to completely differentiate into macrophages, or cultured for 6 days in the presence of 50 ng / ml GMCSF and 50 ng / ml IL-4 (Peprotech, Rocky Hill, NJ) to completely differentiate into dendritic cells. The culture medium containing cytokines was changed every 3 days.

[0107] HEK Blue h Dectin-1-α and HEK Blue h Dectin-1-β cells (Invivogen, San Diego, CA) were maintained in DMEM / 10% FBS supplemented with mormocin and puromycin according to the manufacturer's instructions. Freestyle 293F cells were transiently transfected according to the manufacturer's instructions (Thermo Fisher, Waltham, MA). In short, viable cell density and viability were measured. The cells reached a final density of 11x10 in Freestyle 293 expression medium. 6The reagent was diluted to a concentration of 1000 viable cells / mL. The Freestyle Max reagent was diluted in OptiPro SFM medium, mixed, and incubated at room temperature for 5 minutes. The diluted Freestyle Max reagent was added to the plasmid DNA diluted in OptiPro SFM medium and mixed. The Freestyle Max reagent / plasmid DNA complex was incubated at room temperature for 10-20 minutes. While gently rotating the culture flask during addition, the complex was slowly transferred to the cells, and the cells were incubated in a 37°C incubator at 80% relative humidity and 8% CO2 on an orbital shaker.

[0108] Binding of Dectin-1 antibody to Dectin-1 expressing cells Dectin-1 expressing cells (HEK Blue h Dectin-1-a, HEK Blue h Dectin-1-b, HEK293F h Dectin-1-a FL, human monocytes, or cynomolgus monkey monocytes) were placed in a 96-well V-bottom plate without tissue culture treatment, with 1 × 10⁶ cells per well. 5 ~2×10 5 Cells were plated. Furthermore, human monocytes were incubated with human FcgR blocking antibody (Biolegend, San Diego, CA) at room temperature for 10 minutes to reduce antibody binding to the Fc receptor. Subsequently, the cells were stained with eFluor 506 survival dye (ThermoFisher, Waltham, MA) at a 1:1000 dilution on ice for 30 minutes, followed by washing with FACS buffer (PBS containing 2% fetal bovine serum). Primary Dectin-1 antibody or isotypes were used for titration at 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41, and 0.14 nM, incubated on ice for 30 minutes, and then further washed with FACS buffer.

[0109] For detection of mouse primary antibodies, cells were incubated with fluorescently labeled AF647 anti-mouse Fc-specific secondary antibody (Jackson Immuno). For detection of human IgG4 primary antibodies, cells were incubated on ice for 30 minutes with either Alexa Fluor 647 anti-human Fc-specific secondary antibody (Jackson Immuno) (detection in HEK cells) or FITC anti-human IgG4 antibody (Sigma) (detection in primary monocytes). Data were acquired using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA) and analyzed using Graphpad Prism 8.4.

[0110] Inhibition of laminarin by dectin-1 antibodies HEK Blue h Dectin-1a cells were placed in a 96-well V-bottom plate without tissue culture treatment, at a rate of 1 × 10⁶ cells per well. 5 Cells were plated. Primary anti-dectin-1 antibody was used for titration at 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.14, 0.05, 0.015, and 0.005 nM concentrations, and incubated on ice for 30 minutes in the presence of 8 μg / biotin laminarin. After washing with FACS buffer, biotin laminarin binding on HEK cells was detected using streptavidin-AF647 for 30 minutes on ice. For analysis, 4000 cellular events were acquired using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA) and analyzed using Graphpad Prism 8.4.

[0111] Labeling of polystyrene beads with pHrodo and conjugation to antibodies Polystyrene beads of different sizes coated with goat anti-mouse IgG (or biotin) (Spherotech, Lake Forest, IL) were washed twice with PBS / Tween® 20 0.05%. pHrodo Red succinimidyl ester (pHrodo Red, SE) (ThermoFisher, Waltham, MA) was added to the beads at 10 μM and incubated at room temperature for 60 minutes with shaking. The beads were then washed with PBS / BSA 0.1% to remove excess pHrodo Red.

[0112] After pHrodo labeling, antibodies were conjugated onto the beads according to the manufacturer's recommendations. In short, based on the beads' ability to bind to antibodies, a 5-fold excess of antibody was added to the beads and incubated with shaking at room temperature for 60 minutes. The beads were then washed with PBS / BSA 0.1% to remove unbound antibody. To assess the quality of the beads, the activation of pHrodo red was evaluated by flow cytometry in a low pH buffer. Antibodies bound to the beads were evaluated using fluorescently labeled AF647 anti-mouse Fc-specific antibody or FITC anti-human IgG4 antibody secondary antibody.

[0113] Antibody-dependent targeted phagocytosis of Phrodo-labeled beads For phagocytic experiments, 50,000 HEK cells or primary cells (macrophages or dendritic cells) overexpressing Dectin-1 were seeded in RPMI containing 10 ultra-low IgG FBS in 96-well plates. pHrodo-labeled beads conjugated with anti-Dectin-1 antibody or isotype were added in desired ratios ranging from 1:1 to 1:3 for cell beads, and the plates were briefly spun down.

[0114] In several experiments, cells were labeled with the cell tracker calcein AM (Thermo Fisher, Waltham, MA). Phagocytosis was monitored by taking images at desired time points using the IncuCyte S3 live imaging system (Germany) and analyzed using IncuCyte S3 software. Phagocytosis was quantified by the overlap between bright red fluorescence (incorporated beads) and calcein AM-positive cells, or by the cumulative red intensity of bright red fluorescence.

[0115] SEAP reporter assay using HEK cells overexpressing Dectin-1 and an anti-Dectin-1 antibody. Anti-dectin-1 monoclonal antibody 2M24 (containing VH and VL domains with SEQ ID NOs. 7 and 8, respectively) or 15E2 and control isotypes were immobilized by coating the well surfaces of untreated 96-well U-bottom polypropylene microtiter plates. For coating, 10, 2, 1, 0.5, and 0.1 μg of anti-dectin-1 antibody, diluted in 50 μl of sterile PBS, were added to each well. The plates were left overnight in a Class II laminar flow cabinet with the lids removed to allow the solution to evaporate. The coated plates were washed twice with 200 μl of sterile PBS to remove salt crystals and unbound antibodies. HEK Blue h dectin-1-a cells were then cultured on the plates for 22 hours in RPM1 containing 10% ultra-low IgG FBS (VWR), and alkaline phosphatase levels were assessed at OD 630 nm in the supernatant using QUANTI Blue Solution (Invivogen, San Diego, CA) according to the manufacturer's instructions.

[0116] To measure HEK cell SEAP secretion induced by beads conjugated with anti-dectin-1 antibody, streptavidin-2M24 (hIgG4) was conjugated onto biotin polystyrene beads of sizes 3, 10, and 16 μm (Spherotech, Lake). The beads were conjugated with antibody by incubating them with Forest (IL) at room temperature for 30 minutes, and then washed twice with PBS to remove unbound antibody. The anti-dectin-1 antibody conjugated beads were then divided into 1x10⁶ beads. 5 After mixing individual HEK Blue h Dectin-1-α cells with RPM1 containing 10% ultra-low IgG FBS in a cell:bead ratio of 1:3 for 22 hours, alkaline phosphatase secretion was assessed in the supernatant at OD 630 nm as described above.

[0117] Cytokine secretion Anti-dectin-1 monoclonal antibody 2M24 or 15E2 clone and control isotype were immobilized by coating 10 μg onto the surface of the wells of an untreated 96-well U-bottom polypropylene microtiter plate as described above. Freshly isolated monocytes or peripheral blood mononuclear cells were then cultured for 24 hours at 200,000 cells / well in RPM1 containing 10% ultra-low IgG FBS on plates with the immobilized antibody. In other wells, cells were treated with 10 μg / ml of dectin-1 antibody in solution instead of the immobilized antibody. TNFα, IL-6, and IFNg in the supernatant were evaluated using the U-PLEX assay platform (Meso Scale Discovery), and their levels were expressed as a multiplier change in dectin-1 antibody-induced cytokine secretion compared to the isotype control. As a positive control, cells were stimulated with 25 μg / ml of zymosan.

[0118] result To produce dectin-1 antibodies, recombinant dectin-1 isoform B protein was subcutaneously inoculated into 4-week-old ATX-Gx Alloy transgenic mice, and the antigen was added once a week. The antibodies produced by this inoculation possess both a human variable domain and a mouse constant domain.

[0119] Of the 56 anti-dectin-1 antibody clone candidates generated in this study, the 2M24 clone was the only one that showed binding to both dectin-1 isoforms A and B on HEK cells, as well as monocytes. As shown in Figure 1A, the 2M24 anti-dectin-1 antibody clone showed high affinity for dectin-1 expressing human monocytes. In contrast, the other clones either bound only to dectin-1 isoform A (e.g., 2M08, 2M12, 2M38) or showed no binding at all (2M49). Furthermore, the affinity of 2M24 for dectin-1 was superior to that shown by the other clones and commercially available dectin-1 antibodies (15E2, 259931, GE2). Figure 1C shows a comparison of binding to human monocytes and to dectin-1 overexpressing HEK cells between the 2M24 clone, other dectin-1 clones identified from immunization of Alloy transgenic mice, and commercially available dectin-1 clones.

