Anti-Dectin-1 Antibody and Method of Use Thereof

Abstract

The present invention relates to anti-human Dectin-1 antibody clone 2M24 (hIgG4) or 15E2, and multispecific binding molecules comprising same, as well as methods for producing and using same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 088,895, filed October 7, 2020, and U.S. Provisional Patent Application No. 63 / 174,439, filed April 13, 2021, the disclosures of each of which are incorporated herein by reference in their entireties.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Format (CRF) (Filename: 186542000440SEQLIST.TXT, Recorded: October 6, 2021, Size: 108,547 bytes).

[0003] The present disclosure relates to antibodies, multispecific (eg, bispecific) binding molecules that bind to human Dectin-1, and related methods of use and production. [Background technology]

[0004] Phagocytosis is the primary mechanism used to remove pathogens and cellular debris. Professional phagocytes, such as monocytes, macrophages, dendritic cells, and granulocytes, specifically recognize and ingest abnormal or disease-causing host or foreign material. The ingested material is destroyed through the phagocyte's endolysosomal pathway. Additionally, dendritic cells and macrophages can present antigens to cells of the adaptive immune system to further facilitate the elimination of disease-causing agents.

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

[0006] It would be beneficial to develop targeted removal and degradation of accumulated disease-causing agents without increasing overall phagocytosis. The present 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 by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention [Means for solving the problem]

[0008] The present disclosure relates to antibodies, multispecific (e.g., bispecific) binding molecules that bind to human Dectin-1, and related methods of use and production. Disclosed herein is a method of targeted phagocytosis for the removal of disease-causing agents, including host cells / host cell products, microorganisms, or their products, by administering a multispecific (e.g., bispecific) binding molecule comprising a Dectin-1-binding arm and a second arm that specifically binds to the agent. The multispecific (e.g., bispecific) binding molecule enables phagocytes to engage with the target agent, form a synapse therebetween, and promote the clustering of Dectin-1 on the phagocyte. This stimulates phagocytosis of the target agent and simultaneously stimulates cytokine secretion by the phagocyte via the Dectin-1 / Syk / NfkB pathway. Furthermore, antigens from the endocytosed agent are presented on the surface of dendritic cells / macrophages, enhancing the adaptive immune response against the disease-causing agent. Overall, Dectin-1 agonist multispecific (e.g., bispecific) binding molecules are believed to promote immunostimulation, targeted phagocytosis, and neoantigen presentation / activation of the adaptive immune system to eliminate disease-causing agents.

[0009] Thus, this disclosure describes, inter alia, the generation and functional characterization of agonistic anti-human Dectin-1 antibodies that exhibit high-affinity binding to Dectin-1 and can promote immune stimulation. Additionally, the generation of bispecific antibody formats comprising anti-human Dectin-1 antibodies and antibodies targeting antigens on disease-causing agents is described, with data supporting target engagement, immune stimulation, phagocytosis, and antigen presentation.

[0010] In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that binds to human Dectin-1, 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 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 the VL domain comprises 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, an antibody or antigen-binding fragment thereof that binds to human Dectin-1, wherein the antibody or fragment comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, and the VH domain has the VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVS Provided herein is an antibody or fragment thereof, wherein the VL domain comprises CDR-H1, CDR-H2, and CDR-H3 from 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, provided herein is an antibody or antigen-binding fragment thereof that binds to human Dectin-1, 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 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 VL domain comprises CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising 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). and / or the VL 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 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, provided herein is an antibody or antigen-binding fragment thereof that binds to human Dectin-1, wherein the antibody or fragment binds 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, provided herein is an antibody or antigen-binding fragment thereof that binds to human Dectin-1, wherein the antibody or fragment is capable of binding to human or cynomolgus Dectin-1. In some embodiments, provided herein is an antibody or antigen-binding fragment thereof that binds to human Dectin-1, wherein the antibody or fragment does not compete with a natural ligand of Dectin-1, e.g., human Dectin-1 (e.g., for binding to human Dectin-1). 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 a CDR-H1 comprising the sequence GYTFTDYY (SEQ ID NO: 1), a CDR-H2 comprising the sequence INPNSGDT (SEQ ID NO: 2), and a CDR-H3 comprising the sequence ARNSGSYSFGY (SEQ ID NO: 3), and a light chain variable (VL) domain comprising a CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), a CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and a CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6); (b) a heavy chain variable (VH) domain comprising a CDR-H1 comprising the sequence DYYI (SEQ ID NO: 88), a CDR-H2 comprising the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and a CDR-H3 comprising the sequence NSGSYSFGY (SEQ ID NO: 90), and a light chain variable (VL) domain comprising a CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), a CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and a CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6). A light chain variable (VL) domain comprising a CDR-L1 comprising the sequence GASSLQS (SEQ ID NO: 92), and a CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6); or (c) a heavy chain variable (VH) domain comprising the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable (VL) domain comprising the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).In some embodiments, the antibody or fragment binds to the same epitope of human Dectin-1 as a reference antibody comprising: (a) a heavy chain variable (VH) domain comprising a CDR-H1 comprising the sequence GYTFTDYY (SEQ ID NO: 1), a CDR-H2 comprising the sequence INPNSGDT (SEQ ID NO: 2), and a CDR-H3 comprising the sequence ARNSGSYSFGY (SEQ ID NO: 3), and a light chain variable (VL) domain comprising a CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), a CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and a CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6); (b) a heavy chain variable (VH) domain comprising a CDR-H1 comprising the sequence DYYI (SEQ ID NO: 88), a CDR-H2 comprising the sequence WINPNSGDTNYAQKFQG (SEQ ID NO: 89), and a CDR-H3 comprising the sequence NSGSYSFGY (SEQ ID NO: 90), and a light chain variable (VL) domain comprising a CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), a CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and a CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6). A light chain variable (VL) domain comprising CDR-L1 comprising the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6); or (c) a heavy chain variable (VH) domain comprising the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable (VL) domain comprising the 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 a 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 binds to human Dectin-1 and a second antigen-binding domain that binds to a target of interest, or comprising a first antigen-binding domain that binds to a target of interest 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-chain variable fragment (scFv) comprising a VH domain and a VL domain that binds to human Dectin-1 and a first antibody arm comprising a first Fc region, an antibody heavy chain comprising the VH domain of a second antigen-binding domain associated with an antibody light chain comprising the VL domain of the second antigen-binding domain, and a second antibody arm comprising a second Fc region connected to the VH domain of the second antigen-binding domain. In some embodiments, the second antigen-binding domain binds CD20 and comprises a VH domain comprising the sequence of SEQ ID NO: 24 and a VL domain comprising the sequence of SEQ ID NO: 25. In some embodiments, the second antigen-binding domain binds Trop-2 and comprises a VH domain comprising the sequence of SEQ ID NO: 42 and a VL domain comprising the sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding domain binds light chain amyloid and comprises a VH domain comprising the sequence of SEQ ID NO: 44 and a VL domain comprising the sequence of SEQ ID NO: 45. In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the first Fc region comprises a T366W substitution according to EU numbering and the second Fc region comprises T366S, L368A, and Y407V substitutions. In some embodiments, the first antibody arm comprises a first linker between the VH domain and the VL domain and a second linker between the VL domain and the first Fc region, hi some embodiments, the first linker comprises one or more repeats of the sequence GGGGS (SEQ ID NO: 26).In some embodiments, the first linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the second linker comprises the sequence EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). In some embodiments, the bispecific antibody comprises a first IgG antibody comprising a first antigen-binding domain covalently linked to a second IgG antibody comprising a second antigen-binding domain. In some embodiments, the bispecific antibody comprises a first antibody arm comprising a first antibody heavy chain comprising a VH domain of the first antigen-binding domain and a first Fc region, and a second antibody arm comprising a second antibody heavy chain comprising a VH domain of the 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 cognate hole-forming mutations. In some embodiments, the first Fc region comprises a T366W substitution according to EU numbering, and the second Fc region comprises T366S, L368A, and Y407V substitutions. In some embodiments, the bispecific antibody comprises a first antibody arm comprising a first antibody heavy chain comprising a VH domain of a first antigen-binding domain and a first Fc region, and a second antibody arm comprising a second antibody heavy chain 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 cognate 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 bispecific antibody comprises a first IgG antibody comprising a first antigen-binding domain linked to a biotin- or avidin-binding derivative thereof, and a second IgG antibody comprising a second antigen-binding domain linked to an avidin-, streptavidin-, neutravidin-, or biotin-binding derivative thereof, wherein the biotin- or avidin-binding derivative is bound to the avidin-, streptavidin-, neutravidin-, or biotin-binding derivative. In some embodiments, the bispecific antibody comprises a first IgG antibody comprising a first antigen-binding domain linked to an avidin-, streptavidin-, neutravidin-, or biotin-binding derivative thereof, and a second IgG antibody comprising a second antigen-binding domain linked to a biotin- or avidin-binding derivative thereof, wherein the biotin- or avidin-binding derivative is bound to the avidin-, streptavidin-, neutravidin-, or biotin-binding derivative. In some embodiments, the target of interest is a disease-causing agent. In some embodiments, the disease-causing 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 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, an eosinophil, an ILC2 cell, or an inflammatory immune cell.In some embodiments, the target of interest is a surface antigen of a virus. In some embodiments, the target of interest is an antigen expressed on the surface of a cancer cell. In some embodiments, the target of interest is CD70, HER2, DLL3, Nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR. In some embodiments, the target of interest 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 comprises an amino acid substitution at one or more of positions 234, 235, and 237 according to EU numbering. In some embodiments, each of the antibody heavy chains comprises an L234A, an L235E, and a G237A substitution according to EU numbering. In some embodiments, the antibody comprises two antibody heavy chains, only one of which comprises an H435R and a Y436F substitution according to EU numbering. In some embodiments, the antibody comprises two arms, only one of which comprises a heavy chain comprising an F126C and a C220V substitution according to EU numbering and a light chain comprising an S121C and a C214V substitution according to EU numbering.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 a first antibody light chain, and the VH domain of the second antibody heavy chain forms an antigen-binding domain with the VL domain of a second antibody light chain, wherein the first antibody heavy chain comprises F126C, C220V, and T366W substitutions according to EU numbering, the first antibody light chain comprises S121C and C214V substitutions, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R, and Y436F substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain further comprise L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain. In some embodiments, the antibody comprises a first antibody heavy chain and a second antibody heavy chain, wherein at least one or two of the first antibody heavy chain and the second antibody heavy chain are non-fucosylated. In some embodiments, the antibody may be produced in a cell line with an α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) knockout. In some embodiments, the antibody may be produced in a cell line overexpressing β1,4-N-acetylglucosaminyltransferase 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 a cell line treated with an inhibitor of mannosidase I, such as kifunensine.

[0011] In some embodiments, provided herein are multispecific binding molecules comprising: (a) a first antibody, or antigen-binding fragment thereof, comprising a first antigen-binding domain that binds to human Dectin-1; and (b) a second antibody, or antigen-binding fragment thereof, comprising a second antigen-binding domain that binds to a target of interest. In some embodiments, the target of interest is a disease-causing agent. In some embodiments, the disease-causing 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 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, an 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 of interest 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 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 the VL domain comprises 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 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). and / or the VL 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 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; binds to human Dectin-1 expressed on the surface of cells with an EC50 of less than 2 nM; is capable of binding to human or cynomolgus Dectin-1; and / or does not compete with the natural ligand of human Dectin-1. In some embodiments, the second antigen-binding domain binds to CD20 and comprises a VH domain comprising the sequence of SEQ ID NO: 24 and a VL domain comprising the sequence of SEQ ID NO: 25. In some embodiments, the second antigen-binding domain binds to Trop-2 and comprises a VH domain comprising the sequence of SEQ ID NO: 42 and a VL domain comprising the sequence of SEQ ID NO: 43. In some embodiments, the second antigen-binding domain binds to light chain amyloid and comprises a VH domain comprising the sequence of SEQ ID NO: 44 and a VL domain comprising 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 antibodies, nanobodies, or scFv fragments. In some embodiments, one or both of the first and second antibodies or fragments further comprise 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, e.g., 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, e.g., each comprising an antibody heavy chain and an antibody light chain. In some embodiments, the multispecific binding molecule comprises a first antibody arm comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain that binds to human Dectin-1 and a first Fc region, and a second antibody arm comprising 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 the second antigen-binding domain, and a second Fc region connected to the VH domain of the second antigen-binding domain.In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the first Fc region comprises a T366W substitution according to EU numbering, and the second Fc region comprises T366S, L368A, and Y407V substitutions. In some embodiments, the first antibody arm comprises 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 comprises one or more repeats of the sequence GGGGS (SEQ ID NO: 26). In some embodiments, the first linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 27) or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 28). In some embodiments, the second linker comprises the sequence EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). In some embodiments, a first antibody or fragment binds to its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative, and a second antibody or fragment is linked to its biotin- or avidin-linked derivative; or the second antibody or fragment is linked to its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative, and the first antibody or fragment is linked to its biotin- or avidin-linked derivative; the first antibody or fragment is linked to the second antibody or fragment via an interaction between its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative and its biotin- or avidin-linked 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 comprising 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 comprises a first IgG antibody comprising a first antigen-binding domain covalently linked to a second IgG antibody comprising a second antigen-binding domain. In some embodiments, a multispecific binding molecule comprises a first antibody arm comprising a first antibody heavy chain comprising a VH domain of a first antigen-binding domain, a first Fc region, and a first antibody light chain comprising a VL domain of the first antigen-binding domain; and a second antibody arm comprising a second antibody heavy chain comprising a VH domain of a second antigen-binding domain, a second Fc region, and a second antibody light chain comprising a VL domain of the 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 cognate hole-forming mutations. In some embodiments, the first Fc region comprises a T366W substitution according to EU numbering, and the second Fc region comprises T366S, L368A, and Y407V substitutions. In some embodiments, a multispecific 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 cognate 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, a multispecific binding molecule comprises two antibody Fc regions, wherein each of the antibody heavy chains comprises an amino acid substitution at one or more of positions 234, 235, and 237 according to EU numbering. In some embodiments, each of the antibody Fc regions comprises L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the multispecific binding molecule comprises two antibody heavy chains, where only one of the antibody heavy chains comprises H435R and Y436F substitutions according to EU numbering, hi some embodiments, only one of the antibody arms comprises a heavy chain that comprises F126C and C220V substitutions according to EU numbering, and a light chain that comprises S121C and C214V substitutions according to EU numbering.In some embodiments, the multispecific 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, wherein the first antibody heavy chain comprises F126C, C220V, and T366W substitutions according to EU numbering, the first antibody light chain comprises S121C and C214V substitutions, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R, and Y436F substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain further comprise L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain. In some embodiments, at least one or two of the antibody heavy chains are nonfucosylated. In some embodiments, the antibody may be produced in a cell line with an α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) knockout. In some embodiments, the antibody may be produced in a cell line overexpressing β1,4-N-acetylglucosaminyltransferase 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 a cell line treated with an inhibitor of mannosidase I, such as kifunensine.

[0012] In some embodiments, the antibody is a combination of (a) a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to a first target of interest; (b) a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to a second target of interest; wherein (i) the first antibody or fragment is linked to an avidin-, streptavidin-, neutravidin-, or biotin-binding derivative thereof, and the second antibody or fragment is linked to a biotin- or avidin-binding derivative thereof; or (ii) the second antibody or fragment is linked to an avidin-, streptavidin-, neutravidin-, or biotin-binding derivative thereof.

[0013] Provided herein are multispecific binding molecules comprising: a first antibody or fragment linked to a first antibody or fragment linked to a biotin- or avidin-conjugated derivative, streptavidin-conjugated derivative, neutravidin-conjugated derivative, or biotin-conjugated derivative, and the first antibody or fragment linked to the biotin- or avidin-conjugated derivative; wherein the first antibody or fragment binds to the second antibody or fragment via an interaction between its avidin-conjugated derivative, streptavidin-conjugated derivative, neutravidin-conjugated derivative, or biotin-conjugated derivative and its biotin- or avidin-conjugated derivative. In some embodiments, the first target of interest 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 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 the VL domain comprises 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 first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH domain comprisesThe 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 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 VL domain comprises CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising 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%, 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 multispecific binding molecule has the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFT a first polypeptide chain comprising: FGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31);Array QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV Provided herein is a multispecific binding molecule comprising a second polypeptide chain comprising the sequence SLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 32), and a third polypeptide chain comprising the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33). In some embodiments, a multispecific binding molecule comprises a first arm comprising a first antigen-binding domain and a second arm comprising 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 of interest; and the first armArrayQVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPC Provided herein are multispecific binding molecules comprising polypeptide chains comprising PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31). 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 is a human antibody or fragment or a humanized antibody or fragment. In some embodiments, one or both of the first and second antibodies or fragments is a 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 comprises an Fc domain. In some embodiments, the first antibody or fragment is a Fab fragment and the second antibody comprises 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 comprising 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 comprising an antibody heavy chain and an antibody light chain. In some embodiments, the second target of interest is a disease-causing agent. In some embodiments, the disease-causing 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 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, an eosinophil, an ILC2 cell, or an inflammatory immune cell. In some embodiments, the target of interest is a surface antigen of a virus. In some embodiments, the target of interest is an antigen expressed on the surface of a cancer cell. In some embodiments, the target of interest is CD70, HER2, DLL3, Nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR.In some embodiments, the target of interest 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 Trop-2 and comprises a VH domain comprising the sequence of SEQ ID NO: 42 and a VL domain comprising the sequence of SEQ ID NO: 43. In some embodiments, the second antigen binding domain binds light chain amyloid and comprises a VH domain comprising the sequence of SEQ ID NO: 44 and a VL domain comprising the sequence of SEQ ID NO: 45.

[0013] In some embodiments, provided herein is a polynucleotide encoding the antibody or multispecific binding molecule of any one of the above embodiments. In some embodiments, provided herein is a vector (e.g., an expression vector) comprising the polynucleotide of any one of the above embodiments. In some embodiments, provided herein is a host cell (e.g., an isolated host cell or cell line) comprising the polynucleotide or vector of any one of the above embodiments. In some embodiments, provided herein is a method of producing an antibody or multispecific binding molecule, the method comprising culturing a host cell of any one of the above embodiments under conditions suitable for production of the antibody or multispecific binding molecule. In some embodiments, the method further comprises recovering the antibody or multispecific binding molecule. In some embodiments, provided herein is a pharmaceutical composition comprising the antibody or multispecific binding molecule of any one of the above embodiments and a pharmaceutically acceptable carrier.