[0120] The 2M24 antibody was also evaluated for cross-reactivity with cynomolgus monkey dectin-1. Binding was assessed by flow cytometry analysis of cynomolgus monkey monocytes derived from PBMCs. As shown in Figure 1B, the anti-human dectin-1 clone 2M24 antibody showed cross-reactivity and high affinity for cynomolgus monkey dectin-1 expressed on monocytes. The 2M24 anti-dectin-1 antibody showed superior affinity compared to the commercially available antibodies tested, exhibiting an EC50 of 0.3 nM. The agonist 15E2 and 255931 commercially available antibodies showed EC50s of 14 nM and 16 nM, respectively, in cynomolgus monkey monocytes. Figure 1C shows a comparison of binding affinity between the 2M24 clone and the commercially available clones 15E2 and 259931 to cynomolgus monkey monocytes.

[0121] To evaluate the functionality of the 2M24 dectin-1 antibody in promoting phagocytosis, polystyrene beads were coated with the 2M24 antibody and mixed with HEK-Blue h dectin-1a cells or primary human monocytes. The 2M24 antibody efficiently induced phagocytosis of the beads. As shown in Figures 2A-2B, the 2M24 anti-dectin-1 antibody linked to the polystyrene beads promoted phagocytosis in both HEK-Blue h dectin-1a cells and human primary monocytes.

[0122] A fully human IgG4 isotype 2M24 antibody was generated from the mIgG1 2M24 clone. This antibody possesses both a human constant region and a variable region. The functionality of hIgG4 2M24 was then evaluated for binding to two dectin-1 expressing cell types: HEK-Blue h dectin-1a cells and human monocytes. As shown in Figures 3A-3B, fully human 2M24 showed high affinity binding to dectin-1 in transfected HEK cells (EC50 = 1.6 nM) and human monocytes (EC50 = 0.7 nM).

[0123] Next, the ability of the hIgG4 2M24 antibody to promote phagocytosis of beads in Dectin-1 expressing cells was tested. As shown in Figure 4, the hIgG4 2M24 antibody showed efficient phagocytic activity in HEK cells, human monocytes, and human macrophages overexpressing Dectin-1. Therefore, the fully human IgG4 2M24 antibody can promote phagocytosis in Dectin-1 expressing cells.

[0124] The fully human 2M24(hIgG4) anti-dectin-1 antibody was further tested for its ability to promote signal transduction via dectin-1. The activation of dectin-1 signaling by the antibody can be evaluated using a secreted alkaline phosphatase assay with HEK-Blue h-dectin-1a cells. HEK-Blue h-dectin-1a cells are engineered to express dectin-1 isoform A and genes involved in the dectin-1 / NF-κB / SEAP signaling pathway, and therefore express secreted alkaline phosphatase (SEAP) in response to stimulation with dectin-1 ligands. As shown in Figures 5A-5B, the 2M24(hIgG4) anti-dectin-1 antibody induced alkaline phosphatase secretion in HEK-Blue h-dectin-1a cells in both immobilized and bead-conjugate forms. These observations support the idea that SEAP secretion is promoted by the binding of the 2M24(hIgG4) antibody to Dectin-1 on the cell surface, demonstrating receptor clustering and agonist activity by this antibody. Furthermore, efficient clustering signaling of Dectin-1 can be promoted by beads conjugated with 2M24(hIgG4). Signaling was better induced with larger beads, reflecting better receptor clustering. This supports the possibility that clustering of Dectin-1 promoted by bispecific antibodies, including anti-Dectin-1 antibodies targeting phagocytic cells and antibodies targeting other cells such as cancer cells, may promote Dectin-1 clustering and signaling on phagocytic cells.

[0125] The native ligand for Dectin-1 clusters its receptors and signals downstream of Dectin-1 / Syk / NFκB, inducing inflammatory gene expression. To evaluate whether engagement with Dectin-1 antibodies in solution could induce cytokine secretion, monocytes or macrophages were treated with 10 μg / ml of commercially available anti-Dectin-1 antibody. As shown in Figures 6A-6B, the commercially available 15E2 anti-Dectin-1 antibody did not induce cytokine secretion in primary human macrophages and monocytes, suggesting insufficient clustering of the Dectin-1 receptor. This data supports the idea that free Dectin-1 antibodies in solution do not induce immunostimulation due to insufficient Dectin-1 clustering.

[0126] To evaluate whether cytokine secretion can be induced by the 2M24(hIgG4) anti-dectin-1 antibody, the antibody was immobilized on beads and cultured with monocytes or PBMCs. As shown in Figures 7A-7B, the 2M24 anti-dectin-1 antibody induced cytokine secretion in primary human monocytes and PBMCs. The 2M24 antibody not only promoted cytokine secretion but also showed superior immune stimulation compared to the immune stimulation promoted by the 15E2 anti-dectin-1 agonist antibody. Of the cytokines measured in this experiment, TNFα and IL6 are secreted by dectin-1 expressing monocytes. In contrast, IFNg is secreted by T cells mainly present in PBMCs. Since T cells do not express dectin-1, they are not directly activated by the anti-dectin-1 antibody, but rather activated by cytokines secreted by monocytes in PBMCs stimulated by the dectin-1 antibody. Therefore, the different effects of the dectin-1 antibody on IFNg were more pronounced in PBMCs than in pure monocytes.

[0127] Finally, we tested the activation of dectin-1 by its natural ligand in the presence of an anti-dectin-1 antibody. HEK-Blue h-dectin-1a cells were incubated in the presence of 8 ug / ml biotinylated laminarin with either 2M24(hIgG4) dectin-1 antibody or commercial 15E2, 259931, GE2 anti-dectin-1 antibodies, starting at 300 nM and continuing with 1 / 3 continuous dose titration. As shown in Figure 8, binding of 2M24(hIgG4) antibody to dectin-1 did not inhibit the binding of laminarin, the natural ligand of dectin-1. Therefore, engagement of dectin-1 with 2M24 anti-dectin-1 antibody does not inhibit pathogen clearance and is unlikely to increase susceptibility to potential fungal infections.

[0128] In conclusion, the 2M24 anti-dectin-1 antibody can induce phagocytosis by dectin-1 expressing cells and activate dectin-1 signaling without competing with the native ligand for dectin-1. The properties of the 2M24 and 15E2 antibodies are summarized in Figure 9.

[0129] Example 2: Bispecific anti-dectin-1 antibody This example describes the preparation and characterization of a bispecific antibody comprising a dectin-1 binding arm and a second arm that binds to a specific tumor antigen.

[0130] Materials and methods Creation of a bispecific body Antibodies were differentially labeled with MTA or FOL reagent according to the manufacturer's (AAT Bioquest) guidelines. The labeled antibodies were mixed and incubated to enable covalent bond construction via MTA and FOL interactions. The following antibodies were used as biotin:streptavidin-inducible bispecific antibodies: Anti-dectin-1 15E2 antibody heavy chain:mSA fusion QWQLQQSGAELARPGASWKMSCKASGYTFTTYTMHWWKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAWYYCARERAVLVPYAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH(Sequence ID 18) Anti-dectin-1 15E2 antibody light chain QIVLTQSPAVMSASPGEKWTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVPTRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 19) Avi-tagged anti-CD20 Fab heavy chain (CH1 domain based on hIgG4 sequence) QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE(Sequence ID 20) Anti-CD20 Fab light chain QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 21) Avi-tagged anti-HER2 Fab heavy chain (CH1 domain based on hIgG4 sequence) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE(Sequence ID 22) Anti-HER2 Fab Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 23)

[0131] Cell binding assay Dectin-1-expressing cells were labeled with calcein green, and target cells were labeled with calcein red. Cells were incubated in the presence of bispecific or isotype control antibodies and analyzed by flow cytometry. Cell binding was indicated by a double-positive signal (green + red +). Binding efficiency was quantified as the percentage of all target cells that formed doublets with Dectin-1-expressing cells.

[0132] Five million effector cells (dectin-1 expressing cells) or target cells (cells expressing the target, e.g., CD20-positive Raji cells or HER2-positive SKBR3 cells) were differentially labeled with either calcein green (0.5 nM) or calcein red / pHrodo-red (0.5 nM). The cells were thoroughly washed with PBS and placed on ice. Next, the effector and target cells were co-cultured in a 3:1 ratio (effector:target) in the presence of 2M24 bispecific antibody or isotype control and incubated at 37°C for 30 minutes. After incubation, the samples were gently resuspended and analyzed by flow cytometry. The PMT voltage was adjusted as appropriate, and cells were gated based on FITC and / or PE fluorescence corresponding to the calcein green or red fluorescence. Coupling efficiency was reported as the number of PE-positive cells (target cells) in the doublet population divided by the total number of PE-positive target cells in the reaction.

[0133] SEAP reporter assay using HEK cells overexpressing Dectin-1 and an anti-Dectin-1 antibody. To measure the amount of SEAP secreted by HEK cells induced by Raji cells (expressing CD20), Raji cells were coated on ice for 30 minutes with 2M24 / anti-hCD20 or hIgG4 / anti-CD20 bispecificates, followed by two washes with PBS to remove unbound bispecificates. Raji cells coated with bispecificates were then subjected to 1x10⁶ sampling in RPM1 containing 10% ultra-low IgG FBS. 5 The HEK Blue h Dectin-1-α cells were mixed with Raji cells in a 1:2 ratio (HEK cells:Raji cells). After 22 hours, the amount of alkaline phosphatase secreted in the supernatant was evaluated at OD 630 nm as described in Example 2.

[0134] result Dectin-1 agonist bispecific antibodies can utilize various modes of activity (e.g., immune activation, phagocytosis, neoantigen presentation, and adaptive immune activation) for targeted depletion of cancer cells (Figures 11A-11B). As a proof-of-concept for engaging a dectin-1 antibody (15E2 or 2M24) with a target antibody, a click chemistry approach was used to create a bispecific antibody containing an anti-dectin-1 targeting arm and a second arm targeting the target protein. This approach enabled the creation of bispecific antibodies for various assays. A schematic diagram of this approach is shown in Figures 10A-10B. Since phagocytosis of the target can be induced by the binding of dectin-1 by a dectin-1 specific antibody (see Examples 1 and 2), the bispecific antibody was evaluated for its ability to promote phagocytosis of specific target cells. First, the bispecific antibody was evaluated for its ability to eliminate CD70-expressing cancer cells by phagocytosis. CD70 is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) superfamily. CD70 is expressed at low levels in normal tissues, but is highly overexpressed in various diseases such as acute myeloid leukemia (AML), renal cell carcinoma, rheumatoid arthritis, and lupus.

[0135] Using click chemistry, a bispecific molecule containing a Dectin-1-targeting arm (anti-Dectin-1; clone 2M24) and a CD70-targeting arm (anti-hCD70; clone 113-16) was constructed. The purity of the bispecific (2M24 / anti-hCD70) antibody was evaluated by SDS-PAGE analysis (Figure 12A), and its binding affinity was evaluated by flow cytometry (Figure 12B). As shown in Figure 12B, cell binding tests revealed that 2M24 / anti-hCD70 bound to Dectin-1-expressing HEK293 cell lines with an EC50 of 1.8 nM, and to CD70-positive renal cancer cell lines with an EC50 of 12.34 nM (A498 cells) or 11.62 nM (786-0 cells). Subsequently, the bispecific molecule was evaluated for its ability to induce cell binding. As shown in Figure 13, the 2M24 / anti-hCD70 bispecificity induces ligation between dectin-1-expressing HEK293 cells and CD70-expressing renal cancer cells, resulting in cell doublets of HEK293 cells (labeled with calcein green) and A498 cells (labeled with calcein red).

[0136] Next, we evaluated the targeting of CD20-expressing cells by the bispecific compound. CD20 is a transmembrane protein present in almost all B cells from the stage in which they become involved in B cell development until it is downregulated when they differentiate into antibody-secreting plasma cells, and is considered a pan-B cell antigen marker. As shown in Figures 14A-14B, the 2M24 / anti-hCD20 bispecific compound induced binding between dectin-1-expressing cells (both dectin-1-expressing HEK293 cells and human M0 macrophages) and CD20-expressing B cells (Raji cell line). This bispecific intercellular binding can induce synapse formation between effector and target cells, alter cytokine signaling, activate phagocytosis, and ultimately lead to target antigen presentation.

[0137] To test the induction of signal transduction resulting from stimulation with a bispecific antibody that binds to Dectin-1, a secreted alkaline phosphatase assay was performed. As shown in Figure 15, Raji cells coated with the anti-Dectin-1 / anti-CD20 bispecific antibody induced alkaline phosphatase secretion in HEK-Blue h Dectin-1a cells. Therefore, ligating target cells to Dectin-1-expressing cells (such as phagocytes) using a bispecific antibody can promote signal transduction by Dectin-1-expressing cells. In the case of phagocytes, signal transduction can lead to cytokine production and immune stimulation.

[0138] It has already been demonstrated that Dectin-1 expression in HEK293 cells is necessary and sufficient to induce phagocytosis of beads of various sizes coated with anti-dectin-1 targeted antibodies (see Examples 1 and 2). To demonstrate phagocytosis of viable target cells, a bispecific compound containing Dectin-1 targeted arms and CD20 targeted arms was created. In a co-culture assay of HEK293 cells and CD20-expressing Raji cells, phagocytosis of cells treated with the anti-dectin-1 / anti-hCD20 bispecific compound was observed, in contrast to the isotype control bispecific compound (Figure 16). Furthermore, pre-incubation of cells with latrunclin A, a phagocytic inhibitor that inhibits actin polymerization, inhibited phagocytosis of cells treated with the anti-dectin-1 / anti-hCD20 bispecific compound. These findings indicate that Dectin-1 expression is sufficient to induce phagocytosis, and that simultaneous targeting of Dectin-1 and target cells with a Dectin-1 agonist bispecificity is sufficient to induce phagocytosis in target cells.

[0139] A proof-of-concept experiment was conducted using an anti-dectin-1 / anti-HER2 bispecific antibody to simultaneously target dectin-1-expressing cells and HER2-positive breast cancer cells. Approximately 20% to 25% of invasive breast cancers overexpress the human epidermal growth factor receptor (HER2 tyrosine kinase receptor). As shown in Figure 17, the anti-dectin-1(15E2) / anti-HER2 bispecific antibody induced linkage between dectin-1 and HER2-expressing cells. This interaction is thought to promote synapse formation between effector and target cells because dectin-1 clustering induces cytokine secretion by effector cells, triggers phagocytosis of target cells, and leads to neoantigen presentation and activation of adaptive immune cells (B and T cells).

[0140] Finally, we also evaluated the anti-dectin-1(2M24) / anti-hCD94 bispecificity antibody. Large granular lymphocytic (LGL) leukemia is a rare chronic lymphoproliferative disorder affecting T cell and natural killer (NK) cell lineages. CD94 / NKG2 is a family of C-type lectin receptors primarily expressed on the surface of NK cells and on a subset of CD8+ T lymphocytes. As shown in Figure 18, the anti-dectin-1(2M24) / anti-hCD94 bispecificity antibody induced ligation between dectin-1-expressing cells and CD94-expressing cells. Therefore, bispecific antibodies that bind to dectin-1 can mediate ligation between dectin-1-expressing cells and various target cells.

[0141] Example 3: Preparation of a bispecific anti-dectin-1 antibody using streptavidin-biotin This example describes the biochemical and functional characterization of a bispecific antibody that binds to Dectin-1, prepared using streptavidin-biotin conjugation.

[0142] Materials and methods Creation of a bispecific body mSA was genetically fused to either Fab 2M24 or full-length 2M24. The chimeric fusion was incubated with a biotinylated target antibody to create a bispecific compound containing a dectin-1 binding arm and a second arm that binds to the target receptor or the protein of interest. A full-length 2M24 sequence fused with mSA: (Sequence ID 15) Fab 2M24 sequence fused to mSA: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH(Sequence ID 17)

[0143] Antibody-dependent targeted phagocytosis of Phrodo-labeled beads Antibody-dependent targeted phagocytosis of Phrodo-labeled beads was performed as described in Example 2. To monitor phagocytosis by flow cytometry, HEK cells overexpressing Dectin-1 were incubated with biotin beads conjugated to Fab 2M24-mSA on ice or at 37°C for 30 minutes, followed by two washes with PBS. Phagocytosis was evaluated by detecting activated Phrodo red within the HEK cell / bead duplet population using flow cytometry in the PE channel with a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA).

[0144] result To enable the efficient production of bispecific antibodies, we developed a novel strategy utilizing the high-affinity interaction between streptavidin and biotin. Monomeric streptavidin (mSA) constructs were fused to the Fc domain of 2M24 or the CH1 domain of Fab 2M24. The recombinant fusion proteins were incubated with various biotinylated antibodies of interest to construct bispecific antibodies. A schematic diagram of this strategy is shown in Figures 19A-19B.

[0145] This fusion technology enables high-throughput production and screening of bispecific antibodies. To test this approach, the Fab 2M24-mSA fusion protein was produced and purified. As shown in Figures 20A-20C, the Fab 2M24-mSA fusion showed high affinity binding to Dectin-1 expressing cells (EC50 = 1.45 nM). This Fab 2M24-mSA fusion protein can be combined with various biotinylated antibodies against the target of interest. Furthermore, the Fab 2M24-mSA fusion also induced bead binding and phagocytosis by Dectin-1 expressing HEK 293 cells (Figures 21A-21B), indicating that the Fab version of the 2M24 antibody can efficiently promote phagocytosis in Dectin-1 expressing cells.