[0014] In some embodiments, a method of making a multispecific binding molecule comprises: (a) providing a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to a first target of interest; (b) providing a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to a second target of interest; wherein (i) the first antibody or fragment is linked to an avidin-, streptavidin-, neutravidin-, or biotin-binding derivative thereof, and the second antibody or fragment is linked to a biotin- or avidin-binding derivative thereof; or (ii) the second antibody or fragment is linked to an avidin-, streptavidin-, neutravidin-, or biotin-binding derivative thereof. (c) 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-, streptavidin-, neutravidin-, or biotin-linked derivative and the biotin- or avidin-linked derivative, thereby creating a multispecific binding molecule.In some embodiments, a method for identifying a multispecific binding molecule that binds a first target of interest and a second target of interest comprises: (a) providing a first antibody, or antigen-binding fragment thereof, comprising a first antigen-binding domain that binds to the first target of interest; (b) providing a second antibody, or antigen-binding fragment thereof, comprising a second antigen-binding domain that binds to the second target of interest; wherein (i) the first antibody or fragment is linked to an avidin-, streptavidin-, neutravidin-, or biotin-linked derivative thereof, and the second antibody or fragment is linked to a biotin- or avidin-linked derivative thereof; or (ii) the second antibody or fragment is linked to an avidin-, streptavidin-, or biotin-linked derivative thereof. , a streptavidin-linked derivative, a neutravidin-linked derivative, or a biotin-linked derivative, and the first antibody or fragment is linked to its biotin-linked or avidin-linked derivative; (c) contacting the first antibody or fragment with a 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 its avidin-linked, streptavidin-linked, neutravidin-linked, or biotin-linked derivative and its biotin-linked or avidin-linked derivative, thereby producing a multispecific binding molecule; and (d) measuring binding between the multispecific binding molecule and at least one of the first target of interest and the second target of interest. In some embodiments, the first target of interest 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 a CDR-H1 comprising the sequence GYTFTDYY (SEQ ID NO: 1), a CDR-H2 comprising the sequence INPNSGDT (SEQ ID NO: 2), and a CDR-H3 comprising the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises a CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), a CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and a CDR-L3 comprising 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 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 VL domain comprises CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising 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). and / or the VL 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 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 antibodies, nanobodies, or scFv fragments. In some embodiments, one or both of the first and second antibodies or fragments further comprise 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, e.g., 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, e.g., each comprising 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 comprising 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 of interest is a disease-causing agent. In some embodiments, the disease-causing 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 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, an 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 of interest is an antigen expressed on the surface of a cancer cell, hi some embodiments, the target of interest is CD70, HER2, DLL3, Nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR.In some embodiments, the target of interest 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 comprises an amino acid substitution at one or more of positions 234, 235, and 237 according to EU numbering. In some embodiments, each of the antibody heavy chains comprises an L234A, an L235E, and a G237A substitution according to EU numbering. In some embodiments, the antibody comprises two antibody heavy chains, only one of which comprises an H435R and a Y436F substitution according to EU numbering. In some embodiments, only one of the antibody arms comprises a heavy chain comprising an F126C and a C220V substitution and a light chain comprising an S121C and a C214V substitution according to EU numbering. In some embodiments, a bispecific antibody comprises two antibody heavy chains and two antibody light chains, wherein the VH domain of a first antibody heavy chain forms an antigen-binding domain with the VL domain of a first antibody light chain, and the VH domain of a second antibody heavy chain forms an antigen-binding domain with the VL domain of a second antibody light chain, wherein the first antibody heavy chain comprises F126C, C220V, and T366W substitutions according to EU numbering, the first antibody light chain comprises S121C and C214V substitutions, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R, and Y436F substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain further comprise L234A, L235E, and G237A substitutions according to EU numbering.In some embodiments, the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain. In some embodiments, at least one or two of the antibody heavy chains are nonfucosylated. In some embodiments, the antibody may be produced in a cell line with an α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) knockout. In some embodiments, the antibody may be produced in a cell line overexpressing β1,4-N-acetylglucosaminyltransferase 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 a cell line treated with an inhibitor of mannosidase I, such as kifunensine.

[0015] In some embodiments, provided herein are methods for treating a disease or disorder, comprising administering to an individual in need thereof an effective amount of any one of the antibodies, multispecific binding molecules, or compositions described in the above embodiments. In some embodiments, the first target of interest is human Dectin-1, and the second target of interest is a disease-causing agent. In some embodiments, the disease-causing 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 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, an 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 disease or disorder is cancer, a bacterial infection, a fungal infection, a viral infection, a mast cell disease or disorder, systemic mastocytosis, amyloidosis (e.g., light chain amyloidosis or Alzheimer's disease), or an age-related disease or disorder. In some embodiments, the target of interest 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 a human.

[0016] In some embodiments, provided herein are methods of treating cancer, comprising administering to an individual in need thereof an effective amount of a composition comprising a multispecific binding molecule, the multispecific binding molecule comprising: (a) a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to human Dectin-1; and (b) a second antibody or antigen-binding fragment thereof 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 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, e.g., 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 a CDR-H1 comprising the sequence GYTFTDYY (SEQ ID NO: 1), a CDR-H2 comprising the sequence INPNSGDT (SEQ ID NO: 2), and a CDR-H3 comprising the sequence ARNSGSYSFGY (SEQ ID NO: 3), and the VL domain comprises a CDR-L1 comprising the sequence QGISSW (SEQ ID NO: 4), a CDR-L2 comprising the sequence GAS (SEQ ID NO: 5), and a CDR-L3 comprising 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 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 VL domain comprises CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising 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). and / or the VL 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 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, a multispecific 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 cognate hole-forming mutations. In some embodiments, the first Fc region comprises a T366W substitution according to EU numbering, and the second Fc region comprises T366S, L368A, and Y407V substitutions. In some embodiments, a multispecific 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 cognate 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 antibody comprises two antibody Fc regions, each of which comprises an amino acid substitution at one or more of positions 234, 235, and 237 according to EU numbering. In some embodiments, each of the antibody heavy chains comprises L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the antibody comprises two antibody heavy chains, only one of which comprises H435R and Y436F substitutions according to EU numbering. In some embodiments, only one of the antibody arms comprises a heavy chain comprising F126C and C220V substitutions and a light chain comprising S121C and C214V substitutions according to EU numbering. In some embodiments, the bispecific antibody comprises two antibody heavy chains and two antibody light chains, wherein the VH domain of a first antibody heavy chain forms an antigen-binding domain with the VL domain of a first antibody light chain, and the VH domain of a second antibody heavy chain forms an antigen-binding domain with the VL domain of a second antibody light chain, wherein the first antibody heavy chain comprises F126C, C220V, and T366W substitutions according to EU numbering, the first antibody light chain comprises S121C and C214V substitutions, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R,and Y436F substitutions. In some embodiments, the first antibody heavy chain and the second antibody heavy chain further comprise L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain. In some embodiments (e.g., the second antigen-binding domain binds to CD20), the antibody comprises the following three polypeptide chains: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQP a first polypeptide chain comprising: 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 heavy chains of the antibody are non-fucosylated. In some embodiments, the antibody may be produced in a cell line with an α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) knockout. In some embodiments, the antibody may be produced in a cell line overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III). In further embodiments, the cell line further overexpresses Golgi μ-mannosidase II (ManII). ...It may be produced in a cell line treated with an inhibitor of mannosidase I, such as kifunensine. In some embodiments, the individual is a human.

[0017] In some embodiments, provided herein is a kit or article of manufacture comprising the antibody, multispecific binding molecule, or composition of any one of the above embodiments and instructions for using the antibody, multispecific binding molecule, or composition according to the method of any one of the above embodiments.

[0018] It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the detailed description that follows. [Brief explanation of the drawings]

[0019] [Figure 1A] Figure 1 shows the binding analysis of anti-human Dectin-1 antibody (clone 2M24) on human monocytes and monkey monocytes derived from peripheral blood mononuclear cells (PBMCs) by flow cytometry. To identify monocytes, single viable CD14+ cells were gated. Cells were incubated with 2M24 anti-Dectin-1 primary antibody or mIgG1 isotype control antibody, followed by incubation with a fluorescent anti-mouse secondary antibody. Primary antibodies were used in serial dose titration. Figure 1 shows the binding analysis of anti-human Dectin-1 clone 2M24 on human monocytes. [Figure 1B] Figure 1 shows the binding analysis of anti-human Dectin-1 antibody (clone 2M24) on human monocytes and monkey monocytes derived from peripheral blood mononuclear cells (PBMCs) by flow cytometry. To identify monocytes, single viable CD14+ cells were gated. Cells were incubated with 2M24 anti-Dectin-1 primary antibody or mIgG1 isotype control antibody, followed by incubation with fluorescent anti-mouse secondary antibody. Primary antibodies were used in serial dose titration. Figure 1 shows the binding analysis of anti-human Dectin-1 clone 2M24 antibody on cynomolgus monkey monocytes. [Figure 1C] Flow cytometric binding analysis of anti-human dectin-1 antibody (clone 2M24) on human and monkey monocytes derived from peripheral blood mononuclear cells (PBMCs). To identify monocytes, single viable CD14+ cells were gated. Cells were incubated with 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 a serial dose titration. Comparison of binding to human monocytes, HEK cells overexpressing human dectin-1, and cynomolgus monkey monocytes is shown between the 2M24 clone and other dectin-1 antibodies identified from immunization of ATX-Gx Alloy transgenic mice and a commercially available anti-dectin-1 antibody. The anti-human dectin-1 clone 2M24 antibody exhibited high affinity for human dectin-1 and cynomolgus monkey dectin-1 expressed on monocytes, demonstrating superior affinity compared to other anti-dectin-1 antibodies, including commercially available antibodies. [Figure 2A] This figure shows the phagocytosis of pHrodo-labeled polystyrene anti-mouse Fc IgG beads conjugated with the anti-Dectin-1 antibody 2M24 or an isotype control antibody by HEK-Blue hDectin-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 the Dectin-1 antibody 2M24 or an isotype control. The beads were then incubated with cultured HEK-Blue hDectin-1a cells or human monocytes at a 1:2 (cell:bead) ratio. HEK-Blue hDectin-1a cells were labeled with the cell-permeable dye calcein AM. Bead engulfment 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. Shown is the phagocytosis of beads by HEK-Blue hDectin-1a cells over a 2.5-hour period (top) and a representative image of pHrodo-positive cells after 2.5 hours of phagocytosis (bottom). [Figure 2B]This figure shows the phagocytosis of pHrodo-labeled polystyrene anti-mouse Fc IgG beads conjugated with the anti-Dectin-1 antibody 2M24 or an isotype control antibody by HEK-Blue hDectin-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 the Dectin-1 antibody 2M24 or an isotype control. The beads were then incubated with cultured HEK-Blue hDectin-1a cells or human monocytes at a 1:2 (cell:bead) ratio. HEK-Blue hDectin-1a cells were labeled with the cell-permeable dye calcein AM. Bead engulfment 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. Shown are phagocytosis of beads by human monocytes over a 4-hour period (top) and representative images of pHrodo-positive cells 2.5 hours after engulfment (bottom). In the representative images, internalized beads are brightly fluorescent within the phagosomes. [Figure 3] Figure 1 shows the binding of the fully human 2M24 anti-Dectin-1 antibody (hIgG4) or an isotype control antibody to HEK-Blue hDectin-1a cells and primary human monocytes. (A) shows the binding analysis of the fully human 2M24 anti-Dectin-1 antibody to HEK cells. (B) shows the binding to primary human monocytes. The primary antibody was used in a serial dose titration, followed by a fluorescent secondary antibody against the primary antibody. The fully human 2M24 anti-Dectin-1 hIgG4 antibody bound with high affinity to Dectin-1-expressing cells. [Figure 4]Figure 1 shows targeted phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated to the fully human 2M24 anti-Dectin-1 antibody (hIgG4) or an isotype control antibody by Dectin-1-expressing cells. Polystyrene biotin beads were labeled with pHrodo Red and conjugated to the anti-Dectin-1 antibody 2M24 or an isotype control antibody via streptavidin. The conjugated beads were mixed with cells at a 1:3 ratio, and bead phagocytosis was monitored by IncuCyte live-cell imaging. Figure 2 shows phagocytosis of pHrodo-biotin beads conjugated to streptavidin 2M24 anti-Dectin-1 hIgG4 antibody for HEK-Blue hDectin-1a cells (top left), human monocytes (top right), and human macrophages (bottom). The fully human 2M24 anti-Dectin-1 antibody (hIgG4) promoted phagocytosis of Dectin-1-expressing cells. [Figure 5A] This figure shows the results of a secreted alkaline phosphatase reporter assay of dectin-1 in HEK-Blue hDectin-1a cells. This figure shows the results of a secreted alkaline phosphatase assay performed using immobilized fully human 2M24 anti-Dectin-1 antibody. Fully human 2M24 (hIgG4) anti-Dectin-1 antibody or an isotype control antibody was immobilized overnight at 0.1–10 μg per well on a U-bottom polypropylene microtiter plate. HEK-Blue hDectin-1a cells were then cultured for 22 hours, and alkaline phosphatase secretion was assessed in the supernatant at OD 630 nm. [Figure 5B]This figure shows the results of a secreted alkaline phosphatase reporter assay of dectin-1 in HEK-Blue hDectin-1a cells. This figure shows the results of a secreted alkaline phosphatase assay performed using bead-conjugated fully human 2M24 anti-Dectin-1 antibody. Biotin beads of 3, 10, and 16.5 μm in size were conjugated to streptavidin 2M24 (hIgG4) anti-Dectin-1 antibody. The antibody-conjugated beads were mixed with HEK-Blue hDectin-1a cells for 22 hours, and the supernatant was evaluated for alkaline phosphatase secretion at OD 630 nm. Bars represent the mean ± standard deviation; n = 2 replicates. The 2M24 (hIgG4) anti-Dectin-1 antibody induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells in both immobilized and bead-conjugated forms. [Figure 6A] Figure 1 shows the amount of cytokine secretion by human primary macrophages stimulated with anti-Dectin-1 (15E2) antibody in solution. Primary human macrophages and primary monocytes were stimulated with 15E2 anti-Dectin-1 antibody or isotype antibody at 10 μg / ml in solution for 24 hours, and the amounts of TNFα and IL-6 secreted were assessed by ELISA analysis of the supernatants. 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 and macrophages. [Figure 6B]Figure 1 shows the amount of cytokine secretion by human primary macrophages stimulated with anti-Dectin-1 (15E2) antibody in solution. Primary human macrophages and primary monocytes were stimulated with 15E2 anti-Dectin-1 antibody or isotype antibody at 10 μg / ml in solution for 24 hours, and the amounts of TNFα and IL-6 secreted were assessed by ELISA analysis of the supernatants. 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 and macrophages. [Figure 7A] Figure 1 shows the amount of cytokine secretion by 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 on U-bottom polypropylene microtiter plates, and human monocytes or human PBMCs were cultured for 24 hours. TNFα, IL6, and IFNg secretion were assessed by ELISA analysis of the supernatants. (A) Cytokine secretion by human monocytes after stimulation with immobilized anti-Dectin-1 antibodies. (B) Cytokine secretion by cultured human PBMCs after stimulation. Bars represent mean ± standard deviation; n = 2 replicates. 2M24 anti-Dectin-1 antibody induced cytokine secretion in both primary human monocytes and PBMCs, demonstrating superior immune stimulation compared with the 15E2 agonist Dectin-1 antibody. [Figure 7B]Figure 1 shows the amount of cytokine secretion by 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 on U-bottom polypropylene microtiter plates, and human monocytes or human PBMCs were cultured for 24 hours. TNFα, IL6, and IFNg secretion were assessed by ELISA analysis of the supernatants. (A) Cytokine secretion by human monocytes after stimulation with immobilized anti-Dectin-1 antibodies. (B) Cytokine secretion by cultured human PBMCs after stimulation. Bars represent mean ± standard deviation; n = 2 replicates. 2M24 anti-Dectin-1 antibody induced cytokine secretion in both primary human monocytes and PBMCs, demonstrating superior immune stimulation compared with the 15E2 agonist Dectin-1 antibody. [Figure 8] This figure shows the results of a competition assay performed using the 12M4 anti-Dectin-1 antibody clone and the natural ligand of Dectin-1. HEK-Blue hDectin-1a cells were incubated with 2M24 (hIgG4) anti-Dectin-1 antibody or 15E2, 259931, and GE2 anti-Dectin-1 commercial antibodies at a 1 / 3 dose titration in the presence of 8 μg / ml biotin-laminarin on ice, starting at 300 nM. Binding of laminarin to Dectin-1 was assessed by flow cytometry using Streptavidin-Alexa fluor 647. 2M24 (hIgG4) anti-Dectin-1 antibody did not compete with the natural ligand for binding to Dectin-1. [Figure 9] 1 shows a summary of the functional characterization of the 2M24 and 15E2 anti-Dectin-1 antibodies. [Figure 10] Figure 1 shows a schematic diagram of the generation of bispecific antibodies by click chemistry. (A) Differential labeling of antibodies with MTA or FOL reagents. (B) Covalent cross-linking of antibodies via specific MTA-FOL interactions. [Figure 11A]We demonstrate the potential modes of action deployed by anti-Dectin-1 agonist bispecific antibodies to eliminate targeted cancer cells, including immunostimulation, phagocytosis, neoantigen presentation, and activation of T and B lymphocytes of the adaptive immune system. [Figure 11B] A list of potential targets for cancer cell depletion is presented. [Figure 12A] Figure 1 shows the characterization of a click chemistry-conjugated bispecific containing anti-Dectin-1 (clone 2M24) and anti-hCD70 arms. SDS-PAGE analysis of the covalently conjugated antibody pair (2M24 / anti-hCD20, 2M24 / anti-hCD70, and isotype control) under non-reducing and reducing conditions. [Figure 12B] Characterization of a click chemistry-conjugated bispecific containing anti-Dectin-1 (clone 2M24) and anti-hCD70 arms is shown. Flow cytometry-based characterization of bispecific (2M24 / anti-hCD70 or isotype control) binding 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 binds to Dectin-1- or CD70-expressing cells with affinities of 1.8 nM or 12.34 nM, respectively. [Figure 13]Figure 1 shows the ligation of dectin-1-expressing HEK293 and A498 renal carcinoma cells induced by the 2M24 / anti-hCD70 bispecific. Flow cytometry analysis of cocultures of HEK293 cells (labeled with calcein green) and A498 cells (labeled with calcein red) in the presence of the 2M24 / anti-hCD70 bispecific or isotype control is shown (left). Engagement between HEK293 and A498 cells is indicated by a double-positive signal (green + red + square box). Also shown is the ligation efficiency, quantified as the percentage of total target cells (A498) that form doublets with HEK293 cells (right). Bars represent the mean ± standard deviation; n = 3 replicates. The 2M24 / anti-hCD70 bispecific antibody induced ligation between dectin-1-expressing HEK293 and A498 renal carcinoma cells. [Figure 14A] Figure 1 shows the ligation of Dectin-1-expressing cells to B cells induced by anti-Dectin-1 / anti-hCD20 bispecific antibody. Figure 2 shows the ligation of Dectin-1-expressing HEK293 cells to B cells induced by anti-Dectin-1 / anti-hCD20 bispecific antibody. Flow cytometry analysis of cocultures of HEK293 cells (labeled with calcein green) and Raji cells (labeled with calcein red) in the presence of 2M24 / anti-hCD70 bispecific or isotype control (left). ligation of HEK293 to Raji cells is indicated by a double-positive signal (green + red +; square box). Ligation efficiency, quantified as the percentage of total target cells (Raji) that form doublets with HEK293 cells, is also shown (right). Bars represent the mean ± standard deviation; n = 2 replicates. [Figure 14B] Figure 1 shows the ligation of Dectin-1-expressing cells to B cells induced by an anti-Dectin-1 / anti-hCD20 bispecific antibody. Figure 2 shows the results of a similar experiment performed to evaluate the ligation of human M0 macrophages to Raji cells induced by an anti-Dectin-1 / anti-hCD20 bispecific. Bars represent the mean ± standard deviation; n = 2 replicates. 2M24 / anti-hCD20 bispecific induced the ligation of Dectin-1-expressing cells to CDC20-positive B cells (Raji cells). [Figure 15]This figure shows the results of a Dectin-1-induced secreted alkaline phosphatase reporter assay in HEK-Blue hDectin-1a cells using an anti-Dectin-1 / anti-CD20 bispecific in the presence of Raji cells. The 2M24 (hIgG4) / anti-CD20 bispecific antibody was incubated with Raji cells, followed by two washes to remove unbound bispecific antibody. The Raji cells were then mixed with HEK-Blue hDectin-1a cells at a ratio of 200,000 Raji cells to 100,000 HEK cells for 22 hours. Secreted alkaline phosphatase was assessed in the supernatant at OD 630 nm. Bars represent the mean ± standard deviation; n = 2 replicates. Raji cells coated with the anti-Dectin-1 / anti-CD20 bispecific induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells. [Figure 16] Figure 1 shows the induction of Raji cell phagocytosis by Dectin-1-expressing HEK293 cells with anti-Dectin-1 / anti-hCD20 bispecific antibody. Representative Incucyte images showing phagocytosis of Raji cells (arrows) by HEK cells at 16 h and 0 h are shown (left). Colocalization is indicated by yellow fluorescence. The decrease in calcein red signal in Raji cells after 16 h indicates cell death due to phagocytosis. Quantification of overlap or colocalization of HEK (calcein green) and Raji (calcein red) in different treatment groups is shown (right). Preincubation of HEK cells with the ADCP inhibitor latrunculin A inhibits 15E2 / anti-hCD20 bispecific antibody-mediated phagocytosis (n = 2 replicates). [Figure 17]Figure 1 shows the ligation of dectin-1-expressing cells to HER2-expressing cells induced by an anti-Dectin-1 / anti-hHER2 bispecific antibody. Flow cytometry analysis of cocultures of dectin-1-expressing HEK293 cells (labeled with calcein green) and HER2-expressing SKBR3 cells (labeled with pHrodo red) in the presence of 15E2 / anti-hHER2 bispecific or isotype control is shown (left). ligation between HEK293 and SKBR3 cells is indicated by a double-positive signal (green + red +; square box). Ligation efficiency, quantified as the percentage of total target cells (SKBR3) that form doublets with dectin-1-expressing cells, is also shown (right). Bars represent the mean ± standard deviation; n = 2 replicates. The anti-Dectin-1 / anti-hHER2 bispecific induces ligation of dectin-1 to HER2-positive cancer cells. [Figure 18] Figure 1 shows the ligation of dectin-1-expressing HEK293 cells to CD94-expressing BaF3 cells induced by anti-Dectin-1 / anti-hCD94 bispecific. Flow cytometry analysis of cocultures of HEK293 cells (labeled with calcein green) and BaF3 cells (labeled with pHrodo red) in the presence of 2M24 / anti-hCD94 bispecific or isotype control is shown (left). Engagement of HEK293 cells to BaF3 cells is indicated by a double-positive signal (green + red +; square box). Also shown is the ligation efficiency, quantified as the percentage of total target cells (BaF3) that form doublets with HEK293 cells (right). Bars represent the mean ± standard deviation; n = 2 replicates. Anti-Dectin-1 / anti-hCD94 bispecific induced ligation of dectin-1-expressing cells to CD94-expressing cells. [Figure 19]Figure 1 shows a schematic diagram of Fab 2M24-mSA or full-length 2M24-mSA bound to a biotinylated target antibody. A shows a chimeric fusion of monomeric streptavidin (mSA) with Fab 2M24 or full-length 2M24. mSA is genetically fused to either Fab 2M24 or full-length 2M24. B shows the linkage of Fab 2M24-mSA or 2M24-mSA to a biotinylated target antibody. The chimeric fusion was incubated with a biotinylated target antibody to generate a bispecific comprising a Dectin-1-binding arm and a second arm that binds to a target receptor or protein of interest. [Figure 20] Biochemical and functional characterization of Fab 2M24-mSA fusion protein. A. HPLC characterization of recombinant Fab 2M24-mSA. B. SDS-PAGE analysis of purified Fab 2M24-mSA under reducing conditions. C. Flow cytometry characterization of Fab 2M24-mSA binding to HEK293 cells stably overexpressing human Dectin-1 (EC50 = 1.45 nM). Fab 2M24 fusion to monomeric streptavidin bound to Dectin-1-expressing cells with an affinity of 1.45 nM. [Figure 21A] Figure 1 shows the phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated with monomeric streptavidin-tagged Fab-2M24 anti-Dectin-1 antibody (Fab-2M24-mSA). Figure 2 shows the duplet formation of HEK-Blue hDectin-1a cells with Fab-2M24-mSA conjugated to biotin beads and the phagocytosis of the beads, as assessed by flow cytometry. [Figure 21B]Phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated with Fab-2M24 anti-Dectin-1 antibody tagged with monomeric streptavidin (Fab-2M24-mSA) is shown. Phagocytosis of pHrodo biotin beads (approximately 3 μm) conjugated to Fab-2M24-mSA is assessed by IncuCyte live imaging (top). Representative images of pHrodo-positive cells (incorporated beads emitting bright red fluorescence in phagosomes) versus no-bead controls after 3 hours of phagocytosis are shown (bottom). Fab 2M24-mSA fusions induced bead binding and phagocytosis by Dectin-1-expressing HEK293 cells. [Figure 22] Panels A–D show bispecific complexes containing Fab 2M24-mSA and a target biotinylated antibody. HPLC analysis of Fab 2M24-mSA complexed with biotinylated anti-hCD20 (A), biotinylated anti-hCD19 (B), biotinylated anti-hCD70 (C), or biotinylated anti-Aβ 1-42 (D) is shown. The panels include overlaid A280 traces containing Fab 2M24-mSA alone, the target biotinylated antibody alone, and Fab 2M24-mSA complexed with a biotinylated target antibody. [Figure 23] Figure 1 shows the ligation of Dectin-1-expressing HEK293 cells to CD20-expressing Raji cells induced by Fab 2M24-mSA / biotin anti-hCD20 bispecific antibody. Flow cytometry analysis of cocultures of HEK293 cells (labeled with calcein green) and Raji cells (labeled with calcein red) in the presence of Fab 2M24-mSA / biotin anti-hCD20 bispecific or an isotype bispecific control is shown (left). Cocultures were incubated at 4°C or 37°C. ligation between HEK293 and Raji cells is indicated by a double-positive signal (green + red +; dotted square). Ligation efficiency, quantified as the percentage of total target cells (Raji) that formed doublets, is also shown (right). Bars represent the mean ± standard deviation; n = 4 replicates. The Fab 2M24-mSA / biotin anti-hCD20 bispecific induced binding between Dectin-1-expressing HEK293 cells and Raji cells. [Figure 24] FIG. 1 is a schematic diagram of targeted phagocytosis of amyloid deposits in amyloidosis using a Dectin-1 agonist bispecific antibody. [Figure 25] Figure 1 shows a strategy for targeted depletion of mast cells using Dectin-1 agonist bispecific antibodies. (A) Schematic diagram of mast cell depletion with Dectin-1 agonist bispecific antibodies. (B) List of potential targets for mast cell depletion. [Figure 26] Phagocytosis of large (approximately 16.2 μm) pHrodo-labeled beads by human dendritic cells is shown. Quantification of bead engulfment over 12 hours (left) and a representative image of pHrodo-positive cells after 3 hours of engulfment (engulfed beads fluoresce bright red in phagosomes; right). Dectin-1 antibody promoted directed engulfment of beads in cultured monocyte-derived dendritic cells. [Figure 27] Schematic diagram of targeted depletion of microorganisms by Dectin-1 agonist bispecific antibodies. A bispecific antibody with a Dectin-1-binding arm and a microbial agent-binding arm was engineered to target bacteria, viruses, or fungi (top). The Dectin-1 bispecific antibody recruited Dectin-1-expressing phagocytes to eliminate bacterial, viral, or fungal pathogens (bottom). [Figure 28A] Figure 1 shows 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. Figure 2 shows the binding analysis of an anti-Dectin-1 / anti-Hemagglutinin bispecific antibody to H3N2 influenza virus, assessed by ELISA. A 96-well microtiter plate was coated with H3N2 influenza virus particles, followed by incubation with single antibodies, bispecific antibodies, and an isotype control. After extensive washing, the primary antibody was detected with a secondary anti-mouse FcgR HRP antibody. [Figure 28B]Figure 1 shows 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. Flow cytometric analysis of the binding of the anti-dectin-1 / anti-hemagglutinin bispecific antibody to HEK cells expressing dectin-1 was performed. HEK cells were incubated with the primary antibody, followed by secondary fluorescent antibody detection against either the anti-dectin-1 antibody (anti-mIgG2a APC) or the hemagglutinin antibody (anti-mIgG2b PB). The anti-dectin-1 / anti-hemagglutinin bispecific antibody efficiently bound to both H3N2 influenza virus and HEK cells expressing dectin-1. [Figure 29A] Schematic diagrams of antigen delivery using anti-Dectin-1 antibodies for vaccine generation are shown, showing the use of anti-Dectin-1 antibodies fused to target antigens for delivery to APCs (A), or the use of anti-Dectin-1 bispecific antibodies for targeted delivery of disease-causing agents (e.g., cells, microorganisms, proteins, etc.) to APCs (B). [Figure 29B] Schematic diagrams of antigen delivery using anti-Dectin-1 antibodies for vaccine generation are shown, showing the use of anti-Dectin-1 antibodies fused to target antigens for delivery to APCs (A), or the use of anti-Dectin-1 bispecific antibodies for targeted delivery of disease-causing agents (e.g., cells, microorganisms, proteins, etc.) to APCs (B). [Figure 30]Figure 30 shows the phagocytosis of pHrodo-labeled polystyrene anti-mouse Fc IgG beads (approximately 3.4 μm) conjugated with Dectin-1 antibody (15E2) or an isotype control antibody by human dendritic cells. 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 at a 1:3 (cell:bead) ratio. Bead engulfment was monitored by IncuCyte live-cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the total integrated intensity (summed fluorescence intensity) of red objects (pHrodo) in the image. Figure 30 shows quantification of bead engulfment over 9 hours (top) and a representative image of a pHrodo-positive cell 3 hours after engulfment (engulfed beads emitting bright red fluorescence in the phagosome; bottom). [Figure 31A] Phagocytosis of SARS-CoV-2 spike protein-coated beads by Dectin-1-expressing HEK293 cells. Schematic diagram of the experiment. Beads coated with spike protein from SARS-CoV-2 were linked to Dectin-1-expressing HEK293 cells by an anti-Dectin-1 bispecific antibody containing both a Dectin-1 protein-binding arm and a spike protein-binding arm. [Figure 31B] Phagocytosis of SARS-CoV-2 spike protein-coated beads by Dectin-1-expressing HEK293 cells. Flow cytometry characterization of effector (HEK293 cells) and target (spike-coated beads) engagement by bispecifics and isotype controls (Panel A) and quantification of ligation efficiency based on doublet population (Panel B). [Figure 31C]Phagocytosis of SARS-CoV-2 spike protein-coated beads by Dectin-1-expressing HEK 293 cells is shown. Phagocytosis of SARS-CoV-2 spike protein-coated beads by HEK 293 cells in a coculture experiment is shown. Phagocytosis of pHrodo-labeled beads was monitored by changes in pHrodo fluorescence as a result of the acidic pH within the phagosome. Quantification of phagocytosis, quantified by Incucyte analysis software and expressed as the number of red overlapping objects (pHrodo) relative to calcein-positive cells (left), is shown, along with a representative image of pHrodo-positive cells 2 hours after phagocytosis (engulfed beads fluorescing bright red within the phagosome; right). [Figure 32A]