[0146] Using anti-dectin-1-streptavidin fusions, bispecific antibodies were created for various targets (e.g., CD20, CD19, CD70, amyloid B(1-42)). As shown in Figures 22A-22D, these bispecific antibodies exhibited high homogeneity based on HPLC analysis. These data demonstrate the robust feasibility of this technology for the production of bispecific antibodies.

[0147] Next, we evaluated the ability of anti-dectin-1 bispecifics, which were created using Fab 2M24-mSA fusion proteins, to induce cell ligation. As shown in Figure 23, Fab The 2M24-mSA / biotin anti-hCD20 bispecificity induced ligation between dectin-1 expressing HEK293 cells and CD20 expressing B cells (Raji cell line). This interaction can promote dectin-1 clustering, which induces cytokine secretion by effector cells, triggers phagocytosis of target cells, and leads to neoantigen presentation and activation of adaptive immune cells (B and T cells).

[0148] Example 4: Targeted phagocytosis of amyloid deposition using an anti-dectin-1 bispecific antibody This example describes the use of a bispecific antibody that binds to dectin-1 in the targeted delivery of pathogen antigens to phagocytic cells.

[0149] Dectin-1-induced targeted phagocytosis can be used to mediate amyloid clearance via antigen presentation (e.g., monocytes, macrophages, dendritic cells, and neutrophils), maximizing the depletion of circulating amyloid precursors (free light chains) and deposited amyloid fibrils. This targeted phagocytosis relies on bispecific antibodies that target dectin-1 (by 2M24) and AL amyloid (by amyloid-reactive antibodies or serum amyloid protein P antibodies).

[0150] To test this approach, we first demonstrated the recruitment or presence of antigen-presenting cells to amyloid deposition sites. We determined the phenotype of immune cell populations in newly isolated amyloid-accumulating tissues from AL amyloidosis patients. We measured the frequency of antigen-presenting cells (e.g., macrophages, monocytes, neutrophils, and dendritic cells). We evaluated Dectin-1 expression on APCs in AL amyloidosis patient tissues.

[0151] Next, bispecific antibodies were constructed, each containing a Dectin-1 binding arm (e.g., clone 2M24) and an amyloid binding arm (based on an external antibody). In proof-of-concept studies, phagocytosis of AL amyloid fibrils by circulating monocytes, monocyte-derived macrophages (differentiated in vitro), or patient-derived macrophages (in situ) was demonstrated with the Dectin-1 bispecific antibody prepared with the external antibody (Figure 24). An antibody search campaign was launched to identify high-affinity binders for amyloid fibrils or amyloid precursors, and the obtained antibodies were used to further construct Dectin-1 bispecific antibodies targeting amyloid deposits.

[0152] Example 5: Targeted phagocytosis of mast cells using an anti-dectin-1 bispecific antibody This example describes the use of a bispecific antibody that binds to dectin-1 in the targeted delivery of pathogen antigens to phagocytic cells.

[0153] Materials and methods Bead phagocytosis assay Large (approximately 16.5 μm) polystyrene anti-mouse Fc IgG beads were labeled with a pH-sensitive fluorescent dye (pHrodo red) and conjugated with an anti-dectin-1 antibody or isotype control. For phagocytic assays, the beads were incubated with cultured dendritic cells in a 1:3 (cell:bead) ratio. Phagocytosis of the beads was monitored by IncuCyte live-cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the total integrated intensity (sum fluorescence intensity) of red objects (pHrodo fluorescence) in the image.

[0154] Mastocyte diseases are characterized by the pathological accumulation of mast cells in one or more organs. Because mast cells are resident in tissues and therapeutic access to these cells is difficult, they can be engaged with tissue-resident macrophages to cooperate in the depletion and reduction of mast cells at pathological levels. As described in Example 1, engagement with Dectin-1 can promote phagocytosis of particles of similar size to cells, such as mast cells. Therefore, a Dectin-1-induced targeted phagocytosis platform can be applied for the targeted depletion of mast cells.

[0155] First, bispecific antibodies with macrophage-targeting arms (by dectin-1 binding) and mast cell-targeting arms (by mast cell surface antigens) were constructed as shown in Figure 25A. Mast cell surface antigens / receptors that could potentially be used as lead candidates for bispecific antibody construction are summarized in Figure 25B. Next, these bispecific antibodies were evaluated for their ability to bind to and target mast cells for phagocytosis by dectin-1 expressing cells and to deplete patient mast cells in situ. Phagocytosis of in vitro differentiated mast cells or mast cell lines by monocyte-derived macrophages (differentiated in vitro) or patient-derived macrophages (in situ) was demonstrated using dectin-1 bispecific antibodies.

[0156] The presence or recruitment of antigen-presenting cells (e.g., macrophages, monocytes, neutrophils, and dendritic cells) was demonstrated in patient tissues / organs with a large number of mast cells. The phenotype of immune cell populations was determined in fresh tissues isolated from mastocytosis patients. Dectin-1 expression was evaluated on APCs. Binding of external antibodies was assessed in donor mast cells.

[0157] Considering the large size of mast cells, we evaluated whether Dectin-1 could promote phagocytosis of large entities by conjugating an anti-dectin-1 antibody onto large beads (approximately 16.5 μm) that are similar in size to large cells. Since macrophages are large phagocytic cells capable of taking up large targets, we used macrophages differentiated from monocytes and performed a phagocytosis assay of large beads for 6 days in the presence of MCSF. As shown in Figure 26, the anti-dectin-1 antibody promoted targeted phagocytosis of large beads in cultured human macrophages. The anti-dectin-1 conjugated beads were taken up more readily by macrophages compared to isotype control conjugated beads. These data support the idea that targeting of Dectin-1 can target large cells such as mast cells (size: 16-20 μm) for phagocytosis.

[0158] Example 6: Targeted depletion of microorganisms using anti-dectin-1 bispecific antibodies This example describes the use of a bispecific antibody that binds to dectin-1 in targeted phagocytosis of microorganisms.

[0159] Materials and methods ELISA binding to H3N2 influenza virus To evaluate the binding of anti-dectin-1 / α-hemagglutinin bispecific antibodies to H3N2 influenza virus, H3N2 influenza particles at concentrations of 2.5, 5, and 10 ug / mL were coated overnight on high-binding 96-well plates. The plates were washed twice with PBS, blocked with 3% BSA in PBS / Tween-20 0.05% for 1 hour at room temperature, and then washed again with PBS / Tween-20 0.05%. Primary antibodies containing anti-dectin-1 (15E2), anti-hemagglutinin (12CA5), anti-dectin-1 / anti-hemagglutinin bispecificity, and isotype controls were incubated at 20 nM for 1 hour at room temperature. The plates were then washed twice with PBS / Tween-20 0.05% and incubated with 1:5000 secondary anti-mouse Fcg:HRP for 1 hour at room temperature. Finally, the plate was washed, incubated with the TMB substrate for 30 minutes, and the reaction was stopped with 2N H2SO4. The plate was read at 450 nm with a plate reader.

[0160] Innate immune cells play a crucial role in the recognition and elimination of microbial pathogens. To assist phagocytic cells in attacking bacterial, viral, or fungal pathogens, dectin-1-induced targeted phagocytosis can be performed using bispecific antibodies that include a dectin-1 targeting arm that binds to antigen-presenting cells (macrophages, monocytes, dendritic cells, and neutrophils) and a second arm that targets antigens expressed on the pathogen surface (Figure 27). Targeted phagocytosis of pathogens allows effector cells to efficiently recognize the target pathogen and secrete cytokines and proteases that can directly kill the bound pathogen. Furthermore, bispecific antibodies can mediate dectin-1 clustering and induce targeted phagocytosis of the bound target. Finally, after the degradation of the target, the target antigen is presented, regulating the adaptive immune response so that the host organism can further fight off the pathogen.

[0161] As proof of principle, a high-affinity antibody against the target pathogen-specific surface antigen was identified and used to construct a bispecific antibody containing a Dectin-1 binding arm (anti-Dectin-1 antibody 2M24) and a pathogen-targeting arm. The anti-Dectin-1 / anti-pathogen bispecific antibody was tested for target binding, cytokine secretion by phagocytic cells after target engagement, target phagocytosis and pathogen degradation, and target antigen presentation. Furthermore, the high-affinity antibody was constructed to validate the pathogen target and subsequently used to construct a Dectin-1 bispecific antibody from lead candidates.

[0162] To test the targeted delivery of pathogens to phagocytic antigens, a bispecific antibody was constructed having a dectin-1 binding arm and a second arm that binds to hematoglutinin of the influenza H3N2 virus. The anti-dectin-1 / anti-hemagglutinin bispecific antibody was then tested for binding using both ELISA and flow cytometry. As shown in Figures 28A-28B, the anti-dectin-1 / anti-hemagglutinin bispecific antibody efficiently bound to both the H3N2 influenza virus and HEK cells expressing dectin-1. This antibody configuration can be used to target influenza viruses or influenza virus antigens to antigen-presenting cells (e.g., dendritic cells, macrophages).