[0023] Figure 1 shows a bispecific antibody design of a human bispecific antibody (e.g., a human IgG1 bispecific antibody) targeting Dectin-1 and a disease target or antigen. A schematic diagram of the design is provided. One arm (2M24A.X) with VH domain A and VL domain B targets human Dectin-1, and the other arm (2M24B.X) with VH domain C and VL domain D targets the disease target or antigen. [Figure 32B]

[0023] Figure 1 shows bispecific antibody designs of human bispecific antibodies (e.g., human IgG1 bispecific antibodies) targeting Dectin-1 and disease targets or antigens.

[0024] Figure 2 provides a schematic diagram of an exemplary mechanism of action of an anti-Dectin-1 bispecific antibody with an active Fc domain that targets hDectin-1 on myeloid cells (via the first arm), an antigen on target cells / disease-causing agents (via the second arm), and Fc receptors on myeloid cells and NK cells, inducing robust immune stimulation and phagocytosis. [Figure 33A]Figure 1 shows that a bispecific antibody with one arm targeting hDectin-1 and the other targeting hCD20 (using the variable domain of rituximab) binds to cells expressing human Dectin-1 or human CD20. The top panel shows the binding of a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) or a bispecific antibody targeting hDectin-1 and RSV (2M24 / RSV) to HEK293 cells stably expressing human Dectin-1, as assessed by flow cytometry. The bottom panel shows the binding of the bispecific antibody 2M24 / RSV hIgG1-FITC conjugate and 2M24 bivalent hIgG1-FITC conjugate to PBMCs, as assessed by flow cytometry. [Figure 33B] Figure 1 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. Figure 2 shows the binding of rituximab (human IgG1), 2M24 / CD20 with activating human IgG1 Fc, 2M24 / CD20 with inactivating human IgG1 Fc, 2M24 / RSV with activating human IgG1 Fc, or 2M24 / RSV with inactivating human IgG1 Fc to the CD20-expressing B cell lymphoma Raji cell line. [Figure 34A] This shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) induces ligation between Dectin-1 and CD20-expressing cells. To assess ligation between Dectin-1-expressing HEK293 cells (effector) and CD20-expressing Raji cells (target), cells were differentially labeled with calcein green (effector) or calcein red (target) dye. Labeled cells were cocultured 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 34B]Figure 1 shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) induces ligation of Dectin-1 to CD20-expressing cells. Figure 2 shows dose titration of the bispecific in effector:target cell co-cultures. Ligation efficiency was quantified as the percentage of total target cells that bound or ligated to effector cells. [Figure 35A] This shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an activating hIgG1 Fc does not induce monocyte depletion by antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). PBMCs from two healthy donors, donor 76 (A) and donor 77 (B), were treated with increasing concentrations of 2M24 / CD20 bispecific antibody (hIgG1 activating or inactive isotype) and rituximab for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD14+ monocytes (as a percentage of isotype control). [Figure 35B] This shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an activating hIgG1 Fc does not induce monocyte depletion by antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). PBMCs from two healthy donors, donor 76 (A) and donor 77 (B), were treated with increasing concentrations of 2M24 / CD20 bispecific antibody (hIgG1 activating or inactive isotype) and rituximab for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD14+ monocytes (as a percentage of isotype control). [Figure 36A] Figure 1 shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an activating hIgG1 Fc induces superior B cell depletion compared to rituximab. PBMCs from two healthy donors, donor 83 (A) and donor 84 (B), were treated with increasing concentrations of the indicated antibodies for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as % of B cells in PBMCs treated with isotype control). [Figure 36B] Figure 1 shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an activating hIgG1 Fc induces superior B cell depletion compared to rituximab. PBMCs from two healthy donors, donor 83 (A) and donor 84 (B), were treated with increasing concentrations of the indicated antibodies for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as % of B cells in PBMCs treated with isotype control). [Figure 37A] This shows that rituximab induces greater B cell shaving (CD19 downregulation) than the 2M24 / CD20 activating 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 hours of incubation with increasing concentrations of the 2M24 / CD20 hIgG1 (activating isotype) bispecific antibody, rituximab, or an isotype control. The mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugated) was used to assess the effect of the 2M24 / CD20 bispecific and rituximab on CD19 expression on B cells. EC50 values ​​were calculated based on nonlinear regression analysis. [Figure 37B] This shows that rituximab induces greater B cell shaving (CD19 downregulation) than the 2M24 / CD20 activating 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 hours of incubation with increasing concentrations of the 2M24 / CD20 hIgG1 (activating isotype) bispecific antibody, rituximab, or an isotype control. The mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugated) was used to assess the effect of the 2M24 / CD20 bispecific and rituximab on CD19 expression on B cells. EC50 values ​​were calculated based on nonlinear regression analysis. [Figure 38] Figure 1 shows the difference in cytokine release induced by the 2M24 / CD20 activating IgG1 bispecific antibody compared to rituximab. ELISA-based (mesoscale discovery) cytokine quantification was performed on supernatants isolated from healthy donor PBMCs treated with the 2M24 / CD20 activating hIgG1 bispecific, rituximab, or an isotype control. PBMCs were stimulated overnight with the antibodies, and the supernatants were subsequently analyzed by 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 the amount of 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 figure shows that the 2M24 / CD20 hIgG1 (activating isotype) bispecific antibody induces superior B cell depletion and less CD19 shaving compared to rituximab in cocultures of human macrophages and GFP-expressing Raji B cells. Flow cytometry analysis of cocultures of human macrophages and Raji-GFP cells (3:1 ratio) in the presence of the 2M24 / CD20 hIgG1 (activating isotype) bispecific, 2M24 / RSV control, fucosylated rituximab, or isotype hIgG1 control. Cocultures 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 assessed at the end of the experiment. Primary antibodies were used in serial dose titrations. [Figure 39B]Figure 1 shows that the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody induces superior B cell depletion and lower CD19 shaving compared to rituximab in cocultures of human macrophages and GFP-expressing Raji B cells. CD19 assessment on Raji-GFP cells after 24 hours. B cell receptor shaving is shown as a decrease in CD19 MFI in the presence of anti-Dectin-1 / anti-hCD20 bispecific or rituximab. [Figure 40A] This shows that the 2M24 / CD20 activating IgG1 bispecific antibody induces superior tissue B cell depletion compared to rituximab in single-cell suspensions of renal cancer specimens. Single-cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of the 2M24 / CD20 hIgG1 (activating or inactivating) 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 antibodies (2 μg / ml) at 37°C for 24 hours. Immune cell populations were analyzed by flow cytometry. Cells were first gated for live cells and further separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells). Within the CD45+ population, CD19+ (B cells) and CD3+ (T cells) cells were identified (Figures A and B). The number of remaining B cells was assessed by anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 40B]This study demonstrates that the 2M24 / CD20 activating IgG1 bispecific antibody induces superior tissue B cell depletion compared with rituximab in single-cell suspensions of renal cancer specimens. Single-cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of the 2M24 / CD20 hIgG1 (activating or inactivating) 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 antibodies (2 μg / ml) at 37°C for 24 hours. Immune cell populations were analyzed by flow cytometry. Cells were first gated for live cells and further separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells). Within the CD45+ population, CD19+ (B cells) and CD3+ (T cells) cells were identified (Figures A and B). The number of remaining B cells was assessed by anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 40C] This shows that the 2M24 / CD20 activating IgG1 bispecific antibody induces superior tissue B cell depletion compared to rituximab in single-cell suspensions of renal cancer specimens. Single-cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of the 2M24 / CD20 hIgG1 (activating or inactivating) 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 antibodies (2 μg / ml) at 37°C for 24 hours. Immune cell populations were analyzed by flow cytometry. Cells were first gated for live cells and further separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells). Within the CD45+ population, CD19+ (B cells) and CD3+ (T cells) cells were identified (Figures A and B). The number of remaining B cells was assessed by anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 41]Figures A–C show that anti-Dectin-1 antibody (clone 2M24) induces dectin-1 clustering and TNFα secretion from human macrophages. Cytokine secretion was examined by cultured macrophages and single-cell suspensions of renal cancer specimens stimulated with immobilized anti-Dectin-1 antibody (clone 2M24) or 2M24 / CD20 bispecific antibody. Anti-Dectin-1 antibody (clone 2M24), an isotype control, or 2M24 / CD20 bispecific antibody was immobilized overnight at 10 μg per well on a U-bottom polypropylene microtiter plate, followed by incubation with human monocyte-derived macrophages (A and B) or single-cell suspensions from renal cancer specimens (C). Cells were cultured for 24 hours, and the amount of TNFα secreted in the supernatant was assessed by ELISA. As a positive control, cells were stimulated with zymosan. [Figure 42] This figure shows that immobilized anti-Dectin-1 antibody (clone 2M24) promotes immune stimulation in single-cell suspensions of kidney cancer specimens. Single-cell suspensions from kidney cancer specimens were treated with immobilized anti-Dectin-1 antibody (clone 2M24) or an 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. Each plot shows the amount of cytokine (pg / mL) as a function of antibody treatment. Results are shown for kidney cancer donor 3 (left) or donor 4 (right) treated with anti-Dectin-1 antibody (clone 2M24) or an isotype control hIgG4 antibody. [Figure 43] Figure 1 shows the effect of the 2M24 / CD20 bispecific antibody on CD16 expression on 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 compared to rituximab. [Figure 44]Figure 1 shows the effect of the 2M24 / CD20 bispecific antibody on CD19 expression on human B cells compared to rituximab or an isotype control (2M24 / RSV bispecific). The results show that CD19 antigen levels are better maintained in B cells treated with the 2M24 / CD20 bispecific compared to rituximab. [Figure 45] Figure 1 shows depletion of human B cells by the 2M24 / CD20 bispecific antibody derived from rituximab or the 2M24 / CD20 bispecific antibody derived from obinutuzumab. The results demonstrate that the 2M24 / CD20 bispecific derived from the rituximab arm is superior in depleting B cells compared to the bispecific derived from obinutuzumab. [Figure 46] 1 shows the design of an exploratory study of the safety and efficacy of 2M24 / CD20 bispecific antibodies in non-human primates. [Figure 47] Figure 1 shows depletion of circulating B cells in cynomolgus monkeys by the 2M24 / CD20 hIgG1 bispecific antibody generated in cells treated with kifunensine (KIF). Figure 1 shows depletion of B cells in monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF (top) or inactive 2M24 / CD20 hIgG1 (bottom). [Figure 48] Figure 1 shows depletion of circulating B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody generated on cells treated with kifunensine (KIF). Figure 2 shows depletion of B cells in monkeys treated with 5 mg / kg rituximab hIgG1 KIF. [Figure 49A] Figure 1 shows depletion of tissue-resident B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody generated from cells treated with kifunensine (KIF). Figure 2 shows depletion of B cells in bone marrow from monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF. [Figure 49B]Figure 1 shows depletion of tissue-resident B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody generated from cells treated with kifunensine (KIF). Figure 2 shows depletion of B cells in lymph nodes from monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF. [Figure 50] Figure 1 shows ex vivo depletion of B cells from cynomolgus monkey PBMCs. [Figure 51] The format of a bispecific molecule (2M24 scFv / CD20) that pairs an anti-CD20 conventional half antibody with an anti-Dectin-1 single-chain variable fragment (scFv) Fc fusion arm using knob-into-hole technology is shown. H is the 2M24 VH domain, and L is the 2M24 VL domain. [Figure 52A] Figure 1 shows the purification and functional characterization of the 2M24 / CD20 bispecific antibody. Figure 2 shows the purification of the molecule by size exclusion chromatography (SEC). [Figure 52B]

[0023] Figure 1 shows the purification and functional characterization of 2M24 / CD20 bispecific antibodies. The purified bispecific antibodies promoted targeted immune stimulation as assessed by an NFκB reporter assay. [Figure 52C] 1 shows purification and functional characterization of 2M24 / CD20 bispecific antibody. 2 shows depletion of human B cells by 2M24 scFv / CD20 bispecific antibody. [Figure 53A] Figure 1 shows the generation and characterization of anti-Dectin-1 (2M24) / anti-Trop-2 bispecific antibodies. 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]

[0023] Figure 1 shows the generation and characterization of an anti-Dectin-1 (2M24) / anti-Trop-2 bispecific antibody, demonstrating high affinity binding of the molecule to Dectin-1-expressing HEK cells. [Figure 53C]

[0023] Figure 1 shows the generation and characterization of an anti-Dectin-1 (2M24) / anti-Trop-2 bispecific antibody, which exhibits moderate affinity binding to the Trop-2-expressing A431 cancer cell line. [Figure 54] 1 shows the expression level of Trop-2 in cancer cells. [Figure 55A] Figure 1 shows the binding of the 2M24 / Trop-2 bispecific antibody to the Trop-2-expressing cell line HeLa. The binding EC50 for the cell line was calculated using four-parameter logistic (4PL) nonlinear regression. [Figure 55B] Figure 1 shows the binding of 2M24 / Trop-2 bispecific antibodies to BxPC-3. Binding EC50s calculated using four-parameter logistic (4PL) nonlinear regression for the cell lines are shown. [Figure 55C] Figure 1 shows the binding of 2M24 / Trop-2 bispecific antibodies to SiHa. Binding EC50s calculated using four-parameter logistic (4PL) nonlinear regression for cell lines are shown. [Figure 55D] Figure 1 shows the binding of 2M24 / Trop-2 bispecific antibodies to Capan-2. Binding EC50s calculated using four-parameter logistic (4PL) nonlinear regression for cell lines are shown. [Figure 56A] Figure 1 shows depletion of a Trop-2 expressing cell line (SKBR3 cells) using the 2M24 / Trop-2 bispecific antibody. [Figure 56B] Figure 1 shows depletion of a Trop-2 expressing cell line (A431 cells) using the 2M24 / Trop-2 bispecific antibody. [Figure 57A] 1 shows Trop-2 and Dectin-1 expression in lung cancer specimens. [Figure 57B] 1 shows Trop-2 and Dectin-1 expression in lung cancer specimens. [Figure 58] Depletion of Trop-2 positive cancer cells in lung cancer specimens. [Figure 59A] Figure 1 shows the activity of the 2M24 / Trop-2 bispecific antibody in an NFκB reporter assay. [Figure 59B] 1 shows 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]These results demonstrate that the 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 2M24 / Trop-2 hIgG1 or control 2M24 / RSV hIgG1 bispecific antibody. Phagocytosis or depletion of SKBR3 cells was assessed by flow cytometry by staining for EPCAM expression on SKBR3 cells. Data are reported relative to the control bispecific 2M24 / RSV. [Figure 59D] Figure 1 shows that the 2M24 / Trop-2 bispecific antibody promotes antigen presentation and T cell activation. IFNγ levels in the supernatants were quantified using the BD OptiEIA kit. [Figure 59E] We demonstrate that the 2M24 / Trop-2 bispecific antibody enhances antigen presentation and T cell activation. Expression of CD69, an early activation marker, on T cells was assessed by flow cytometry. Data are reported relative to total CD3+ T cells. [Figure 60A] 1 shows the design and production of 2M24 / Nectin-4 bispecific antibodies. A diagram of the bispecific molecule is shown. [Figure 60B] 1 shows the design and production of 2M24 / Nectin-4 bispecific antibodies. 2 shows the purification of the bispecific antibodies using Protein A chromatography. [Figure 61A] 1 shows Nectin-4 expression in cancer cell lines. [Figure 61B] 1 shows Nectin-4 expression in cancer cells from primary tumor specimens. [Figure 62] Binding of the 2M24 / Nectin-4 bispecific antibody to Dectin-1-expressing HEK cells (top) or Nectin-4-expressing A431 cells (bottom) is shown. [Figure 63] Figure 1 shows stimulation of Dectin-1 in an NFκB reporter assay by the 2M24 / Nectin-4 bispecific antibody. The upper panel shows a diagram of the assay. The lower panel shows the quantification results based on SEAP levels in the medium. [Figure 64A]Figure 64A shows the depletion of Nectin-4-expressing cancer cells by the 2M24 / Nectin-4 bispecific antibody. Figure 64B shows the detection of phagocytosis / depletion by flow cytometry. [Figure 64B] Figure 64B shows depletion of Nectin-4-expressing cancer cells by 2M24 / Nectin-4 bispecific antibody. Figure 64B shows depletion relative to the RSV control. [Figure 65A] 2 shows that the 2M24 / 11-1F4 bispecific antibody binds to light chain amyloid. Purification by SEC of the parent anti-amyloid antibody 11-1F4 (top) and the 2M24 / 11-1F4 bispecific antibody (bottom) is shown. [Figure 65B] Figure 1 shows that the 2M24 / 11-1F4 bispecific antibody binds to light chain amyloid. Octet shows binding of the 11-1F4 parent antibody (B) or the 2M24 / 11-1F4 bispecific antibody (C) to recombinant light chain amyloid from different patients (AL30, AL47, AL48, and AL55). [Figure 65C] Figure 1 shows that the 2M24 / 11-1F4 bispecific antibody binds to light chain amyloid. Octet shows binding of the 11-1F4 parent antibody (B) or the 2M24 / 11-1F4 bispecific antibody (C) to recombinant light chain amyloid from different patients (AL30, AL47, AL48, and AL55). [Figure 66] This shows the phagocytosis of light chain amyloid fibrils by monocytes. DETAILED DESCRIPTION OF THE INVENTION

[0020] Some aspects are described below with reference to illustrative example applications. It should be understood that numerous specific details, relationships, and methods are described to provide a thorough understanding of the features described herein. However, those skilled in the art will readily recognize that the features described herein can be implemented in other ways without one or more of the specific details. The features described herein are not limited to the illustrated order of acts or events, as some actions may occur in different orders and / or simultaneously with other actions or events. Furthermore, not all illustrated acts or events are required to implement a method in accordance with the features described herein.

[0021] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in the same manner as the term "comprising." As used herein, the term "comprising" is synonymous with "including" or "containing" and is inclusive or open-ended.

[0022] References to "or" herein are intended to encompass "and / or" unless otherwise specified. As used herein, the term "about" with respect to a numerical value means a numerical value of +10% or -10% of that numerical value. The term "about" with respect to a range refers to the range minus 10% of its minimum value and plus 10% of its maximum value.

[0023] I. Antibodies and Multispecific Binding Proteins In certain aspects, the present disclosure provides antigen-binding domains, antibodies, and antibody fragments that bind to human Dectin-1, as well as multispecific (e.g., bispecific) binding molecules comprising same.

[0024] In some embodiments, antibody and immunoglobulin are used interchangeably and are used herein in the broadest sense to 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), so 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 a native antibody structure, or a protein having heavy and light chain variable regions having structures substantially similar to native heavy and light chain variable region structures. A native antibody refers to naturally occurring immunoglobulin molecules with diverse structures. For example, the IgG class of native immunoglobulins is a heterotetrameric glycoprotein of approximately 150,000 daltons composed of two disulfide-bonded 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 a heavy chain constant region. 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 configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies (immunoglobulins) are assigned to different classes. There are five major classes of antibodies: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an immunoglobulin can be assigned to 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 immunoglobulin hinge region.

[0026] In some embodiments, Fc, Fc region, or Fc domain refers to the C-terminal region of an antibody heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. Fc can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and optionally all or part of the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may also include the J chain. An IgG Fc region includes the IgG CH2 and IgG CH3 domains, and optionally includes the hinge. Unless otherwise specified herein, the numbering of amino acid residues in 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 the present disclosure and can be Fc domains derived from human IgG1, IgG2, or IgG4.

[0027] Multiple definitions of CDR sequences of 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 definitions of IMGT. 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 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 the VL domain comprises 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 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 mentioned above, the Kabat definition of CDR sequences can also be used. In some embodiments, an 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 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 VL domain comprises CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising 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 has the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and and / or a VL domain comprising 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 comprises a VH domain comprising the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and / or a VL domain comprising 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 comprising the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7) and a VL domain comprising 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, an antibody that binds to human Dectin-1 has the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT a heavy chain comprising the sequence ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11), and / or a light chain comprising the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIERTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).In some embodiments, an antibody that binds to human Dectin-1 has the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE and a light chain comprising 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 isoforms. In some embodiments, human Dectin-1 isoform A comprises the amino acid sequence MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVKTTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO: 9). In some embodiments, human dectin-1 isoform B comprises 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 surface of a cell with 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, e.g., cynomolgus 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 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 a light chain variable (VL) domain comprising CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising the sequence GASSLQS (SEQ ID NO: 92), 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: 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 epitope of human Dectin-1 as 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 binds to the same epitope of human Dectin-1 as a reference antibody comprising: 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 a light chain variable (VL) domain comprising CDR-L1 comprising the sequence RASQGISSWLA (SEQ ID NO: 91), CDR-L2 comprising the sequence GASSLQS (SEQ ID NO: 92), and CDR-L3 comprising the sequence QQAYSFPFT (SEQ ID NO: 6). In some embodiments, the antigen-binding domain, antibody, or antibody fragment binds to the same epitope of human Dectin-1 as a reference antibody comprising: a heavy chain variable (VH) domain comprising the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), and a light chain variable (VL) domain comprising 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 portion of an antibody that binds to an antigen and comprises the variable region and CH1 of a heavy chain linked to a light chain via an interchain disulfide bond.

[0038] In some embodiments, antibodies of the present disclosure comprise an Fc region. The antibody can 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 molecules that trigger targeted phagocytosis can play an important role in the process by binding to Fc receptors and inducing further phagocytosis. In some embodiments, the molecules have a modified Fc region (e.g., containing one or more mutations that reduce effector function as described herein) that has reduced ADCC activity compared to wild-type human IgG1.

[0039] In some embodiments, an antibody of the present disclosure comprises an Fc region, wherein the carbohydrate structures attached to the Fc region are fucose-reduced or fucose-deficient, e.g., at least one or two of the antibody's heavy chains are afucosylated. In some embodiments, a composition is provided herein comprising an antibody of the present disclosure comprising an Fc region, wherein the carbohydrate structures attached to the Fc region are fucose-reduced or fucose-deficient, e.g., at least one or two of the antibody's heavy chains are afucosylated. In some embodiments, less than 50% of the N-glycoside-linked carbohydrate chains in the composition comprise fucose residues. In some embodiments, none of the N-glycoside-linked carbohydrate chains comprise substantially fucose residues. In some embodiments, the fucose-reduced or fucose-deficient antibody has improved ADCC function.

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

[0041] Fucosylation or fucosylated antibodies can refer to the presence of fucose residues in the oligosaccharides attached to the peptide backbone of an antibody. Specifically, a fucosylated antibody contains an α(l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or both N-linked oligosaccharides attached to the Fc region of the antibody, e.g., at 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 297, i.e., between 294 and 300, depending on slight sequence variations in immunoglobulins. Nonfucosylated or fucose-deficient antibodies have reduced fucose compared to the amount of fucose in the same antibody produced in a cell line. Antibody fucosylation can be measured, for example, in an antibody composition treated with N-glycosidase F, assessed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI TOF MS).

[0042] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate fucosylation, e.g., a substitution at Asn297 (EU numbering of Fc region residues) of human IgG1 Fc region. Optionally, the Fc region further comprises one or more amino acid substitutions therein that further improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of 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. al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).

[0043] In some embodiments, defucosylated or nonfucosylated antibodies are produced in cell lines with genetic modifications that result in defucosylated or nonfucosylated antibodies. Examples of cell lines that produce defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. 2003 / 0157108 A1 (Presta, L), and WO2004 / 056312 A1 (Adams et al.) (especially in Example 11)), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. al. Biotech. Bioeng. 87:614 (2004)), β1,4-N-acetylglucosaminyltransferase III (GnT-III) and also Golgi μ-mannosidase II (ManII), as well as cells in which mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1; see Byrne, G. et al. (2018) PLoS Biol. 16:e2005817) has been knocked out.

[0044] In some embodiments, defucosylated or nonfucosylated antibodies are produced in cell lines treated with inhibitors of sugar-processing enzymes, such as kifunensine, 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 kifunensine (e.g., at 250 μg / mL), cultured, and used for antibody production.

[0045] In certain aspects, the present disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments comprising a first antigen-binding domain that binds to a first target of interest and a second antigen-binding domain that binds to a second target of interest. In some embodiments, the present 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 of interest.

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

[0047] Exemplary avidin-, streptavidin-, neutravidin-, or biotin-conjugated derivatives thereof are known in the art. In some embodiments, the streptavidin is monomeric streptavidin (mSA). Exemplary biotin- or avidin-conjugated derivatives thereof are known in the art. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure is biotinylated. Kits for biotinylating an antibody of interest are known in the art and 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 is linked to mSA via a linker comprises 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 has the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVK PSAASGS (SEQ ID NO: 16) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH (SEQ ID NO: 17).

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

[0050] In some embodiments, one or both of the first and second antigen-binding domains, antibodies, or fragments comprises a tag, e.g., 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 antibodies, nanobodies, or scFv fragments. In some embodiments, one or both of the first and second antibodies or fragments further comprise 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, the antibody or fragment is linked to its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative, or to its biotin- or avidin-linked derivative, via a linker. Linkers for creating antibody fusion proteins are known in the art. In some embodiments, the linker comprises, consists of, or consists essentially of glycine and / or serine residues. In some embodiments, the linker is 15-20 amino acids in length. 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). Additional linker sequences are described in Chen, X. et al. (2013) Adv. Drug Deliv. Rev. 65:1357-1369. In some embodiments (e.g., in scFvs of the present disclosure), the scFv comprises one type of linker between the VH and VL domains and another type of linker connecting the VL domain to the remainder of the half antibody, e.g., the Fc region. For example, in some embodiments, the linker between the VH and VL domains comprises 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 comprises EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). In some embodiments, the linker between the VH and VL domains comprises glycine and / or serine residues and is 15 to 20 amino acids in length.

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

[0054] In certain aspects, the present 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 antigen-binding domains that bind to human Dectin-1 of the present 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 of interest (e.g., those described herein). In some embodiments, the target of interest is a disease-causing agent.

[0055] In some embodiments, a multispecific (e.g., bispecific) binding molecule comprises a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1 and a first antibody arm comprising a first Fc region, and a second antibody arm comprising an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second Fc region connected to the VH domain. In some embodiments, the scFv arm binds to Dectin-1, and a conventional antibody arm having the VH domain and VL domain on separate polypeptides binds to a target of interest (e.g., those described herein), such as a disease-causing agent. In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the first antibody arm comprises a first linker (e.g., 15-20 amino acids in 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 comprises 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 comprises the sequence EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30). Additional 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-causing agent is a bacterial cell, a fungal cell, a virus, a senescent cell, a tumor cell, a protein aggregate, an LDL particle, a mast cell, an 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, an 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 of interest is a protein aggregate or a monomer thereof, 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, e.g., in oncology applications, the second target of interest is CD70, HER2, DLL3, nectin-4, TROP-2, mesothelin, LIV-1, c-MET, FOLR1, CD20, CCR8, CD33, or EGFR, e.g., expressed on the surface of a cancer cell.

[0057] In some embodiments, the target of interest is CD20, e.g., human CD20. In some embodiments, the antigen binding domain that binds to CD20 comprises 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 comprises a VH domain comprising the sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24), and / or comprises a VL domain comprising the sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25). In some embodiments, the antigen binding domain that binds to CD20 comprises VH domain and VL domain sequences derived from rituximab. In some embodiments, the antigen binding domain that binds to CD20 comprises VH domain and VL domain sequences derived from obinituzumab. In some embodiments, the antigen binding domain that binds to CD20 comprises a VH domain comprising the sequence of SEQ ID NO: 46 and / or a VL domain comprising the sequence of SEQ ID NO: 47.