[0163] Example 7: Targeted delivery of antigens for vaccine production This example describes the targeted delivery of viral antigens to phagocytic cells.

[0164] Materials and methods Bead phagocytosis assay Small (approximately 3.4 μm) polystyrene anti-mouse Fc IgG beads were labeled with a pH-sensitive fluorescent dye (pHrodo red) and conjugated with an anti-dectin-1 antibody or isotype control. For phagocytic assays, the beads were incubated with cultured dendritic cells in a 1:3 (cell:bead) ratio. Phagocytosis of the beads was monitored by IncuCyte live-cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the total integrated intensity (sum fluorescence intensity) of red objects (pHrodo fluorescence) in the image.

[0165] Labeling of polystyrene beads with pHrodo and conjugation to antibodies As described above, polystyrene beads were labeled with pHrodo and conjugated to antibodies.

[0166] To coat polystyrene beads with the SARS-CoV-2 spike protein S1, goat anti-rabbit IgG(Fc) beads (Spherotech) were labeled with pHrodo Red as described above and conjugated with rabbit anti-Flag antibody (Cell Signaling). Then, flag-tagged spike protein (Genscript) was ligated to the pHrodo / anti-Flag beads, and unbound spike protein was washed away with PBS.

[0167] Antibody-dependent targeted phagocytosis of Phrodo-labeled beads To enable phagocytosis of SARS-CoV-2 spike protein coated beads, the beads were pre-incubated at room temperature for 60 minutes with anti-dectin-1 / anti-SARS-CoV-2 spike protein bispecificity (anti-SARS-CoV-2 spike protein antibody purchased from Genscript), and the unbound antibody was washed away. The beads were then mixed with HEK cells.

[0168] result Dendritic cells are specialized antigen-presenting cells. Targeting antigens expressed on disease-causing substances (cancer cells, pathogens, or protein aggregates), or the antigens themselves, to dendritic cells via Dectin-1 can potentially trigger a protective immune response against the antigen and the antigen-derived disease-causing substance. This response includes T cell activation and proliferation, cytokine secretion, and B cell activation. Therefore, Dectin-1 antibody-targeted vaccines can be designed to deliver antigens to dendritic cells and promote the recognition and elimination of disease-causing substances (e.g., cancer cells or pathogens). Target antigens can be fused to anti-Dectin-1 antibodies for delivery to APCs (Figure 29A), or anti-Dectin-1 bispecific antibodies can be used to target the delivery of disease-causing substances to APCs (Figure 29B).

[0169] To measure whether the phagocytic activity of human dendritic cells can be enhanced by antibody engagement with Dectin-1, purified monocytes (CD14+) derived from human PBMCs were differentiated into dendritic cells in the presence of IL4 / GMCSF. After 6 days, the dendritic cells were incubated with pHrodo-labeled polystyrene beads conjugated with 15E2 anti-dectin-1 antibody or an isotype control. As shown in Figure 30, the anti-dectin-1 antibody enhanced the targeted phagocytosis of the beads in cultured monocyte-derived dendritic cells. Significantly greater phagocytosis was observed with anti-dectin-1 conjugated beads than with isotype-conjugated beads, as evidenced by the bright red pHrodo particles within the cells. Dendritic cells are specialized antigen-presenting cells. By targeting antigens on disease-causing substances (e.g., cancer cells, pathogens, or protein aggregates), or the antigens themselves, to dendritic cells via Dectin-1, a protective immune response against the antigen and the antigen-derived disease-causing substances may be triggered. This response may include T cell activation and proliferation, cytokine secretion, and B cell activation. Therefore, dectin-1 antibody-targeted vaccines can be designed to deliver antigens to dendritic cells to promote the recognition and elimination of disease-causing substances (e.g., cancer cells or pathogens).

[0170] We also evaluated the targeting of another virus, SARS-CoV-2, using a dectin-1 agonist bispecific antibody. Using click chemistry, bispecific antibodies targeting dectin-1 and the SARS-CoV-2 spike S1 protein were constructed. The spike protein was coated onto beads, which were then taken up in the presence of the anti-dectin-1 / anti-SARS-CoV-2 spike S1 bispecific antibody (Figure 31A). The anti-dectin-1 / anti-SARS-CoV-2 spike S1 bispecific antibody induced ligation between dectin-1-expressing HEK293 cells and the spike-coated beads (Figure 31B), and promoted phagocytosis of the spike-coated beads by dectin-1-expressing cells (Figure 31C). Based on these results, the anti-dectin-1 / anti-SARS-CoV-2 spike S1 bispecific antibody may be able to mediate the targeted delivery of the SARS-CoV-2 spike protein to macrophages.

[0171] conclusion The bispecific antibody described in this embodiment, comprising a dectin-1 binding arm and a second arm that binds to an antigen from a pathogen such as influenza virus or SARS-CoV-2, can promote the uptake of the target pathogen, antigen presentation, subsequent T cell activation and proliferation, and antibody production by B cells. The adaptive immune response can promote the elimination of the virus and virus-infected cells. The dectin-1 antibody-targeted vaccine approach may also work against other pathogens of bacterial or viral origin.

[0172] We produced various anti-dectin-1 (e.g., 2M24) / antigen-specific bispecific antibodies and demonstrated their efficient binding to APCs. We evaluated the internalization of target antigens from APCs. Methods have been developed to evaluate antigen presentation of target antigens on the surface of dendritic cells. Activation of CD4+ and CD8+ T cells (T cell proliferation and cytokine secretion) was evaluated from dendritic cells that received antigens via dectin-1. We evaluated B cell activation and antibody production against antigens. Mice were vaccinated to protect against diseases caused by pathogens / malignant cells, and their adaptive immune responses were evaluated in vivo.

[0173] Example 8: Design of bispecificity for producing human bispecific antibodies targeting Dectin-1 and disease targets or antigens To enable the construction and efficient production of highly purified, active bispecific antibodies, design principles were adopted based on previously reported strategies such as "knob-into-hole" (Ridgway, 1996; patent US8679785B2), DuetMab (Mazor, 2015; patent EP3452089A2), single-step protein A and G avidity purification (Ollier, 2019; AU2018204314B2), and mutations to eliminate FcR binding (patent WO2016 / 081746A2). Construction of complete bispecific antibodies involves the expression of four distinct subunits cloned into an expression vector such as pFUSE. A diagram of an exemplary anti-dectin-1 bispecific antibody is shown in Figure 32A.

[0174] As shown in Table 1, bispecific antibodies were constructed using this design for proof-of-concept testing. These bispecific antibodies have one arm targeting h-dectin-1 and a second arm targeting proteins on hCD20, hHER2, hCD70, or RSV. The bispecific antibodies listed in Table 1 were prepared by expressing all four chains and purifying them to 95% purity and uniformity. All bispecific antibodies were confirmed to bind to their respective targets. [Table 2]

[0175] The variable domains of the antibody arm opposite to anti-dectin-1 in Table 1 were as follows: CD20 VH:QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (Sequence ID 24) CD20 VL:QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK(Sequence ID 25) HER2 VH:EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS(Sequence ID 34) HER2 VL:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 35) CD70 VH:EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS(Sequence ID 38) CD70 VL:QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVL(Sequence ID 39) RSV VH:QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSANQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSS(Sequence ID 36) RSV VL:DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTAFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK(Sequence ID 37).

[0176] These h-dectin-1 bispecific antibodies engage with three targets: dectin-1 on bone marrow cells, antigens on target cells or disease-causing substances, and Fc receptors on bone marrow cells and NK cells, inducing robust immune stimulation and phagocytosis (Figure 32B). In particular, bispecific antibodies with an unfucosylated active hIgG1 Fc domain can recruit bone marrow cells (e.g., monocytes, macrophages, and dendritic cells) and natural killer (NK) cells to eliminate disease-causing target cells, such as tumor cells, that express specific antigens. In the context of cancer, while we do not wish to be bound by theory, the dual engagement of Dectin-1 and Fcγ receptors on myeloid cells and NK cells is thought to induce a potent immune response that ultimately eliminates cancer cells through the following mechanisms: (1) Bispecific antibody-induced crosslinking of Dectin-1 and Fcγ receptors leads to ITAM-dependent activation of downstream inflammatory pathways and the release of immunomodulatory cytokines and cytotoxic proteins (proteases, perforins) that can modulate the tumor microenvironment and directly kill target cells; (2) Bispecific antibody-induced clustering of Dectin-1 and Fcγ receptors leads to phagocytosis and elimination of targeted cancer cells by monocytes, macrophages, and dendritic cells; and (3) the phagocytosed antigens are presented by macrophages and DCs (this is the process that triggers a T-cell immune response aimed at eliminating cancer cells).