[0058] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to a target of interest (e.g., a disease-causing agent of the present disclosure). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).

[0059] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of SEQ ID NO:24 and a VL domain comprising the sequence of SEQ ID NO:25. In some embodiments, the second antibody arm is QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE a second polypeptide comprising the sequence PQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 32); and a third polypeptide comprising the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0060] In some embodiments, provided herein is a multispecific (e.g., bispecific) binding molecule comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 34), and a VL domain comprising the sequence of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 35). In some embodiments, the antigen-binding domain that binds to HER2 comprises VH and VL domain sequences derived from trastuzumab.

[0061] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1 and 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 connected to the VH domain, wherein the VH and VL domains 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain, and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm comprises QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYTDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCARGGFGSSYWYFDVWGQGSLVTVSS (SEQ ID NO: 42) and a VL domain comprising the sequence DIQLTQSPSSLSASVGDRVSITCKASQDVSIAVAWYQQKPGKAPKLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFAVYYCQQHYITPLTFGAGTKVEIK (SEQ ID NO: 43). In some embodiments, the second antibody arm comprises a VH domain comprising the sequence QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTKTGEPTYAEEFKGRFAFSLETSASTAYLQINNLKKEDTATYFCGRGGYGSSYWYFDVWGAGTTVTVSS (SEQ ID NO: 56) and a VL domain comprising the sequence DIVMTQSHKFMSTSVGDRVSITCKASQDVSIAVAWYQQKPGQSPKVLIYSASYRYTGVPDRFTGSGSGTDFTFTISRVQAEDLAVYYCQQHYITPLTFGAGTKLELK (SEQ ID NO: 57). In some embodiments, the second antibody arm comprises a VH domain comprising the sequence QVQLQQSGPELVRPGTSVRISCKASGYTFTIYWLGWVKQRPGHGLEWIGNIFPGSAYINYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCAREGSNSGYWGQGTTLTVSS (SEQ ID NO: 58) and a VL domain comprising the sequence DIVMTQSPSSLSVSAGEKVTMTCKSSQSLLNSGNQQNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCQSDHIYPYTFGGGTKLEIK (SEQ ID NO: 59).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of QVQLQESGPGLVKPSETLSLTCTVSGGSISSYGVHWIRQPPGKGLEWIGVIWTGGSTDYNSALMSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGDYDRYTMDYWGQGTLVTVSS (SEQ ID NO: 66), and a VL domain comprising the sequence of DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPYTFGQGTKLEIK (SEQ ID NO: 67). In some embodiments, the antigen binding domain that binds to Trop-2 comprises VH and VL domain sequences from sacituzumab, AR47A6.4.2, h7E6, or Pr1E11.

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

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

[0064] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH and VL domains 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS (SEQ ID NO: 38), and a VL domain comprising the sequence of QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVL (SEQ ID NO: 39). In some embodiments, the antigen binding domain that binds CD70 comprises VH and VL domain sequences from 4ID12.

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

[0066] In some embodiments, provided herein is a multispecific (e.g., bispecific) binding molecule comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (SEQ ID NO: 62) and a VL domain comprising the sequence DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK (SEQ ID NO: 63). In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGVGTFDYWGQGTLVTVSS (SEQ ID NO: 64), and a VL domain comprising the sequence of EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAEIK (SEQ ID NO: 65). In some embodiments, the antigen-binding domain that binds to EGFR comprises VH and VL domain sequences derived from cetuximab or necitumumab.

[0067] In some embodiments, provided herein is a multispecific (e.g., bispecific) binding molecule comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH and VL domains 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARIGDSSPSDYWGQGTLVTVSS (SEQ ID NO: 68) and a VL domain comprising the sequence EIVMTQSPATLSVSPGERATLSCKASQSVSNDVVWYQQKPGQAPRLLIYYASNRYTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQDYTSPWTFGQGTKLEIK (SEQ ID NO: 69). In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYVYYSGTTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASIAVTGFYFDYWGQGTLVTVSS (SEQ ID NO: 70), and a VL domain comprising the sequence of EIVLTQSPGTLSLSPGERVTLSCRASQRVNNNYLAWYQQRPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDRSPLTFGGGTKLEIK (SEQ ID NO: 71). In some embodiments, the antigen binding domain that binds to DLL3 comprises VH domain and VL domain sequences from rovalpituzumab or DLL3-4.

[0068] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1 and 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 connected to the VH domain, wherein the VH and VL domains 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain, and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm comprises QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSS (SEQ ID NO: 72) and a VL domain comprising the sequence of DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIK (SEQ ID NO: 73). In some embodiments, the antigen-binding domain that binds mesothelin comprises VH and VL domain sequences derived from amatuximab.

[0069] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH and VL domains 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS (SEQ ID NO: 74), and a VL domain comprising the sequence of DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVK (SEQ ID NO: 75). In some embodiments, the antigen binding domain that binds CD33 comprises VH and VL domain sequences derived from gemtuzumab.

[0070] In some embodiments, provided herein is a multispecific (e.g., bispecific) binding molecule comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of EVQLVESGGRLVQPKGSLKLSCAASGFAFNTYALYWIRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRARFYYSDYGYAMDYWGQGTSVTVSS (SEQ ID NO: 76), and a VL domain comprising the sequence of DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPLTFGAGTKLELK (SEQ ID NO: 77). In some embodiments, the antigen binding domain that binds to CCR8 comprises VH domain and VL domain sequences from 10A11.

[0071] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH and VL domains 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS (SEQ ID NO: 78), and a VL domain comprising the sequence of EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 79). In some embodiments, the antigen binding domain that binds to CTLA4 comprises VH and VL domain sequences from ipilimumab.

[0072] In some embodiments, provided herein are multispecific (e.g., bispecific) binding molecules comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and 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 connected to the VH domain, wherein the VH and VL domains 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence of EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 80), and a VL domain comprising the sequence of DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIK (SEQ ID NO: 81). In some embodiments, the antigen-binding domain that binds to cMET comprises VH and VL domain sequences derived from onartuzumab.

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

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

[0075] In some embodiments, provided herein is a multispecific (e.g., bispecific) binding molecule comprising a single-chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1 and 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 connected to the VH domain, wherein the VH domain and VL domain of the second antibody arm form an antigen-binding domain that binds to 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 cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain, and a second linker of the present disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm comprises QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm comprises a VH domain comprising the sequence QVQLVQSGAEVKKPGSSVKVSCKASGFTFATYNMHWVRQAPGQGLEWMGYIYPGDGNANYNQQFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGDFDYDGGYYFDSWGQGTLVTVSS (SEQ ID NO: 86), and a VL domain comprising the sequence DIQMTQSPSSLSASVGDRVTITCRASENIYSYLAWYQQKPGKAPKLLIHNAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGAPLTFGQGTKLEIK (SEQ ID NO: 87). In some embodiments, the antigen-binding domain that binds to SAP comprises VH and VL domain sequences from desamizumab.

[0076] Multispecific antibodies have binding specificities for at least two different epitopes (usually 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 agents by phagocytosis, the antigen-binding domain of the present disclosure may be selected from IgG, intrabodies, peptibodies, nanobodies, single domain antibodies, SMTPs, and multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFvs, tandem tri-scFvs, ADAPTIRs).

[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 cognate interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. In some embodiments, the interface can be engineered so that a "knob" or "protuberance" located at the interface of one immunoglobulin polypeptide corresponds to a cognate "hole" or "cavity" located at the interface of the other immunoglobulin polypeptide. In some embodiments, knobs can be constructed by replacing small amino acid side chains with larger side chains. In some embodiments, holes can be constructed by replacing large amino acid side chains with smaller side chains. The knobs or holes may be present within the original interface or may be synthetically introduced. Polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding knob- or hole-forming mutations can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; 8,679,785; 8,844,834; U.S. Patent Application Publication No. 2013 / 0089553; Spiess et al., Nature Biotechnology 31:753-758, 2013; and Ridgway and Carter (1996) Protein Eng. 9:617-621. The modified immunoglobulin polypeptides can be produced using prokaryotic host cells such as E. coli, or eukaryotic host cells such as mammalian cells (eg, CHO cells) or yeast cells.Immunoglobulin polypeptides with corresponding knobs and holes can be expressed in host cells in co-culture and purified together as heteromultimers, or expressed in monoculture, purified separately, and assembled in vitro. Exemplary cognate knob and hole mutations are shown below (numbering according to the 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" can refer to a pair of antibody heavy and light chains, 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, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences.

[0080] In some embodiments, a multispecific (e.g., bispecific) antibody further comprises 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, e.g., Mazor, Y. et al. (2015) MAbs 7:377-389 and EP3452089A2. In some embodiments, a multispecific or bispecific antibody comprises two antibody light chains and two antibody heavy chains, wherein only one of the antibody heavy chains comprises the amino acid substitutions F126C and C220V according to EU numbering, and only the corresponding or cognate light chain comprises the amino acid substitutions S121C and C214V.

[0081] Multispecific (e.g., bispecific) antibodies also include cross-linked or "heteroconjugate" antibodies. Techniques for generating bispecific antibodies from antibody fragments are also described in this document. For example, bispecific antibodies can be prepared using chemical linkage. In some embodiments, a bispecific antibody comprises a first IgG antibody comprising a first antigen-binding domain covalently linked to a second IgG antibody comprising a second antigen-binding domain.

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

[0083] In some embodiments, the monospecific or multispecific (e.g., bispecific) antibody further comprises one or more mutations to reduce effector function, e.g., to reduce or eliminate binding of the Fc region to an Fc receptor. In some embodiments, the antibody comprises two antibody Fc regions, wherein the antibody Fc regions comprise amino acid substitutions at one or more of positions 234, 235, and 237 according to EU numbering. In some embodiments, the antibody comprises two antibody Fc regions, wherein the antibody Fc regions comprise 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 a first antibody heavy chain forms an antigen-binding domain with the VL domain of a first antibody light chain, and the VH domain of a second antibody heavy chain forms an antigen-binding domain with the VL domain of a second antibody light chain, wherein the first antibody heavy chain comprises F126C, C220V, and T366W substitutions according to EU numbering, the first antibody light chain comprises S121C and C214V substitutions, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R, and Y436F substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain further comprise L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain.

[0085] In some embodiments, provided herein is a polynucleotide encoding the antibody or multispecific binding molecule of any one of the above embodiments. In some embodiments, provided herein is a vector (e.g., an expression vector) comprising the polynucleotide of any one of the above embodiments. In some embodiments, provided herein is a host cell (e.g., an isolated host cell or cell line) comprising the polynucleotide or vector of any one of the above embodiments. In some embodiments, provided herein is a pharmaceutical composition comprising the antibody or multispecific binding molecule of any one of the above embodiments and a pharmaceutically acceptable carrier. Any of these can be used in the production and / or processing methods disclosed herein.

[0086] In some embodiments, provided herein is a method of producing an antibody or multispecific binding molecule, comprising culturing a host cell of any one of the above embodiments under conditions suitable for production of the antibody or multispecific binding molecule. In some embodiments, the method further comprises recovering the antibody or multispecific binding molecule. The antibody or multispecific binding molecule can be produced using standard recombinant techniques, as described herein and / or exemplified below.

[0087] Antibodies and antibody fragments can be produced using recombinant methods. For example, nucleic acids encoding the antibody / fragment can be isolated and inserted into a replicable vector for further cloning or for expression. DNA encoding the antibody / fragment can be easily isolated and sequenced using conventional procedures (e.g., via oligonucleotide probes capable of specifically binding to 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 a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryote, yeast, or higher eukaryote cells. When using recombinant techniques, the antibody / fragment can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody / fragment is produced intracellularly, particulate debris (either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Where the antibody / fragment is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter.

[0088] In some embodiments, an antibody or multispecific binding molecule of the disclosure is part of a pharmaceutical composition, e.g., comprising an antibody and one or more pharmaceutically acceptable carriers. The pharmaceutical compositions and formulations described herein can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing the active ingredient (e.g., a fusion protein) having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers, e.g., phosphate, citrate, 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 dextrins; 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 non-ionic surfactants, e.g., polyethylene glycol (PEG).

[0089] Certain aspects of the present disclosure relate to kits or articles of manufacture comprising any of the antibodies or multispecific binding molecules disclosed herein. In some embodiments, the article of manufacture comprises a container and a label or package insert on or associated with the container. In some embodiments, the kit or article of manufacture further comprises instructions for using the antibody or multispecific binding molecule according to any of the methods disclosed herein to treat a disease or disorder, such as cancer.

[0090] Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating a condition and may have a sterile access port (e.g., the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or multispecific binding molecule described herein. The label or package insert indicates that the composition is used for treating a particular condition. The label or package insert further comprises instructions for administering the antibody or multispecific binding molecule composition to a subject. Articles of manufacture and kits containing the combination therapies described herein are also contemplated.

[0091] II. Methods of Production and Identification In certain aspects, the present disclosure provides methods for producing or generating multispecific (e.g., bispecific) antibodies and antibody fragments. In some embodiments, the methods include providing a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to a first target of interest; providing a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to a second target of interest; 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 an interaction between its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative and its biotin- or avidin-linked derivative, thereby generating a multispecific binding molecule. In some embodiments, a first antibody or fragment is linked to its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative, and a second antibody or fragment is linked to its biotin- or avidin-linked derivative. In some embodiments, a second antibody or fragment is linked to its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative, and a first antibody or fragment is linked to its biotin- or avidin-linked derivative. Any antigen-binding domain, antibody, and antibody fragment of the present disclosure (e.g., described above in Section I) can be produced or generated using the methods for producing or generating multispecific (e.g., bispecific) antibodies and antibody fragments disclosed herein. Advantageously, this platform provides a modular format for generating 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 aspects, the present disclosure provides methods for identifying multispecific (e.g., bispecific) binding molecules that bind to a first target of interest and a second target of interest. In some embodiments, the methods include: providing a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to the first target of interest; providing a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to the second target of interest; 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 its avidin-, streptavidin-, neutravidin-, or biotin-binding derivative and its biotin- or avidin-binding derivative, thereby creating a multispecific binding molecule; and measuring binding between the multispecific binding molecule and at least one of the first target of interest and the second target of interest. In some embodiments, a first antibody or fragment is linked to its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative, and a second antibody or fragment is linked to its biotin- or avidin-linked derivative. In some embodiments, a second antibody or fragment is linked to its avidin-, streptavidin-, neutravidin-, or biotin-linked derivative, and a first antibody or fragment is linked to its biotin- or avidin-linked derivative. Advantageously, this platform allows for screening of various antigen-binding domains for binding to targets of interest in a multispecific (e.g., bispecific) format.

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

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

[0095] Assays for measuring binding between a multispecific binding molecule and at least one of a first target of interest and a second target of interest are known in the art. In some embodiments, binding between a multispecific binding molecule and a purified antigen is measured, for example, using an ELISA or SPR binding assay. In some embodiments, binding between a multispecific binding molecule and cells expressing an antigen on their surface is measured, for example, using a flow cytometry-based binding assay. In some embodiments, binding between a multispecific binding molecule and antigen-coated beads or other solid substrate is measured. In some embodiments, functional assays are 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) that have been brought together by binding of the multispecific binding molecule, for example, by measuring cytokine production, cell death / phagocytosis, etc.

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

[0097] Any of the antigen-binding domains, antibodies, and antibody fragments of the present disclosure (e.g., described above in Section I) can be used in the methods of treatment and use disclosed herein, as well as in compositions (e.g., pharmaceutical compositions) related thereto. For example, in some embodiments, the methods involve using a multispecific (e.g., bispecific) binding molecule of the present 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 agent. In some embodiments, the disease-causing 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 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, an eosinophil, an ILC2 cell, or an inflammatory immune cell. In some embodiments, the target of interest is a surface antigen of a virus. In some embodiments, the target of interest is CD70, HER2, DLL3, Nectin-4, TROP-2, mesothelin, LIV-1, C-MET, FOLR1, CD20, CCR8, CD33, or EGFR. Binding of molecules that mediate targeted removal of disease-causing agents by phagocytosis can be with or without avidity, i.e., with or without induction of dimerization of a phagocytic receptor, such as Dectin-1, or a target antigen present on the disease-causing agent.

[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, such as disease-associated tumors, lymphomas, dead cells, necrotic cells, apoptotic cells, dying cells, infected cells, and damaged cells, accumulate and are not cleared. Additionally, various cellular products, such as aggregated proteins (β-amyloid plaques, tau aggregates, or antibody lambda or kappa light chain amyloid), lipoprotein particles, and the like, can cause disease if accumulated in large amounts. Disease-causing cells may possess glycoproteins, surface proteins, or glycolipids typical of abnormal cells associated with disease, disorder, or other undesirable conditions. In addition to host-produced substances, various foreign pathogens, such as infectious microorganisms (e.g., viruses, fungi, and bacteria) and microbial-produced products and debris (e.g., viral particle envelopes, endotoxins, etc.), may not be adequately cleared from patients. In some embodiments, the virus is influenza virus. In some embodiments, the virus is SARS-CoV-2.

[0099] The above abnormalities 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 human subjects. In addition to beneficial removal of disease-causing substances through phagocytosis, these molecules can induce the production of inflammatory mediators to alter the microenvironment of diseases such as tumors, cancer, and lymphoma. Without wishing to be bound by theory, it is believed that molecules that perform targeted phagocytosis may show clear benefits to patients with, for example, Alzheimer's disease, Parkinson's disease, cancer, infectious diseases (viral, bacterial, fungal, or protozoal infections), inflammatory or immune diseases (e.g., autoimmune diseases, inflammatory bowel disease, and multiple sclerosis), degenerative diseases (e.g., joint and cartilage diseases), rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, and IBD. Additionally, targeted phagocytic antibody therapy may be more effective at depleting cells in tissues than NK cell-dependent ADCC. This therapy targets myeloid cells and may have more selective activity for eliminating specific disease-causing agents than therapies that improve phagocytosis in general. For example, targets of interest for cancer therapy 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 presented to enable any person skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided merely as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples 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 examples described and shown herein, but are to be accorded the scope consistent with the appended claims. [Example]

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

[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 5 weeks and boosted weekly with antigen. Antibody titers in mouse serum were assessed before and after the booster inoculation via ELISA and flow cytometry. Mice with the highest serum antibody titers were selected to provide B cells for hybridoma generation.

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

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

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

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

[0107] HEK Blue hDectin-1-a and HEK Blue hDectin-1-b 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). Briefly, viable cell density and viability were measured. Cells were grown in Freestyle 293 expression medium to a final density of 11 x 10 cells. 6The Freestyle Max reagent was diluted to 10 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. The complex was slowly transferred to the cells, gently rotating the culture flask during the addition. The cells were then incubated on an orbital shaker in an incubator at 37°C, 80% relative humidity, and 8% CO2.

[0108] Binding of Dectin-1 Antibody to Dectin-1-Expressing Cells Dectin-1-expressing cells (HEK Blue hDectin-1-a, HEK Blue hDectin-1-b, HEK293F hDectin-1a FL, human monocytes, or cynomolgus monkey monocytes) were plated at 1 × 10 per well in a non-tissue-culture-treated 96-well V-bottom plate. 5 ~2×10 5 Human monocytes were plated with 1000-well plated human monocytes (HMCs). Human monocytes were further incubated with a human FcγR-blocking antibody (Biolegend, San Diego, CA) for 10 minutes at room temperature to reduce antibody binding to Fc receptors. Cells were then stained with a 1:1000 dilution of eFluor 506 viability dye (ThermoFisher, Waltham, MA) for 30 minutes on ice, followed by a wash step with FACS buffer (PBS containing 2% fetal bovine serum). Primary Dectin-1 antibodies or isotypes were used at titrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41, and 0.14 nM. After a 30-minute incubation on ice, the cells were 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 with 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) on ice for 30 minutes. Data were acquired using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA) and analyzed using Graphpad Prism 8.4.

[0110] Dectin-1 antibody inhibition of laminarin HEK Blue hDectin-1a cells were plated at 1 × 10 per well in non-tissue culture treated 96-well V-bottom plates. 5 The cells were plated with primary anti-Dectin-1 antibody at titrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.14, 0.05, 0.015, and 0.005 nM and incubated in the presence of 8 μg / ml biotin-laminarin for 30 min on ice. After a wash step with FACS buffer, binding of biotin-laminarin to HEK cells was detected using streptavidin-AF647 for 30 min on ice. For analysis, 4000 cellular events were acquired on 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 to the beads according to the manufacturer's recommendations. Briefly, based on the antibody-binding capacity of the beads, 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, pHrodo red activation was assessed in low pH buffer by flow cytometry. Bead-bound antibodies were assessed using fluorescently labeled AF647 anti-mouse Fc-specific antibody or FITC anti-human IgG4 antibody secondary antibodies.

[0113] Antibody-dependent targeted phagocytosis of Phrodo-labeled beads For phagocytosis 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 a 96-well plate. pHrodo-labeled beads conjugated to anti-Dectin-1 antibodies or isotypes were added at the desired ratio ranging from 1:1 to 1:3 cells / beads, and the plate was spun down briefly.

[0114] In some 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 an IncuCyte S3 live imaging system (Germany) and analyzed using IncuCyte S3 software. Phagocytosis was quantified as the overlap of bright red fluorescence (engulfed beads) with calcein AM-positive cells or the integrated red intensity of bright red fluorescence.

[0115] SEAP reporter assay using dectin-1-overexpressing HEK cells and anti-dectin-1 antibody Anti-Dectin-1 monoclonal antibody 2M24 (VH domain and VL domain contain SEQ ID NOs: 7 and 8, respectively) or 15E2 and a control isotype were immobilized by coating onto the surface 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 lid 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 antibody. HEK Blue hDectin-1-a cells were then cultured on the plates in RPM1 containing 10% ultra-low IgG FBS (VWR) for 22 hours, and alkaline phosphatase levels were assessed in the supernatants at OD 630 nm using QUANTI Blue Solution (Invivogen, San Diego, CA) according to the manufacturer's instructions.

[0116] To measure the amount of SEAP secretion from HEK cells induced by beads conjugated with anti-Dectin-1 antibody, streptavidin-2M24 (hIgG4) was conjugated to biotin polystyrene beads (Spherotech, Lake Forest, IL) of 3, 10, and 16 μm sizes by incubating the beads with the antibody at room temperature for 30 min and then washing twice with PBS to remove unbound antibody. Anti-Dectin-1 antibody-conjugated beads were added at 1 × 10 5 After mixing with HEK Blue hDectin-1-a cells at a cell:bead ratio of 1:3 in RPM1 containing 10% ultra-low IgG FBS 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 antibodies (2M24 or 15E2 clones) and control isotypes were immobilized by coating 10 μg onto the surface of wells of untreated 96-well U-bottom polypropylene microtiter plates as described above. Freshly isolated monocytes or peripheral blood mononuclear cells were then cultured on the immobilized antibody-containing plates at 200,000 cells / well in RPM1 containing 10% ultra-low IgG FBS for 24 hours. In other wells, cells were treated with 10 μg / ml of dectin-1 antibody in solution instead of immobilized antibody. TNFα, IL-6, and IFNγ levels in the supernatants were assessed using the U-PLEX assay platform (Meso Scale Discovery), and their levels were expressed as fold changes in dectin-1 antibody-induced cytokine secretion relative to the isotype control. As a positive control, cells were stimulated with 25 μg / ml zymosan.

[0118] result To generate Dectin-1 antibodies, 4-week-old ATX-Gx Alloy transgenic mice were subcutaneously inoculated with recombinant Dectin-1 isoform B protein and boosted weekly with antigen. The antibodies generated by this inoculation have human variable domains and mouse constant domains.

[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 clone exhibited high affinity for dectin-1-expressing human monocytes. In contrast, 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 exhibited by other clones and commercially available dectin-1 antibodies (15E2, 259931, GE2). Figure 1C shows a comparison of the binding of the 2M24 clone to human monocytes and HEK cells overexpressing dectin-1 with 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 dectin-1. Binding was assessed by flow cytometry analysis of PBMC-derived cynomolgus monocytes. As shown in Figure 1B, the anti-human dectin-1 clone 2M24 antibody exhibited cross-reactivity and high affinity for cynomolgus dectin-1 expressed on monocytes. The 2M24 anti-dectin-1 antibody exhibited superior affinity to the commercially available antibodies tested, exhibiting an EC50 of 0.3 nM. The agonist 15E2 and 255931 commercial antibodies exhibited EC50s of 14 nM and 16 nM, respectively, on cynomolgus monocytes. Figure 1C shows a comparison of binding to cynomolgus monocytes between the 2M24 clone and the commercially available clones 15E2 and 255931.

[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 hDectin-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 coupled to polystyrene beads promoted phagocytosis in both HEK-Blue hDectin-1a cells and primary human monocytes.

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

[0123] Next, the hIgG4 2M24 antibody was tested for its ability to promote phagocytosis of beads in dectin-1-expressing cells. As shown in Figure 4, the hIgG4 2M24 antibody exhibited efficient phagocytosis in HEK cells, human monocytes, and human macrophages that overexpress dectin-1. Thus, the fully human IgG4 2M24 antibody can promote phagocytosis of dectin-1-expressing cells.

[0124] The fully human 2M24 (hIgG4) anti-Dectin-1 antibody was further tested for its ability to promote signaling through Dectin-1. Activation of Dectin-1 signaling by the antibody was assessed in a secreted alkaline phosphatase assay using HEK-Blue hDectin-1a cells. HEK-Blue hDectin-1a cells were 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 ligand. As shown in Figures 5A-5B, the 2M24 (hIgG4) anti-Dectin-1 antibody induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells in both immobilized and bead-conjugated forms. These observations support the idea that binding of the 2M24(hIgG4) antibody to cell surface dectin-1 promotes SEAP secretion and demonstrate receptor clustering and agonistic activity by this antibody. Furthermore, efficient dectin-1 clustering signaling can be promoted by beads conjugated to 2M24(hIgG4). Signaling was more effectively induced with larger beads, reflecting better receptor clustering. This supports the idea that dectin-1 clustering promoted by bispecific antibodies containing an anti-dectin-1 antibody targeting phagocytes and an antibody targeting another cell type, such as cancer cells, may promote clustering and signaling by dectin-1 on phagocytes.

[0125] The natural ligand of dectin-1 clusters receptors, signaling downstream of dectin-1 / Syk / NFkB and inducing inflammatory gene expression. To assess whether engagement of dectin-1 antibodies in solution can induce cytokine secretion, monocytes or macrophages were treated with 10 μg / ml of a commercially available anti-dectin-1 antibody. As shown in Figures 6A-6B, the 15E2 commercial anti-dectin-1 antibody did not induce cytokine secretion in primary human macrophages and monocytes, suggesting insufficient clustering of dectin-1 receptors. This data supports the conclusion that free dectin-1 antibodies in solution do not induce immune stimulation due to insufficient dectin-1 clustering.

[0126] To evaluate whether the 2M24 (hIgG4) anti-Dectin-1 antibody could induce cytokine secretion, 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 demonstrated superior immune stimulation compared to that promoted by the 15E2 anti-Dectin-1 agonist antibody. Among the cytokines measured in this experiment, TNFα and IL-6 were secreted by monocytes expressing dectin-1. In contrast, IFNγ was primarily secreted by T cells present in PBMCs. Because T cells do not express dectin-1, they are not directly activated by the anti-Dectin-1 antibody, but are activated by cytokines secreted by monocytes within PBMCs stimulated by the dectin-1 antibody. Therefore, the differential effect of the dectin-1 antibody on IFNγ was 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 anti-dectin-1 antibodies. HEK-Blue hDectin-1a cells were incubated with 2M24 (hIgG4) dectin-1 antibody or 15E2, 259931, and GE2 anti-dectin-1 commercial antibodies in the presence of 8 μg / ml biotinylated laminarin, using a serial dose titration starting at 300 nM and extending to 1 / 3. As shown in Figure 8, binding of 2M24 (hIgG4) antibody to dectin-1 did not inhibit 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 can induce activation of Dectin-1 signaling without competing with the natural ligand of Dectin-1. The properties of the 2M24 and 15E2 antibodies are summarized in Figure 9.

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

[0130] Materials and Methods Creation of bispecifics Antibodies were differentially labeled with MTA or FOL reagents according to the manufacturer's guidelines (AAT Bioquest). The labeled antibodies were mixed and incubated to allow covalent binding via MTA and FOL interactions. The following antibodies were used for biotin:streptavidin-derived bispecific antibodies: Anti-Dectin-1 15E2 antibody heavy chain:mSA fusion QWQLQQSGAELARPGASWKMSCKASGYTFTTYTMHWWKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAWYYCARERAVLVPYAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH (SEQ ID NO: 18) Anti-Dectin-1 15E2 antibody light chain QIVLTQSPAVMSASPGEKWTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVPTRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) Avi-tagged anti-CD20 Fab heavy chain (CH1 domain based on hIgG4 sequence) QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE (SEQ ID NO: 20) Anti-CD20 Fab light chain QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21) Avi-tagged anti-HER2 Fab heavy chain (CH1 domain based on hIgG4 sequence) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE (SEQ ID NO: 22) Anti-HER2 Fab light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 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 + ). Engagement efficiency was quantified as the percentage of total target cells that formed doublets with Dectin-1-expressing cells.

[0132] Five million effector (Dectin-1-expressing cells) or target cells (cells expressing a target of interest, 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). Cells were washed extensively with PBS and placed on ice. Effector and target cells were then co-cultured at a 3:1 ratio (effector:target) in the presence of 2M24 bispecific antibody or an isotype control and incubated at 37 °C for 30 min. After incubation, samples were gently resuspended and analyzed by flow cytometry. PMT voltages were adjusted accordingly, and cells were gated based on FITC and / or PE fluorescence corresponding to calcein green or red fluorescence. Engagement 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 dectin-1-overexpressing HEK cells and anti-dectin-1 antibody To measure the amount of SEAP secretion from HEK cells induced by Raji cells (expressing CD20), Raji cells were coated with 2M24 / anti-hCD20 or hIgG4 / anti-CD20 bispecifics for 30 minutes on ice, followed by two washes with PBS to remove unbound bispecifics. Bispecific-coated Raji cells were cultured at 1x10 in RPM1 containing 10% ultra-low IgG FBS. 5 The cells were mixed with HEK Blue hDectin-1-a cells at a ratio of 1:2 (HEK cells:Raji cells). After 22 hours, the amount of alkaline phosphatase secreted in the supernatant was assessed 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 targeting antibody, we used a click chemistry approach to create bispecifics containing an anti-dectin-1 targeting arm and a second arm targeting a protein of interest. This approach enabled the creation of bispecifics for various assays. A schematic of this approach is shown in Figures 10A-10B. Because binding of dectin-1 by a dectin-1-specific antibody can induce phagocytosis of the target (see Examples 1 and 2), we evaluated the bispecific antibodies for their ability to promote phagocytosis of specific target cells. We first evaluated the bispecifics for their 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 a variety of diseases, including acute myeloid leukemia (AML), renal cell carcinoma, rheumatoid arthritis, and lupus.

[0135] Click chemistry was used to generate a bispecific molecule containing a Dectin-1-targeting arm (anti-Dectin-1; clone 2M24) and a CD70-targeting arm (anti-hCD70; clone 113-16). The purity of the bispecific (2M24 / anti-hCD70) antibody was assessed by SDS-PAGE analysis (Figure 12A), and binding was assessed by flow cytometry analysis (Figure 12B). As shown in Figure 12B, cell binding studies revealed that 2M24 / anti-hCD70 bound to the Dectin-1-expressing HEK293 cell line 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). The bispecific was then evaluated for its ability to induce cell binding. As shown in Figure 13, the 2M24 / anti-hCD70 bispecific induced binding between Dectin-1-expressing HEK293 cells and CD70-expressing renal carcinoma 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. CD20 is a transmembrane protein present on almost all B cells, from their commitment to B cell development until it is downregulated during differentiation into antibody-secreting plasma cells. It is considered a pan-B cell antigen marker. As shown in Figures 14A-14B, the 2M24 / anti-hCD20 bispecific 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 cell-cell binding mediated by the bispecific can induce synapse formation between effector and target cells, alter cytokine signaling, activate phagocytosis, and ultimately perform target antigen presentation.

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

[0138] We have previously 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-targeting antibodies (see Examples 1 and 2). To demonstrate phagocytosis of live target cells, we generated a bispecific comprising a dectin-1-targeting arm and a CD20-targeting arm. In a coculture assay of HEK293 cells and CD20-expressing Raji cells, we observed phagocytosis of cells treated with the anti-dectin-1 / anti-hCD20 bispecific, in contrast to an isotype control bispecific (Figure 16). Furthermore, preincubation of cells with latrunculin A, an inhibitor of phagocytosis that inhibits actin polymerization, inhibited phagocytosis of cells treated with the anti-dectin-1 / anti-hCD20 bispecific. These findings indicate that expression of Dectin-1 is sufficient to induce phagocytosis and that co-targeting Dectin-1 and a target of interest with a Dectin-1 agonist bispecific is sufficient to induce phagocytosis of target cells.

[0139] We conducted a proof-of-concept experiment 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 induced the association of Dectin-1 with HER2-expressing cells. This interaction is thought to promote synapse formation between effector and target cells, as 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 bispecific antibody. Large granular lymphocytic (LGL) leukemia is a rare chronic lymphoproliferative disorder of the T cell and natural killer (NK) cell lineages. CD94 / NKG2 is a family of C-type lectin receptors expressed primarily on the surface of NK cells and a subset of CD8+ T lymphocytes. As shown in Figure 18, the anti-Dectin-1 (2M24) / anti-hCD94 bispecific antibody induced ligation between Dectin-1-expressing cells and CD94-expressing cells. Thus, bispecific antibodies that bind to Dectin-1 can mediate ligation between Dectin-1-expressing cells and various target cells.

[0141] Example 3: Generation of bispecific anti-Dectin-1 antibodies using streptavidin-biotin This example describes the biochemical and functional characterization of bispecific antibodies that bind to Dectin-1, generated using streptavidin-biotin conjugation.

[0142] Materials and Methods Creation of bispecifics mSA was genetically fused to either Fab 2M24 or full-length 2M24. The chimeric fusion was incubated with a biotinylated target antibody to generate a bispecific containing a Dectin-1-binding arm and a second arm that binds to the target receptor or protein of interest. Full length 2M24 sequence fused to mSA: (SEQ ID NO: 15) Fab 2M24 sequence fused to mSA: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH (SEQ ID NO: 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 assessed by detecting activated Phrodo red within the HEK cell / bead duplet population by flow cytometry in the PE channel using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA).

[0144] result To enable efficient generation of bispecific antibodies, we developed a novel strategy that exploits the high-affinity interaction between streptavidin and biotin. A monomeric streptavidin (mSA) construct was fused to the Fc domain of 2M24 or the CH1 domain of Fab 2M24. The recombinant fusion protein was incubated with various biotinylated antibodies of interest to construct bispecifics. 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, we generated and purified the Fab 2M24-mSA fusion protein. As shown in Figures 20A-20C, the Fab 2M24-mSA fusion exhibited 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 targets 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 cells expressing Dectin-1.

[0146] Anti-Dectin-1-streptavidin fusions were used to generate bispecific antibodies against 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 generating bispecific antibodies.

[0147] Next, the anti-Dectin-1 bispecific generated using the Fab 2M24-mSA fusion protein was evaluated for its ability to induce cell ligation. As shown in Figure 23, the Fab 2M24-mSA / biotin anti-hCD20 bispecific induced ligation between Dectin-1-expressing HEK293 cells and CD20-expressing B cells (Raji cell line). This interaction can induce cytokine secretion by effector cells, trigger phagocytosis of target cells, and promote Dectin-1 clustering, which leads to neoantigen presentation and activation of adaptive immune cells (B and T cells).

[0148] Example 4: Targeted phagocytosis of amyloid deposits using anti-Dectin-1 bispecific antibodies This example describes the use of a bispecific antibody that binds Dectin-1 in the targeted delivery of pathogen antigens to phagocytes.

[0149] Dectin-1-induced targeted phagocytosis can be used to mediate amyloid clearance by antigen-presenting cells (e.g., monocytes, macrophages, dendritic cells, and neutrophils) and maximize depletion of circulating amyloid precursors (free light chains) and deposited amyloid fibrils. This targeted phagocytosis relies on bispecific antibodies targeting dectin-1 (with 2M24) and AL amyloid (with 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 sites of amyloid deposition. We determined the phenotype of immune cell populations in amyloid-laden tissue freshly isolated from patients with AL amyloidosis. We measured the frequency of antigen-presenting cells (e.g., macrophages, monocytes, neutrophils, and dendritic cells). We also assessed dectin-1 expression on APCs in AL amyloidosis patient tissue.

[0151] We then generated bispecific antibodies containing a Dectin-1-binding arm (e.g., clone 2M24) and an amyloid-binding arm (based on an external antibody). In a proof-of-concept study, phagocytosis of AL amyloid fibrils by circulating monocytes, monocyte-derived macrophages (differentiated in vitro), or patient-derived macrophages (in situ) was demonstrated with the external antibody and the prepared Dectin-1 bispecific antibody (Figure 24). We initiated an antibody discovery campaign to identify high-affinity binders for amyloid fibrils or amyloid precursors, and used the resulting antibodies to further generate Dectin-1 bispecific antibodies targeting amyloid deposits.

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

[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 anti-Dectin-1 antibody or an isotype control. For the phagocytosis assay, the beads were incubated with cultured dendritic cells at a 1:3 (cell:bead) ratio. Bead phagocytosis was monitored by IncuCyte live-cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the total integrated intensity (summed fluorescence intensity) of red objects (pHrodo fluorescence) in the image.

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

[0155] First, bispecific antibodies with a macrophage-targeting arm (via Dectin-1 binding) and a mast cell-targeting arm (via mast cell surface antigens) were generated, as shown in Figure 25A. Potential mast cell surface antigens / receptors that could be used as lead candidates for bispecific generation are summarized in Figure 25B. These bispecific antibodies were then evaluated for their ability to bind 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 tissues / organs from patients with high numbers of mast cells. Phenotypes of immune cell populations were determined in fresh tissues isolated from patients with mastocytosis. Dectin-1 expression was assessed on APCs. Binding of external antibodies was assessed on donor mast cells.