[0177] The 2M24 / CD20 and 2M24 / RSV bispecific antibodies listed in Table 1 were tested for binding to cells expressing human dectin-1 or CD20. 2M24 / RSV was used in all assays as an isotype control for the target binding arm. The bispecific variants tested here included mutations in the hIgG1 Fc domain (hIgG1 inactive form) that eliminate Fc binding to the Fc receptor (EU numbering L234A, L235E, and G237A). Binding of 2M24 / CD20 or 2M24 / RSV bispecific antibodies to HEK293 cells stably expressing human dectin-1 was evaluated by flow cytometry (Figure 33A). The 2M24 / CD20 and 2M24 / RSV hIgG1 inactive bispecific antibodies were able to bind to human dectin-1 expressing cells with similar affinity (cell-based binding EC50 values ​​of 1.4 and 1.7 nM, respectively). Therefore, bispecific antibodies for 2M24 / CD20 or 2M24 / RSV showed high affinity binding to Dectin-1 expressing HEK293 cells.

[0178] The binding of rituximab, 2M24 / CD20, or 2M24 / RSV bispecific antibodies (active or inactive hIgG1 isotypes) was also evaluated using the CD20-expressing B-cell lymphoma Raji cell line (Figure 33B). While the 2M24 / CD20 bispecific (active or inactive hIgG1 isotype) antibodies were able to bind to CD20-expressing Raji cells, their affinity was at least 10-fold lower compared to rituximab. Although we do not wish to be constrained by theory, the difference in CD20 binding affinity between the 2M24 / CD20 bispecific antibody and rituximab is thought to be due to a loss of avidity (monovalent vs. divalent binding) in the bispecific antibody.

[0179] Next, the ability of a 2M24 / CD20 bispecific antibody to induce ligation between cells expressing hdectin-1 and cells expressing hCD20 was assayed. Dectin-1 expressing HEK293 cells (effector) and CD20 expressing Raji cells (target) were differentially labeled with calcein green (effector) or calcein red (target) dye. Labeled cells were co-cultured and treated with hIgG1-inactive 2M24 / CD20 or 2M24 / RSV (control) bispecific antibodies to induce effector:target ligation. Successful effector:target cell ligation was indicated by double positive staining (calcein green+, calcein red+, square box; Figure 34A). Ligation efficiency (quantified as the percentage of all target cells that bind or ligate to effector cells) was assayed using dose titration of the bispecific antibody in co-cultures of effector:target cells (Figure 34B).

[0180] These results demonstrate that the 2M24 / CD20 bispecific antibody can ligate Dectin-1 expressing "effector" cells and CD20 expressing "target" cells with a potent EC50 of 0.17 nM. Despite the low affinity binding of the 2M24 / CD20 bispecific antibody to CD20 on Raji cells (Figure 33B), 2M24 / CD20 ligated very efficiently. These findings suggest that the binding affinity of 2M24 / CD20 is enhanced by high expression of Dectin-1 or CD20 in both effector and target cells (avidence), thereby promoting efficient ligation of the two cells. Based on these findings, the 2M24 / CD20 bispecific antibody is thought to be able to effectively engage Dectin-1 expressing monocytes, macrophages, or dendritic cells with target disease cells such as B-cell lymphoma cells that express high levels of CD20. Effector:Target engagement is the first step in the MOA of the 2M24 bispecific antibody.

[0181] The active human IgG1 isotype binds to Fcγ receptors on NK cells or monocytes. Therefore, we evaluated whether the active hIgG1 isotype of 2M24 / CD20 can induce monocyte killing by NK cells (antibody-dependent cell-mediated cytotoxicity, via ADCC) or other monocytes (fructoridine or antibody-dependent cell-mediated phagocytosis, via ADCP). In this scenario, the active hIgG1 domain of 2M24 / CD20 engages with Fcγ receptors on NK cells or monocytes and with Dectin-1 receptors on monocytes, thereby inducing Fcγ-mediated activation and target depletion. PBMCs from two healthy donors, Donor 76 (Figure 35A) and Donor 77 (Figure 35B), were treated for 24 hours with increasing concentrations of 2M24 / CD20 bispecific hIgG1 (active or inactive isotype) and rituximab. The levels of remaining viable CD14+ monocytes were then quantified by flow cytometry (as a percentage of the isotype control). No decrease in monocyte count was observed in either donor, indicating that 2M24 / CD20 active IgG1 did not induce monocyte depletion. While we do not wish to be constrained by theory, it appears that 2M24 / CD20 hIgG1 (active isotype) does not affect monocyte levels, and therefore the risk of infection is minimal.

[0182] Based on the proposed MOA of the 2M24 / CD20 bispecific antibody (shown in Figure 32B), B cell depletion by the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody or rituximab was evaluated to compare B cell depletion. PBMCs from two healthy donors, Donor 83 (Figure 36A) and Donor 84 (Figure 36B), were treated with the indicated antibody at increasing concentrations for 24 hours, and then analyzed by flow cytometry to quantify the level of remaining viable CD19+ B cells (reported as a percentage of B cells in treated PBMCs of isotype control). Thus, in the two healthy donors, high concentrations of the 2M24 / CD20 bispecific antibody significantly induced B cell depletion (approximately 80% reduction) compared to rituximab (approximately 40% reduction), despite the divalent binding affinity of rituximab and an approximately 10-fold difference in binding affinity (shown in Figure 33B). The unique mechanism of action of 2M24 / CD20-activated IgG1 (Figure 32B), including binding to Dectin-1 on bone marrow cells, Fcγ receptors on NK cells and monocytes, and CD20 on target B cells, results in overall superior B cell depletion compared to rituximab. These data support the concept that Dectin-1-induced immunostimulation via 2M24 / CD20 bispecificity promotes target cell depletion.

[0183] The ability of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody or rituximab (hIgG1) to downregulate CD19 expression on B cells in a process known as shaving or trogocytosis was evaluated. CD19+ expression on B cells from two healthy donors, donor 83 (Figure 37A) and donor 84 (Figure 37B), was quantified by flow cytometry after 24-hour incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab, or isotype control. The effect of 2M24 / CD20 bispecific antibody and rituximab on CD19 expression on B cells was evaluated using mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugate). In PBMCs derived from donor 83, the EC50 for CD19 expression was 0.014 nM with rituximab and 0.080 nM with the 2M24 / CD20 hIgG1 bispecific antibody (Figure 37A). In PBMCs derived from donor 84, the EC50 for CD19 expression was 0.013 nM with rituximab and 0.090 nM with the 2M24 / CD20 hIgG1 bispecific antibody (Figure 37B). Both the 2M24 / CD20 active IgG1 bispecific antibody and rituximab caused downregulation of CD19 expression on B cells. Interestingly, rituximab showed at least 5-fold stronger shaving compared to the 2M24 / CD20 bispecific antibody. Downregulation of target CD20 on B cells has already been reported as a mechanism by which malignant B cells evade rituximab-mediated depletion (Beum, PV et al. (2006) J. Immunol. 176:2600-2609). Therefore, these findings suggest that the 2M24 / CD20-activated IgG1 bispecificity may be superior to rituximab in B cell depletion due to its reduced shaving ability.

[0184] Immunostimulation induced by the 2M24 / CD20-activated hIgG1 bispecific antibody resulted in the secretion of a unique cytokine repertoire compared to rituximab (Figure 38). ELISA-based (mesoscale discovery) quantification of cytokines was performed on the supernatant isolated from healthy donor PBMCs treated with 2M24 / CD20-activated hIgG1 bispecific, rituximab, or isotype control. PBMCs were stimulated with the antibody overnight, followed by analysis of the supernatant in MSD. The cytokines tested were IFNγ, IL-12p70, IL-6, TNFα, IL-1β, IL-4, IL-13, IL-10, and IL-8. The results indicated that 2M24 / CD20-activated IgG1 induced higher levels and more distinct cytokine activation in PBMCs compared to rituximab. Furthermore, engagement of Dectin-1 and Fc receptors by the 2M24 / RSV bispecific antibody alone did not induce cytokine release, ruling out the possibility of systemic cytokine activation. These findings highlight a unique MOA that distinguishes the 2M24 / CD20-activated IgG1 bispecific antibody from rituximab. While we do not wish to be constrained by theory, these findings further suggest that 2M24 / CD20 may be able to induce the release of Th1 and Th2 type responses and promote immune stimulation of the tumor microenvironment.

[0185] The 2M24 / CD20 hIgG1 (active isotype) bispecific antibody was also found to induce superior B cell depletion and lower CD19 shaving compared to rituximab in co-cultures of human macrophages and GFP-expressing Raji B cells. Co-cultures of human macrophages and Raji-GFP cells (3:1 ratio) were analyzed by flow cytometry in the presence of the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, 2M24 / RSV control, fucosylated rituximab, or isotype hIgG1 control. Co-cultures were incubated at 37°C for 24 hours and then stained with PE anti-CD206 antibody to label macrophages and BV-605 anti-CD19 antibody to label Raji cells. The number of remaining viable / Raji-GFP+ cells was evaluated at the end of the experiment. The primary antibody was used in continuous dose titration. CD19 was evaluated on Raji-GFP cells after 24 hours (Figure 39B), and B cell receptor levels were shown as a decrease in CD19 MFI in the presence of anti-dectin-1 / anti-hCD20 bispecificity or rituximab. EC50 for CD19 expression was 0.020 nM with rituximab and 0.95 nM with 2M24 / CD20 hIgG1 bispecificity. These results indicate that B cell depletion (mediated by the Fcγ receptor) was accelerated by the 2M24 / CD20 bispecific antibody compared to rituximab. Rituximab reduced B cell receptor CD19 surface levels more potently than the anti-dectin-1 / anti-hCD20 bispecific antibody. Similarly, B cell receptor shaving against CD20 was observed with rituximab, and the reduction in CD20 limits the B cell depletion effect of rituximab. While we do not wish to be constrained by theory, these data suggest that the superiority of 2M24 / CD20 hIgG1 (active isotype) bispecificity over B cell depletion is due to lower B cell receptor shaving compared to rituximab. This highlights a different mechanism of cell depletion by 2M24 / CD20 hIgG1 (active isotype) bispecificity.