[0157] Considering the large size of mast cells, we evaluated whether dectin-1 could promote phagocytosis of large entities by conjugating anti-dectin-1 antibodies to large beads (approximately 16.5 μm) similar in size to these cells. Because macrophages are large phagocytes and can ingest large targets, we performed a large-bead phagocytosis assay using macrophages differentiated from monocytes in the presence of MCSF for 6 days. As shown in Figure 26, anti-dectin-1 antibodies promoted directed phagocytosis of large beads in cultured human macrophages. Anti-dectin-1-conjugated beads were more readily ingested by macrophages compared to isotype control-conjugated beads. These data support the idea that dectin-1 targeting 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 Dectin-1 in targeted phagocytosis of microorganisms.

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

[0160] Innate immune cells play a crucial role in the recognition and elimination of microbial pathogens. To help phagocytes attack bacterial, viral, or fungal pathogens, bispecific antibodies containing 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 can be used for Dectin-1-induced targeted elimination (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 target degradation, the target antigen is presented, modulating the adaptive immune response to further repel the pathogen.

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

[0162] To test targeted delivery of pathogens to phagocyte antigens, we generated a bispecific antibody with a Dectin-1-binding arm and a second arm that binds to the hematoglutinin of 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 H3N2 influenza virus and HEK cells expressing Dectin-1. This antibody format can be used to target influenza virus 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 phagocytes.

[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 anti-Dectin-1 antibody or an isotype control. For the phagocytosis assay, the beads were incubated with cultured dendritic cells at a 1:3 (cell:bead) ratio. Bead phagocytosis was monitored by IncuCyte live-cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the total integrated intensity (summed fluorescence intensity) of red objects (pHrodo fluorescence) in the image.

[0165] Labeling of polystyrene beads with pHrodo and conjugation to antibodies Labeling of polystyrene beads with pHrodo and conjugation to antibodies was performed as described above.

[0166] To coat polystyrene beads with SARS-CoV-2 spike protein S1, goat anti-rabbit IgG (Fc) beads (Spherotech) were labeled with pHrodo Red and conjugated to rabbit anti-Flag antibody (Cell Signaling) as described above. Flag-tagged spike protein (Genscript) was then 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 For phagocytosis of SARS-CoV-2 spike protein-coated beads, the beads were preincubated with anti-Dectin-1 / anti-SARS-CoV-2 spike protein bispecific antibody (anti-SARS-CoV-2 spike protein antibody purchased from Genscript) at room temperature for 60 minutes, and then 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 agents (cancer cells, pathogens, or protein aggregates) or the antigen itself to dendritic cells through Dectin-1 can elicit a protective immune response against the antigen and the disease-causing agent derived from it. 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 agents (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 agents to APCs (Figure 29B).

[0169] To determine whether phagocytosis of human dendritic cells can be promoted by antibody engagement of Dectin-1, purified monocytes (CD14+) derived from human PBMCs were differentiated into dendritic cells in the presence of IL4 / GMCSF. After 6 days, 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 promoted directed phagocytosis of beads by cultured monocyte-derived dendritic cells. Significantly more phagocytosis of anti-Dectin-1 conjugated beads was observed than of isotype-conjugated beads, as evidenced by the presence of bright red pHrodo particles within the cells. Dendritic cells are specialized antigen-presenting cells. Targeting antigens on disease-causing agents (e.g., cancer cells, pathogens, or protein aggregates), or the antigen itself, to dendritic cells via Dectin-1 may elicit a protective immune response against the antigen and the disease-causing agent derived from the antigen. This response can 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 and promote the recognition and elimination of disease-causing agents (e.g., cancer cells or pathogens).

[0170] We also evaluated the use of a dectin-1 agonist bispecific antibody to target another virus, SARS-CoV-2. Using click chemistry, we created a bispecific antibody targeting both dectin-1 and the SARS-CoV-2 spike S1 protein. The spike protein was coated onto beads, which were then internalized in the presence of an anti-dectin-1 / anti-SARS-CoV-2 spike S1 bispecific (Figure 31A). The anti-dectin-1 / anti-SARS-CoV-2 spike S1 bispecific induced binding of dectin-1-expressing HEK293 cells to the spike-coated beads (Figure 31B) and promoted phagocytosis of the spike-coated beads by the dectin-1-expressing cells (Figure 31C). Based on these results, the anti-dectin-1 / anti-SARS-CoV-2 spike S1 bispecific may mediate targeted delivery of the SARS-CoV-2 spike protein to macrophages.

[0171] conclusion The bispecific antibodies described in this example, which contain 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 uptake of the target pathogen and antigen presentation, followed by T cell activation and proliferation and antibody production by B cells. The adaptive immune response can promote elimination of the virus and virus-infected cells. This Dectin-1 antibody-targeted vaccine approach may also work against other pathogens of bacterial or viral origin.

[0172] Various anti-Dectin-1 (e.g., 2M24) / antigen-specific bispecific antibodies were generated and demonstrated to efficiently bind to APCs. Internalization of target antigens from APCs was assessed. A method for assessing antigen presentation of target antigens on the surface of dendritic cells was developed. CD4+ and CD8+ T cell activation (T cell proliferation and cytokine secretion) was assessed from dendritic cells that received antigen through Dectin-1. B cell activation and antibody production against antigens were assessed. Mice were vaccinated and protected against disease caused by pathogens / malignant cells, and adaptive immune responses were assessed in vivo.

[0173] Example 8: Bispecific design to generate human bispecific antibodies targeting Dectin-1 and a disease target or antigen To enable the construction and efficient production of highly purified and active bispecific antibodies, we employed design principles based on previously reported strategies, such as "knobs-into-holes" (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 the complete bispecific antibody involves expression of the four individual 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] Bispecific antibodies using this design were constructed for proof-of-concept studies, as shown in Table 1. These bispecific antibodies have one arm that targets hDectin-1 and a second arm that targets hCD20, hHER2, hCD70, or a protein on RSV. The bispecific antibodies listed in Table 1 were produced by expressing all four chains and purifying them to 95% purity and homogeneity. All bispecifics were confirmed to bind to their respective targets. [Table 2]

[0175] The variable domains of the opposing antibody arms of the anti-Dectin-1 in Table 1 were as follows: CD20 VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24) CD20 VL:QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25) HER2 VH: EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (SEQ ID NO: 34) HER2 VL:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (SEQ ID NO: 35) CD70 VH: EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKGRFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS (SEQ ID NO: 38) CD70 VL: QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHSGVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVL (SEQ ID NO: 39) RSV VH: QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLTISKDTSANQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSS (SEQ ID NO: 36) RSV VL:DIQMTQSPSTLSASVGDRVTITCKCQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTAFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK (SEQ ID NO: 37).

[0176] These hDectin-1 bispecific antibodies engage three targets: Dectin-1 on myeloid cells, antigens on target cells or disease-causing agents, and Fc receptors on myeloid cells and NK cells, inducing robust immune stimulation and phagocytosis (Figure 32B). In particular, bispecific antibodies with a nonfucosylated, activated hIgG1 Fc domain can recruit myeloid 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, without wishing to be bound by theory, dual engagement of dectin-1 and Fcγ receptors on myeloid and NK cells is thought to elicit potent immune responses that ultimately eliminate cancer cells through the following actions: (1) bispecific antibody-induced crosslinking of dectin-1 with Fcγ receptors leads to ITAM-dependent activation of downstream inflammatory pathways and the release of immunomodulatory cytokines and cytotoxic proteins (proteases, perforin) 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) presentation of phagocytosed antigens by macrophages and DCs, a process that triggers T cell immune responses 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 contained mutations (L234A, L235E, and G237A according to EU numbering) in the hIgG1 Fc domain (hIgG1 inactive) that abolish Fc binding to Fc receptors. Binding of the 2M24 / CD20 or 2M24 / RSV bispecifics to HEK293 cells stably expressing human Dectin-1 was assessed by flow cytometry (Figure 33A). The 2M24 / CD20 and 2M24 / RSV hIgG1 inactive bispecific antibodies were able to bind to cells expressing human Dectin-1 with similar affinity (cell-based binding EC50 values ​​of 1.4 and 1.7 nM, respectively). Thus, the 2M24 / CD20 or 2M24 / RSV bispecific antibodies exhibited high affinity binding to Dectin-1-expressing HEK293 cells.

[0178] The binding of rituximab, 2M24 / CD20, or 2M24 / RSV hIgG1 active or inactive bispecific antibodies was also evaluated using the CD20-expressing B-cell lymphoma Raji cell line (Figure 33B). The 2M24 / CD20 bispecific (active or inactive hIgG1 isotype) antibodies were able to bind to CD20-expressing Raji cells, but with at least 10-fold lower affinity than rituximab. Without wishing to be bound by theory, the difference in CD20 binding affinity between the 2M24 / CD20 bispecific and rituximab is thought to be due to a loss of avidity (monovalent vs. bivalent binding) in the bispecific antibodies.

[0179] Next, we assayed the ability of the 2M24 / CD20 bispecific antibody to induce ligation between cells expressing hDectin-1 and cells expressing hCD20. Dectin-1-expressing HEK293 cells (effector) and CD20-expressing Raji cells (target) were differentially labeled with calcein green (effector) or calcein red (target) dye. The labeled cells were cocultured 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 total target cells that bound or ligated to effector cells) was assayed using dose titration of the bispecific antibody in the effector:target cell coculture (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 to CD20 on Raji cells (Figure 33B), 2M24 / CD20 ligated very efficiently. These findings suggest that the binding affinity of 2M24 / CD20 is enhanced (avidity) by high expression of dectin-1 or CD20 on both effector and target cells, thereby promoting efficient ligation of the two cells. Based on these findings, the 2M24 / CD20 bispecific antibody is likely to effectively engage dectin-1-expressing monocytes, macrophages, or dendritic cells with target disease cells, such as B-cell lymphoma, that express high levels of CD20. Effector:target engagement is the first step in the MOA of the 2M24 bispecific antibody.

[0181] The human IgG1 activating isotype binds to Fcγ receptors on NK cells or monocytes. Therefore, we evaluated whether the hIgG1 activating isotype of 2M24 / CD20 could trigger monocyte killing by NK cells (via antibody-dependent cellular cytotoxicity, ADCC) or other monocytes (via fratricide or antibody-dependent cellular phagocytosis, ADCP). In this scenario, the activating hIgG1 domain of 2M24 / CD20 engages Fcγ receptors on NK cells or monocytes and the Dectin-1 receptor 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 with increasing concentrations of 2M24 / CD20 bispecific (hIgG1 active or inactive isotype) and rituximab for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD14+ monocytes (as a percentage of isotype control). No reduction in monocyte numbers was observed in either donor, indicating that 2M24 / CD20 active IgG1 did not induce monocyte depletion. Without wishing to be bound by theory, 2M24 / CD20 hIgG1 (active isotype) is believed to have no effect on monocyte levels and therefore minimal risk of infection.

[0182] Based on the proposed MOA of the 2M24 / CD20 bispecific antibody (shown in Figure 32B), B cell depletion with 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 increasing concentrations of the indicated antibodies for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as % of B cells in isotype control-treated PBMCs). Thus, in two healthy donors, high concentrations of the 2M24 / CD20 bispecific antibody induced superior B cell depletion (approximately 80% reduction) compared to rituximab (approximately 40% reduction), despite the bivalent binding of rituximab and the approximately 10-fold difference in binding affinity (shown in Figure 33B). The unique mechanism of action of 2M24 / CD20 activating IgG1 (Figure 32B), which involves binding to Dectin-1 on myeloid cells, Fcγ receptors on NK cells and monocytes, and CD20 on target B cells, results in an overall superior B cell depletion compared to rituximab. These data support the concept that Dectin-1-induced immune stimulation via the 2M24 / CD20 bispecific promotes target cell depletion.

[0183] The ability of the 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 hours of incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab, or an isotype control. The mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugated) was used to assess the effect of the 2M24 / CD20 bispecific and rituximab on CD19 expression on B cells. In PBMCs from donor 83, the EC50 for CD19 expression was 0.014 nM for rituximab and 0.080 nM for the 2M24 / CD20 hIgG1 bispecific (Figure 37A). In PBMCs from donor 84, the EC50 for CD19 expression was 0.013 nM for rituximab and 0.090 nM for the 2M24 / CD20 hIgG1 bispecific (Figure 37B). Both the 2M24 / CD20 activating IgG1 bispecific antibody and rituximab caused downregulation of CD19 expression on B cells. Interestingly, rituximab showed at least 5-fold more potent shaving compared to the 2M24 / CD20 bispecific antibody. Downregulation of the target CD20 on B cells has previously 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 activating IgG1 bispecific may have an advantage in B cell depletion due to its reduced shaving ability compared to rituximab.

[0184] Immune stimulation induced by the 2M24 / CD20 activating IgG1 bispecific antibody resulted in the secretion of a unique repertoire of cytokines compared with rituximab (Figure 38). ELISA-based (mesoscale discovery) cytokine quantification was performed on supernatants isolated from healthy donor PBMCs treated with the 2M24 / CD20 activating hIgG1 bispecific, rituximab, or an isotype control. PBMCs were stimulated overnight with the antibody, and the supernatants were subsequently analyzed by MSD. The cytokines tested were IFNγ, IL-12p70, IL-6, TNFα, IL-1β, IL-4, IL-13, IL-10, and IL-8. The results showed that the 2M24 / CD20 activating IgG1 induced higher levels and more distinct cytokine activation in PBMCs compared with rituximab. Furthermore, engagement of Dectin-1 and Fc receptors by the 2M24 / RSV bispecific alone did not induce cytokine release, precluding the possibility of systemic cytokine activation. These findings highlight the unique MOA that distinguishes the 2M24 / CD20 activating IgG1 bispecific antibody from rituximab. Without wishing to be bound by theory, these findings further indicate that 2M24 / CD20 may be able to trigger the release of Th1- and Th2-type responses and promote immune stimulation of the tumor microenvironment.

[0185] The 2M24 / CD20 hIgG1 (activating isotype) bispecific antibody was also found to induce superior B cell depletion and less CD19 shaving compared with rituximab in cocultures of human macrophages and GFP-expressing Raji B cells. Cocultures of human macrophages and Raji-GFP cells (3:1 ratio) were analyzed by flow cytometry in the presence of the 2M24 / CD20 hIgG1 (activating isotype) bispecific, 2M24 / RSV control, fucosylated rituximab, or an isotype hIgG1 control. The cocultures 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 assessed at the end of the experiment. Primary antibodies were used in serial dose titrations. CD19 was assessed on Raji-GFP cells after 24 hours (Figure 39B), and B cell receptor expression was demonstrated as a decrease in CD19 MFI in the presence of the anti-Dectin-1 / anti-hCD20 bispecific or rituximab. The EC50 for CD19 expression was 0.020 nM for rituximab and 0.95 nM for the 2M24 / CD20 hIgG1 bispecific. These results demonstrate enhanced B cell depletion (mediated by Fcγ receptors) by the 2M24 / CD20 bispecific antibody compared with 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 of CD20 by rituximab was observed, limiting the effectiveness of rituximab in depleting B cells. Without wishing to be bound by theory, these data suggest that the superiority of the 2M24 / CD20 hIgG1 (activating isotype) bispecific in B cell depletion is due to reduced B cell receptor shaving compared to rituximab, highlighting a different mechanism of cell depletion by the 2M24 / CD20 hIgG1 (activating isotype) bispecific.

[0186] B cell depletion was also analyzed in single-cell suspensions from kidney cancer tissue specimens. Single-cell suspensions from two kidney cancer tissue specimens were analyzed by flow cytometry in the presence of the 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, the 2M24 / RSV hIgG1 control, fucosylated rituximab, and the respective isotype controls. The kidney cancer tissue specimens were dissociated into single-cell suspensions and treated with primary antibodies (2 μg / ml) at 37°C for 24 hours. Immune cell populations were analyzed by flow cytometry (Figures 40A and 40B). The number of remaining viable B cells was assessed using an anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (Figure 40C). The 2M24 / CD20 active IgG1 bispecific antibody significantly depleted tissue B cells compared to rituximab in single-cell suspensions from kidney cancer specimens. The 2M24 / CD20 hIgG1 (activating isotype) bispecific antibody depleted B cells by 44% and 46%, respectively, in two kidney cancer donor specimens, whereas rituximab induced B cell depletion by 33% and 18%, respectively (Figure 40C). This data supports the ability of the 2M24 / CD20 hIgG1 (activating isotype) bispecific antibody to deplete cancer tissue cells via dectin-1-induced immune stimulation and Fcγ receptor engagement. Without wishing to be bound by theory, because dectin-1 is predominantly expressed on tumor-associated macrophages (TAMs) in these specimens, the 2M24 / CD20 hIgG1 (activating isotype) bispecific antibody may engage TAMs to promote target cell depletion.

[0187] Cytokine secretion by 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 examined. Anti-Dectin-1 antibody (clone 2M24), an isotype control, or 2M24 / CD20 bispecific antibody was immobilized overnight at 10 μg per well on U-bottom polypropylene microtiter plates, followed by incubation with human monocyte-derived macrophages (Figures 41A and 41B) or single-cell suspensions from renal cancer specimens (Figure 41C). Cells were cultured for 24 hours, and the amount of TNFα secreted in the supernatant was assessed 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 immune stimulation in primary macrophage cultures and single-cell homogenates of cancer specimens. Because Dectin-1 is expressed in myeloid cells, tumor-associated macrophages in cancer specimens are expected to produce cytokines in response to anti-Dectin-1 antibody stimulation. This promotes the transition of tumor-associated macrophages from an anti-inflammatory to a pro-inflammatory state, resulting in potent antitumor effects. Furthermore, monovalent binding of the 2M24 / CD20 bispecific antibody to Dectin-1 was sufficient to promote Dectin-1 clustering and immune stimulation of macrophages.

[0188] We also analyzed immune stimulation by immobilized anti-Dectin-1 antibody in single-cell suspensions from renal cancer specimens (Figure 42). Single-cell suspensions from renal cancer specimens were treated with immobilized anti-Dectin-1 antibody (clone 2M24) or an 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 primarily expressed by tumor-associated macrophages, or TAMs) triggered the release of a specific repertoire of cytokines downstream of the Dectin-1 signaling pathway, either directly or indirectly through activation of other immune cells. Without wishing to be bound by theory, we believe that engagement of Dectin-1 by the 2M24 bispecific antibody promotes immune stimulation and modulates the tumor microenvironment to support the elimination of target-expressing cancer cells.

[0189] Example 9: Characterization of a bispecific antibody targeting Dectin-1 and CD20 This example describes further characterization of a bispecific antibody targeting human Dectin-1 and human CD20. The anti-Dectin-1 arm contained the variable domain of 2M24, and the anti-CD20 arm contained the variable domain of rituximab (see SEQ ID NOs: 24 and 25 for the VH and VL domains, 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 antibody. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. CD16 expression on CD56+ NK cells was quantified and compared with expression levels in the isotype control-treated group.

[0191] CD19 expression on B cells Human PBMCs from healthy donors were treated with 0.1 nM 2M24 / CD20 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. CD19 expression (MFI) on B cells was quantified.

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

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

[0194] result First, we examined the effect of the 2M24 / CD20 bispecific on CD16 expression in human NK cells. Because CD16 is required for ADCC activity by NK cells, loss of CD16 expression may reduce the cytotoxic activity of NK cells. Rituximab induced a potent and robust reduction of CD16 on NK cells compared with 2M24 / CD20 hIgG1 KIF (Figure 43). In contrast, CD16 levels on NK cells were better maintained after 2M24 / CD20 bispecific antibody treatment compared with rituximab treatment. Without wishing to be bound by theory, it is believed that the 2M24 / CD20 bispecific may better preserve the cytotoxic activity of NK cells.