[0186] B cell depletion was also analyzed in single-cell suspensions from kidney cancer tissue samples. Single-cell suspensions from two kidney cancer tissue samples were analyzed by flow cytometry in the presence of 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, 2M24 / RSV hIgG1 control, fucosylated rituximab, and their respective isotype controls. Kidney cancer tissue samples were dissociated into single-cell suspensions and treated with primary antibody (2 μg / ml) at 37°C for 24 hours. The immune cell population was analyzed by flow cytometry (Figures 40A and 40B). The number of remaining viable B cells was evaluated using anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (Figure 40C). The 2M24 / CD20 active IgG1 bispecific antibody significantly induced tissue B cell depletion compared to rituximab in single-cell suspensions from kidney cancer samples. The 2M24 / CD20 hIgG1 (active isotype) bispecific antibody reduced B cells by 44% and 46% (respectively) in samples from two kidney cancer donors, while rituximab induced B cell reductions of 33% and 18%, respectively (Figure 40C). This data supports the function of the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody in depleting cancer cells via dectin-1-induced immunostimulation and Fcγ receptor engagement. While we do not wish to be constrained by theory, given that dectin-1 is primarily expressed in tumor-associated macrophages (TAMs) in the above samples, it is conceivable that the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody could engage TAMs and promote the depletion of target cells.

[0187] Cytokine secretion from cultured macrophages and single-cell suspensions of renal cancer specimens stimulated with immobilized anti-dectin-1 antibody (clone 2M24) or 2M24 / CD20 bispecific antibody was tested. Anti-dectin-1 antibody (clone 2M24), isotype control, or 2M24 / CD20 bispecific antibody was immobilized overnight at 10 μg per well in U-bottom polypropylene microtiter plates, followed by the cultivation of single-cell suspensions from human monocyte-derived macrophages (Figures 41A and 41B) or renal cancer specimens (Figure 41C). Cells were cultured for 24 hours, and TNFα secretion in the supernatant was evaluated by ELISA. As a positive control, cells were stimulated with zymosan. Anti-dectin-1 antibody (clone 2M24) was found to induce dectin-1 clustering and TNFα secretion from human macrophages. These data provide evidence that the parental anti-dectin-1 antibody (clone 2M24) can promote immunostimulation in primary macrophage cultures and single-cell homogenates of cancer samples. Since dectin-1 is expressed in bone marrow cells, tumor-associated macrophages in cancer samples are expected to produce cytokines in response to anti-dectin-1 antibody stimulation. This promotes the transition of tumor-associated macrophages from anti-inflammatory to pro-inflammatory states, resulting in a potent antitumor effect. Furthermore, monovalent binding of the 2M24 / CD20 bispecific antibody to dectin-1 was sufficient to promote dectin-1 clustering and immunostimulation of macrophages.

[0188] Immunostimulation by immobilized anti - dectin - 1 antibody in single - cell suspensions from renal cancer specimens was also analyzed (Figure 42). Single - cell suspensions from renal cancer specimens were treated with immobilized anti - dectin - 1 antibody (clone 2M24) or isotype - control hIgG4 antibody for 24 hours. Supernatants were analyzed by ELISA for the release of various cytokines including IFNγ, IL - 6, TNFα, IL - 23, IL - 12p70, IL - 10, and IL - 13. These results indicate that activation of dectin - 1 in myeloid cells (in this example, dectin - 1 is mainly expressed by tumor - associated macrophages, TAMs) induced the release of a specific repertoire of cytokines either directly downstream of the dectin - 1 signaling pathway or indirectly through the activation of other immune cells. Without wishing to be bound by theory, it is considered that engagement of dectin - 1 by the 2M24 bispecific antibody promotes immunostimulation, modulates the tumor microenvironment, and may support the elimination of target - expressing cancer cells.

[0189] Example 9: Characterization of Bispecific Antibodies Targeting Dectin - 1 and CD20 In this example, further characterization of bispecific antibodies targeting human dectin - 1 and human CD20 will be described. The anti - dectin - 1 arm contained the variable domains of 2M24, and the anti - CD20 arm contained the variable domains of rituximab (see SEQ ID NO: 24 and SEQ ID NO: 25 for the VH domain and VL domain, respectively).

[0190] Materials and Methods CD16 Expression on NK Cells Human PBMCs from healthy donors were treated with serial dilutions of 2M24 / CD20 hIgG1 KIF, rituximab KIF, and isotype - control RSV hIgG1 KIF antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage - specific markers for flow cytometry analysis. CD16 expression on CD56 + NK cells was quantified and compared to the expression levels in the isotype - control treatment group.

[0191] CD19 Expression on B Cells Human PBMCs derived from healthy donors were treated with 0.1 nM 2M24 / CD20 hIgG1 KIF, rituximab KIF, and isotype-controlled RSV hIgG1 KIF antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. CD19 expression (MFI) on B cells was quantified.

[0192] B cell depletion in PBMCs Human PBMCs derived from healthy donors were treated with serial dilutions of the indicated antibody. 24 hours after treatment, the PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified compared to an untreated control group (shown as a dotted line in Figure 45).

[0193] B cell depletion in kidney cancer samples Single-cell suspensions were prepared from kidney cancer specimens, and cells were treated with 2M24 / CD20 hIgG1, 2M24 / RSV hIgG1, rituximab hIgG1, and isotype-controlled RSV hIgG1 antibodies. 24 hours after treatment, cells were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified as the proportion of CD19+ cells within a CD45+ immune cell population.

[0194] result First, we investigated the effect of the 2M24 / CD20 bispecificity on CD16 expression in human NK cells. Since CD16 is required for ADCC activity by NK cells, loss of CD16 expression may reduce the cytotoxicity of NK cells. Rituximab induced a potent and robust reduction of CD16 on NK cells compared to 2M24 / CD20 hIgG1 KIF (Figure 43). In contrast, CD16 levels on NK cells were well maintained after treatment with the 2M24 / CD20 bispecific antibody compared to rituximab treatment. While we do not wish to be constrained by theory, it is thought that the 2M24 / CD20 bispecificity may better preserve the cytotoxicity of NK cells.

[0195] Next, the effect of the 2M24 / CD20 bispecificity on CD19 expression in human B cells was investigated. Maintaining the expression of the target antigen is important for the therapeutic activity of monoclonal antibodies. B cell antigens such as CD20, CD19, and BCMA are validated immuno-oncological targets. CD19 is known to be downregulated by shaving / shedding after binding to anti-CD19 antibodies. A bystander effect was observed when using CD20-targeted antibodies, and CD19 expression decreased after treatment with rituximab, but not with the 2M24 / CD20 hIgG1 KIF bispecificity (Figure 44). CD19 levels on B cells were better maintained with the 2M24 / CD20 bispecificity antibody compared to rituximab. While we do not wish to be constrained by theory, therapeutically, the 2M24 / CD20 bispecificity may exhibit long-term activity because it has minimal impact on target antigen expression.

[0196] To compare rituximab with the anti-CD20 antibody obinutuzumab, 2M24 bispecific antibodies against CD20 were constructed using the variable domain sequences of either rituximab or obinutuzumab. The variable domain sequence of obinutuzumab was as follows: VH:QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS(SEQ ID NO: 46); VL:DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK(SEQ ID NO: 47).

[0197] In the ADCC / ADCP assay, 2M24 / CD20 (derived from the rituximab sequence) showed near-complete depletion of B cells and was superior to 2M24 / CD20 (derived from obinutuzumab) or the parental bivalent antibody and isotype control (Figure 45). These data support the use of the rituximab sequence for the creation of a 2M24 / CD20 bispecificity.

[0198] Example 10: Characterization of bispecific antibodies targeting Dectin-1 and CD20 in exploratory studies in non-human primates. This example describes the results of an exploratory study on the safety and efficacy of the bispecific antibodies targeting human dectin-1 and human CD20 described in Example 9, in cynomolgus monkeys.

[0199] Materials and methods Three groups of cynomolgus monkeys (one male and one female per group) were administered a single dose of the test product (5 mg / kg): A) 2M24 / CD20 hIgG1 KIF, B) 2M24 / CD20 hIgG1 inactive form, and C) rituximab hIgG1 KIF. Blood samples were collected at the indicated time points. The abbreviations for the test products are as follows (2M24 / CD20 KIF, 2M24 / CD20 inactive form, RTX KIF).

[0200] B cell levels were evaluated by flow cytometry. Depletion was quantified by the number of CD19+ B cells remaining in the sample after administration, compared to the level before administration of the test substance. Bone marrow and lymph node aspirates were collected at indicated times, and B cell levels were evaluated by flow cytometry. Depletion was quantified by the number of CD19+ B cells remaining in the sample after administration (day 7), compared to the level before administration of the test substance (days 1-7).

[0201] For the PBMC assay, PBMCs from healthy cynomolgus monkeys were 2M24 / CD20 Cells were treated with serial dilutions of hIgG1 KIF, rituximab KIF, and isotype control RSV hIgG1 KIF antibody. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cell depletion was quantified compared to the isotype control group.

[0202] result This exploratory study was designed to verify the safety and efficacy of a 2M24 / CD20 bispecific antibody in non-human primates. The study design is shown in Figure 46. Cynomolgus monkeys were divided into three treatment groups, each consisting of two monkeys (one male and one female). Each group received a single dose of a specific test product at 5 mg / kg. The test product was as follows: 1) 2M24 / CD20 1) hIgG1 KIF, 2) 2M24 / CD20 hIgG1 inactive form, and 3) rituximab hIgG1 KIF. Animals were monitored daily, and samples such as whole blood, bone marrow, lymph nodes, and colorectal tissue were collected as instructed. This study was planned for 8 weeks.

[0203] As shown in Figure 47 (top), the 2M24 / CD20 hIgG1 KIF bispecific antibody depleted B cells in vivo in cynomolgus monkeys. Nearly complete and sustained B cell depletion (approximately 98%) was observed in both groups of animals treated with a single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF. In the rituximab group (Figure 48), one animal showed complete depletion, while the second showed robust but incomplete depletion (approximately 87%). In the 2M24 / CD20 hIgG1 inactive group (Figure 47 bottom), partial B cell depletion was observed in one animal, but the second animal did not show depletion on day 7. The 2M24 / CD20 hIgG1 KIF bispecific antibody was well-tolerated in cynomolgus monkeys.

[0204] The 2M24 / CD20 hIgG1 KIF bispecific strain also depleted B cells in the bone marrow (Figure 49A) and lymph nodes (Figure 49B) in vivo in cynomolgus monkeys. A single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF induced robust B cell depletion in the bone marrow (approximately 87-88%) and partial depletion in the lymph nodes (60-78%) in both animals. B cell depletion was also observed in both tissues in the rituximab group. In the 2M24 / CD20 hIgG1 inactive strain group, partial B cell depletion was observed, with the exception of animal CB764A, which showed the least B cell depletion in the lymph nodes.

[0205] The 2M24 / CD20 hIgG1 KIF bispecific antibody also induced robust depletion of cynomolgus monkey B cells ex vivo (Figure 50). KIF induced robust B cell depletion compared to rituximab hIgG1 KIF. The maximum depletion achieved by rituximab was approximately 30% of B cells, while the 2M24 / CD20 hIgG1 KIF bispecificity showed a maximum depletion of approximately 50%.

[0206] Example 11: Purification and functional characterization of 2M24 / CD20 bispecific antibody in scFv format. This example describes the preparation, purification, and characterization of a 2M24 / CD20 bispecific antibody in which the Dectin-1 targeting arm (based on the 2M24 variable domain) is fused to a human IgG1 Fc domain with a knob-forming mutation (scFv), and the CD20 targeting arm is based on rituximab hIgG1 with a hole-forming mutation. A diagram of this molecule is shown in Figure 51. The knob-forming mutation on the Dectin-1 targeting arm was T366W, and the hole-forming mutations on the CD20 targeting arm were T366S, L368A, and Y407V. While we do not wish to be constrained by theory, this format is considered to provide a universal platform for preparing anti-Dectin-1 bispecific antibodies with simpler manufacturing requirements (e.g., compared to bispecific antibodies with anti-Dectin-1 arms having multiple polypeptide chains).

[0207] 2M24 scFv / CD20 hIgG1 was expressed in Hek293 cells by transfecting three plasmids (2M24 scFv hIgG1 plasmid, CD20 heavy chain, and CD20 light chain). Four days after expression, the supernatant was collected and purified via Protein A. Aggregates were removed by size exclusion chromatography. As shown in Figure 52A, the 2M24 scFv / CD20 hIgG1 bispecific antibody was purified as a homogeneous molecule on SEC.

[0208] Next, co-cultures of CD20-expressing Raji cells and dectin-1-expressing HEK reporter assay were treated while increasing the concentration of the 2M24 scFv / CD20 hIgG1 bispecific. Activation of the reporter was evaluated by measuring the SEAP level (based on absorbance at 630 nm) in the medium. The bispecific molecule promoted targeted immunostimulation as evaluated by this NFkB reporter assay (Figure 52B). [[ID=⑦]]

[0209] [[ID=⑧]] [[ID=⑨]]To verify B cell depletion, human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. Twenty-four hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified relative to an untreated control group (shown as a dotted line in Figure 52C). The results demonstrated that the 2M24 scFv / CD20 hIgG1 bispecific antibody was able to deplete human B cells, similar to the 2M24 / CD20 hIgG1 KIF molecule (Figure 52C). [[ID=⑩]] [[ID=⑪]]

[0210] [[ID=⑫]] [[ID=⑬]]Example 12: Preparation and Characterization of Anti-dectin-1 / Anti-Trop-2 Bispecific Antibody [[ID=⑭]] Trop-2 is a 323aa type I membrane protein involved in calcium signaling, embryonic and fetal development, tight junction formation, and integrin-dependent signaling. Mutations in Trop-2 are associated with gelatinous drop-like corneal dystrophy, characterized by corneal amyloidosis and blindness. Trop-2 is overexpressed in various epithelial cancers and promotes cell proliferation, invasion, and angiogenesis. High expression is associated with poor prognosis and reduced survival in many cancers (particularly TNBC breast cancer and NSCLC lung cancer). Sacituzumab govitecan (Trodelvy®), a Trop-2-targeted ADC, is the only treatment approved for the treatment of patients with metastatic TNBC. It received accelerated approval from the FDA in 2020.

[0211] Therefore, Trop-2 is a clinically validated oncological target. However, an unmet need exists: Trodelvy® achieved a 33% response rate in a population of metastatic TNBC patients with multiple prior treatment histories. Side effects such as neutropenia, diarrhea, and vomiting are associated with Trodelvy toxin (SN-38) conjugates.

[0212] In contrast, anti-dectin-1 targeted approaches have the potential to limit their antitumor activity to the disease microenvironment. The immunomodulatory and phagocytic activity of 2M24 is tightly controlled by the presence of cancer cells. While Trodelvy® has a single mechanism of action (delivery of a toxin conjugate that induces the killing of target cells), anti-dectin-1 targeted approaches utilize multiple mechanisms of action (targeted immune stimulation, phagocytosis, and antigen presentation) to eliminate cancer cells and promote sustained immunity.

[0213] This example describes the preparation and characterization of an anti-dectin-1 (2M24) / anti-Trop-2 bispecific antibody. The variable domains used for the anti-Trop-2 antibody are as follows: VH:QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 42); VL:DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 43).

[0214] The 2M24 / Trop-2 bispecific antibody was purified by size exclusion chromatography, and the purified antibody was analyzed by SDS-PAGE under non-reducible (NR) or reduced (R) conditions (Figure 53A). The 2M24 / Trop2 bispecific antibody was purified as a monodisperse molecule. It was found to bind with high affinity to Dectin-1 expressing HEK cells (Figure 53B) and with moderate affinity to the Trop-2 expressing A431 cancer cell line (Figure 53C).

[0215] To evaluate Trop-2 expression levels on different cancer cells, cancer cell lines (A431 and SKBR3) were stained with anti-human PE Trop-2 or isotype control antibodies, and Trop-2 expression was assessed. Receptor copy number was determined by comparing the fluorescence intensity of fluorescently dye-labeled microspheres with a known amount of fluorophores with the fluorescence intensity of labeled cells determined by flow cytometry. Trop-2 was highly expressed in cancer cell lines A431 and SKBR3, with receptor copy numbers of 8.4 million and 1.1 million, respectively (Figure 54). These results indicate that the Trop-2 polypeptide is highly expressed on cancer cells. Due to its high expression on cancer cells, Trop-2 is an attractive antigen for targeted killing of cancer cells with 2M24 / Trop-2 bispecific antibodies.

[0216] Next, the binding of the 2M24 / Trop-2 bispecific antibody to Trop-2 expressing cell lines was investigated. Cancer cell lines (HeLa, SiHa, BxPC-3, and Capan-2) were incubated with a single concentration (300 nM) of serial dilutions of either the 2M24 / Trop-2 hIgG1 bispecific antibody or an isotype control antibody. A secondary antibody (AF647 goat anti-human) was used for ...

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[Claim 1] The invention described in the specification.

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