[0195] Next, we examined the effect of the 2M24 / CD20 bispecific on CD19 expression in human B cells. Maintaining target antigen expression is important for the therapeutic activity of monoclonal antibodies. B cell antigens such as CD20, CD19, and BCMA are validated immuno-oncology targets. CD19 is known to be downregulated by shaving / shedding after binding of anti-CD19 antibodies. A bystander effect was observed when using a CD20-targeting antibody, and CD19 expression was reduced upon treatment with rituximab, but not with the 2M24 / CD20 hIgG1 KIF bispecific (Figure 44). CD19 levels on B cells were better maintained by the 2M24 / CD20 bispecific compared to rituximab. Without wishing to be bound by theory, it is believed that the 2M24 / CD20 bispecific may exhibit long-term activity in therapeutic settings due to its minimal effect on target antigen expression.

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

[0197] In ADCC / ADCP assays, 2M24 / CD20 (derived from the rituximab sequence) demonstrated near-complete depletion of B cells, outperforming 2M24 / CD20 (derived from obinutuzumab) or the parental bivalent antibody and isotype control (Figure 45). These data support the use of the rituximab sequence to generate 2M24 / CD20 bispecifics.

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

[0199] Materials and Methods Three groups of cynomolgus monkeys (one male and one female per group) received a single dose of test article (5 mg / kg): A) 2M24 / CD20 hIgG1 KIF, B) 2M24 / CD20 hIgG1 inactive, and C) rituximab hIgG1 KIF. Blood was collected at the indicated time points. Abbreviations for the test articles are as follows (2M24 / CD20 KIF, 2M24 / CD20 inactive, RTX KIF).

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

[0201] For the PBMC assay, PBMCs from healthy cynomolgus monkeys were treated with serial dilutions of 2M24 / CD20 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 test the safety and efficacy of the 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 specific test article at a single dose of 5 mg / kg. The test articles were: 1) 2M24 / CD20 hIgG1 KIF, 2) 2M24 / CD20 hIgG1 inactive, and 3) rituximab hIgG1 KIF. Animals were monitored daily, and samples including whole blood, bone marrow, lymph node, and colorectal tissue were collected as indicated. The study was scheduled 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 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%). Partial B cell depletion was observed in one animal in the 2M24 / CD20 hIgG1 inactive group (Figure 47 bottom), while the second animal showed no depletion at day 7. The 2M24 / CD20 hIgG1 KIF bispecific antibody was well tolerated in cynomolgus monkeys.

[0204] The 2M24 / CD20 hIgG1 KIF bispecific also depleted B cells in the bone marrow (Figure 49A) and lymph nodes (Figure 49B) in cynomolgus monkeys in vivo. 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. In the rituximab group, B cell depletion was also observed in both tissues. In the 2M24 / CD20 hIgG1 inactive group, partial B cell depletion was observed, except for animal CB764A, which showed minimal 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). 2M24 / CD20 hIgG1 KIF induced a more robust depletion of B cells than rituximab hIgG1 KIF. While the maximum depletion achieved by rituximab was approximately 30% of B cells, the 2M24 / CD20 hIgG1 KIF bispecific showed a maximum depletion of approximately 50%.

[0206] Example 11: Purification and functional characterization of 2M24 / CD20 bispecific antibodies in scFv format This example describes the generation, purification, and characterization of a 2M24 / CD20 bispecific antibody in which the Dectin-1 targeting arm (based on the 2M24 variable domain) is an scFv fused to a human IgG1 Fc domain with a knob-forming mutation, and the CD20 targeting arm is based on rituximab hIgG1 with hole-forming mutations. 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. Without wishing to be bound by theory, it is believed that this format provides a universal platform for generating 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). Supernatants were collected four days after expression 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 homogenous molecule on SEC.

[0208] Next, cocultures of CD20-expressing Raji cells and Dectin-1-expressing HEK reporter assays were treated with increasing concentrations of the 2M24 scFv / CD20 hIgG1 bispecific. Reporter activation was assessed by measuring SEAP levels in the medium (based on absorbance at 630 nm). The bispecific molecule promoted targeted immune stimulation, as assessed by this NFkB reporter assay (Figure 52B).

[0209] To verify B cell depletion, human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified compared to the untreated control group (shown by the 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).

[0210] Example 12: Generation and characterization of anti-Dectin-1 / anti-Trop-2 bispecific antibodies Trop-2 is a 323-aa 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 droplet corneal dystrophy, a condition characterized by corneal amyloidosis and blindness. Trop-2 is overexpressed in various epithelial cancers and promotes cell proliferation, invasion, and angiogenesis. High expression correlates with poor prognosis and reduced survival in many cancers, particularly TNBC breast cancer and NSCLC lung cancer. Sacituzumab govitecan (Trodelvy®), a Trop-2-directed ADC, is the only therapeutic agent approved for the treatment of patients with metastatic TNBC. It received accelerated approval from the FDA in 2020.

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

[0212] In contrast, anti-Dectin-1 targeting approaches have the potential to restrict antitumor activity to the disease microenvironment. The immunomodulatory and phagocytic activities of 2M24 are tightly controlled by the presence of cancer cells. While Trodelvy® has a single mechanism of action (delivery of a toxin conjugate that induces target cell killing), anti-Dectin-1 targeting 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 generation and characterization of an anti-Dectin-1 (2M24) / anti-Trop-2 bispecific antibody. The variable domains used for anti-Trop-2 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-reducing (NR) or reducing (R) conditions (Figure 53A). The 2M24 / Trop2 bispecific antibody was purified as a monodisperse molecule and 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 assess the level of Trop-2 expression on different cancer cells, cancer cell lines (A431 and SKBR3) were stained with anti-human PE Trop-2 or an isotype control antibody to assess Trop-2 expression. Receptor copy number was assessed by comparing the fluorescence intensity of fluorescent dye-labeled microspheres with known amounts of fluorophore and that of labeled cells by flow cytometry. Trop-2 was found to be highly expressed in the 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. Because of its high expression on cancer cells, Trop-2 is an attractive antigen for targeted killing of cancer cells with the 2M24 / Trop-2 bispecific antibody.

[0216] Next, we examined the binding of the 2M24 / Trop-2 bispecific antibody to Trop-2-expressing cell lines. Cancer cell lines (HeLa, SiHa, BxPC-3, and Capan-2) were incubated with serial dilutions of the 2M24 / Trop-2 hIgG1 bispecific antibody or an isotype control antibody at a single concentration (300 nM). A secondary antibody (AF647 goat anti-human) was used for detection by flow cytometry. Binding EC50 values ​​were calculated using four-parameter logistic (4PL) nonlinear regression. The results showed that the 2M24 / Trop-2 bispecific antibody bound to different Trop-2-expressing cell lines with variable submicromolar affinity (Figures 55A-55D).

[0217] The 2M24 / Trop-2 bispecific antibody was assayed for its ability to deplete Trop-2-expressing cell lines. Monocytes were cultured with MCSF for 6 days to generate macrophages. After differentiation, macrophages were cocultured with Trop-2-expressing cancer cell lines, SKBR3 or A431, for 24 hours in the presence of 10 μg / ml of 2M24 / Trop-2 hIgG1 or 2M24 / RSV hIgG1. PE-CD206 antibody was used to detect macrophages. Cancer cells were detected either by prestaining with calcein AM (Figure 56A, SKBR3 cells) or by using APC-EPCAM antibody (Figure 56B, A431 cells). Phagocytosis was assessed by flow cytometry as double-positive PE-CD206+calcein+ cells in the single-cell gate for SKBR3 cells (Figure 56A) and as remaining EPCAM+ cells for A431 cells (Figure 56B). The results demonstrated that the 2M24 / Trop-2 bispecific antibody induced robust depletion of both Trop-2-expressing cancer cell lines. The 2M24 / Trop-2 bispecific antibody induced phagocytosis of Trop-2-expressing cell lines by macrophages. The reduction in cancer cells was 56% for SKBR3 (Figure 56A) and 87% for A431 cancer cells (Figure 56B). This data provides strong evidence that the 2M24 / Trop-2 bispecific antibody can instruct macrophages to eliminate Trop-2-expressing cancer cells.

[0218] Trop-2 expression in non-immune cells and Dectin-1 expression in tumor-associated macrophages were assessed in single-cell suspensions of lung cancer biopsy tissue. Trop-2 was assessed in CD45- cancer cells using a PE Trop-2 antibody. EPCAM expression was also confirmed in the same population. Dectin-1 expression was confirmed in tumor-associated macrophages, gated as CD45+CD11b+CD163+ cells after excluding B cells, T cells, and NK cells (Figures 57A and 57B). Dectin-1 is expressed in tumor-associated macrophages, and Trop-2 is expressed in cancer cells in lung cancer specimens.

[0219] To verify whether the 2M24 / Trop-2 bispecific antibody could induce depletion of Trop-2-expressing cancer cells from lung cancer biopsies, single-cell suspensions were prepared from lung cancer specimens and treated with 2M24 / Trop-2 hIgG1 or 2M24 / RSV hIgG1 antibodies. After 24 hours of treatment, the cells were stained with FITC CD45 and APC EPCAM antibodies, and cancer cells were quantified by flow cytometry. The reduction in cancer cells was expressed as the percentage of CD45-EPCAM+ cells within the viable cell population. The results demonstrated that the 2M24 / Trop-2 bispecific antibody depleted Trop-2-expressing cancer cells in lung cancer specimens (Figure 58). The 2M24 / Trop-2 bispecific antibody induced a 50% reduction in Trop-2-expressing cancer cells in lung cancer specimens. This data indicates that the 2M24 / Trop-2 bispecific antibody can induce the ph...

Claims

1. An antibody or antigen-binding fragment thereof that binds to human Dectin-1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; The VH domain comprises CDR-H1, CDR-H2, and CDR-H3 from the VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7). H3, wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8); Each of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 is defined according to the Kabat definition, the IMGT definition, or the Chothia definition of a CDR. The antibody or antigen-binding fragment thereof.

2. the VH domain comprises a sequence with at least 90% identity to the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7); 2. The antibody of claim 1, wherein the VL domain comprises a sequence having at least 90% identity to the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).

3. The antibody described in claim 2, wherein the VH domain comprises the amino acid sequence of SEQ ID NO: 7 and the VL domain comprises the amino acid sequence of SEQ ID NO:

8.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment binds to human Dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, and / or granulocytes.

6. The antibody of any one of claims 1 to 5, wherein the antigen-binding fragment is a Fab, Fab', F(ab')2, Fv, Fab'-SH, F(ab')2, or scFv fragment.

7. The antibody of any one of claims 1 to 5, wherein the antibody further comprises an Fc region.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is a multispecific antibody or antigen-binding fragment thereof.

9. The antibody of claim 8, wherein the antibody or antigen-binding fragment thereof is a bispecific antibody, antigen-binding fragment thereof, or diabody comprising a first antigen-binding domain comprising the VH domain and the VL domain that binds to human Dectin-1, and a second antigen-binding domain comprising a second VH domain and a VL domain that binds to a target of interest.

10. 10. The antibody of claim 9, wherein the target of interest is an antigen expressed on the surface of a disease-causing agent, and the disease-causing agent is a bacterial cell, a fungal cell, a virus, a senescent cell, a tumor cell, a protein aggregate, an LDL particle, a mast cell, an eosinophil, an ILC2 cell, or an inflammatory immune cell.

11. (a) 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, an eosinophil, an ILC2 cell, or an inflammatory immune cell; (b) the target of interest is a surface antigen of the virus; or (c) the target of interest is an antigen expressed on the surface of a cancer cell; The antibody described in claim 10.

12. The antibody according to any one of claims 9 to 11, wherein the antibody comprises two antibody heavy chains, each of which comprises an amino acid substitution at one or more of positions 234, 235, and 237 (EU numbering) of a human IgG1 Fc region.

13. The antibody of claim 12, wherein each of the antibody heavy chains comprises L234A, L235E, and G237A substitutions according to EU numbering in the human IgG1 Fc region. (a) the antibody comprises two antibody heavy chains, only one of which comprises H435R and Y436F substitutions according to EU numbering in a human IgG1 Fc region; and / or (b) the antibody comprises a first antibody arm comprising a first antibody heavy chain comprising the VH domain of the first antigen-binding domain and a first antibody light chain comprising a first Fc region and the VL domain of the first antigen-binding domain, and a second antibody arm comprising a second antibody heavy chain comprising the VH domain of the second antigen-binding domain and a second Fc region and a second antibody light chain comprising the VL domain of the second antigen-binding domain, wherein only one of the first antibody arm or the second antibody arm comprises a heavy chain comprising F126C and C220V substitutions and a light chain comprising S121C and C214V substitutions according to EU numbering of a human IgG1 Fc region; The antibody according to any one of claims 9 to 13.

15. The antibody of any one of claims 9 to 14, wherein the bispecific antibody comprises a first antibody heavy chain and a second antibody heavy chain, wherein the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain, and / or the bispecific antibody comprises a first antibody heavy chain and a second antibody heavy chain, wherein at least one or two of the first antibody heavy chain and the second antibody heavy chain are non-fucosylated.

16. (a) a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to human Dectin-1; (b) a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to the target of interest; and 1. A multispecific binding molecule comprising: the first antigen-binding domain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain; The VH domain comprises CDR-H1, CDR-H2, and CDR-H3 from the VH domain sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7). H3, wherein the VL domain comprises CDR-L1, CDR-L2, and CDR-L3 from the VL domain sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8); a multispecific binding molecule, wherein each of said CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 is defined according to the Kabat definition, the IMGT definition, or the Chothia definition of a CDR.

17. 17. The multispecific binding molecule of claim 16, wherein the target of interest is an antigen expressed on the surface of a disease-causing agent, and the disease-causing agent is a bacterial cell, a fungal cell, a virus, a senescent cell, a tumor cell, a protein aggregate, an LDL particle, a mast cell, an eosinophil, an ILC2 cell, or an inflammatory immune cell.

18. (a) 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, an eosinophil, an ILC2 cell, or an inflammatory immune cell; (b) the target of interest is a surface antigen of the virus; or (c) the target of interest is an antigen expressed on the surface of a cancer cell; 18. The multispecific binding molecule of claim 17.

19. the VH domain of the first antigen-binding domain comprises a sequence having at least 90% identity to the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSS (SEQ ID NO: 7), 19. The multispecific binding molecule of any one of claims 16 to 18, wherein the VL domain of said domain comprises a sequence having at least 90% identity to the sequence DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE (SEQ ID NO: 8).

20. The multispecific binding molecule of claim 19, wherein the VH domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 7 and the VL domain of the first antigen-binding domain comprises the amino acid sequence of SEQ ID NO:

8.

21. the multispecific binding molecule comprises a first antibody arm comprising a single-chain variable fragment (scFv) comprising the VH domain and the VL domain of the first antigen-binding domain and a first Fc region, and a second antibody arm comprising an antibody heavy chain comprising the VH domain of the second antigen-binding domain associated with an antibody light chain comprising the VL domain of the second antigen-binding domain, and a second Fc region connected to the VH domain of the second antigen-binding domain; The multispecific binding molecule of any one of claims 16 to 20. Claim 22: (a) the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations; and / or (b) the first antibody arm comprises a first linker between the VH domain and the VL domain and a second linker between the VL domain and the first Fc region; 22. The multispecific binding molecule of claim 21.

23. The polyspecific binding molecule of claim 22, wherein the first linker comprises one or more repeats of the sequence GGGGS (SEQ ID NO: 26), or the sequence GGGGSGGGGGSGGGGS (SEQ ID NO: 27) or GGGGSGGGGGSGGGGGSGGGGS (SEQ ID NO: 28), and / or the second linker comprises the sequence EPKRSDKTHTCPPC (SEQ ID NO: 29) or SATHTCPPC (SEQ ID NO: 30).

24. (a) the multispecific binding molecule comprises a first antibody arm comprising a first antibody heavy chain comprising the VH domain of the first antigen-binding domain, and a first Fc region, and a first antibody light chain comprising the VL domain of the first antigen-binding domain; and a second antibody arm comprising a second antibody heavy chain comprising the VH domain of the second antigen-binding domain, and a second Fc region, and a second antibody light chain comprising the VL domain of the 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 cognate hole-forming mutations; or (b) the multispecific binding molecule comprises a first antibody arm comprising a first antibody heavy chain comprising the VH domain of the first antigen-binding domain and a first Fc region, and a second antibody arm comprising a second antibody heavy chain comprising the VH domain of the 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 cognate knob-forming mutations; The multispecific binding molecule of any one of claims 16 to 20.

25. The polyspecific binding molecule of claim 24, wherein the Fc region comprising the one or more knob-forming mutations comprises a T366W substitution according to EU numbering in a human IgG1 Fc region, and the Fc region comprising the one or more hole-forming mutations comprises T366S, L368A, and Y407V substitutions according to EU numbering in a human IgG1 Fc region.

26. (a) the first Fc region and / or the second Fc region comprises an amino acid substitution at one or more of positions 234, 235, and 237 (EU numbering) of a human IgG1 Fc region; (b) only one of the Fc regions comprises the H435R and Y436F substitutions according to EU numbering of a human IgG1 Fc region; and / or (c) only one of the antibody arms comprises a heavy chain comprising F126C and C220V substitutions, and a light chain comprising S121C and C214V substitutions, according to EU numbering, in a human IgG1 heavy chain or a human constant light chain (CL) domain, respectively; The multispecific binding molecule of any one of claims 21 to 25.

27. The multispecific binding molecule of claim 26, wherein each of the first Fc region and the second Fc region comprises L234A, L235E, and G237A substitutions according to EU numbering of a human IgG1 Fc region.

28. 17. The multispecific binding molecule of claim 16, wherein the multispecific 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, wherein the first antibody heavy chain comprises F126C, C220V, and T366W substitutions according to EU numbering of a human IgG1 heavy chain or human light chain constant (CL) domain, and the first antibody light chain comprises S121C and C214V substitutions, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R, and Y436F substitutions.

29. The multispecific binding molecule of claim 28, wherein the first antibody heavy chain and the second antibody heavy chain further comprise L234A, L235E, and G237A substitutions according to EU numbering of a human IgG1 heavy chain.

30. 30. The multispecific binding molecule of any one of claims 24 to 29, wherein the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain and / or at least one or two of the first antibody heavy chain and the second antibody heavy chain are afucosylated.

31. 1. A multispecific binding molecule comprising a first arm comprising a first antigen-binding domain and a second arm comprising 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 of interest; wherein the first arm has the sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHW VRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDDTAVFYCARNSGSYSFGYWGQGTLVT VSSGGGGSGGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQS GVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).

32. A polynucleotide encoding the antibody or multispecific binding molecule of any one of claims 1 to 31.

33. A vector comprising the polynucleotide of claim 32.

34. 34. An isolated host cell comprising the polynucleotide of claim 32 or the vector of claim 33.

35. 35. An in vitro method for producing an antibody or multispecific binding molecule, comprising culturing the host cell of claim 34 under conditions suitable for the production of said antibody or multispecific binding molecule.

36. The method of claim 35, wherein the method further comprises recovering the antibody or multispecific binding molecule, and / or wherein the host cells are treated with kifunensine prior to production of the antibody or multispecific binding molecule.

37. A pharmaceutical composition comprising the antibody or multispecific binding molecule of any one of claims 1 to 31 and a pharmaceutically acceptable carrier.

38. 32. A medicament for use in a method for treating a disease or disorder in an individual in need thereof, comprising an antibody according to any one of claims 1 to 15 or a multispecific binding molecule according to any one of claims 16 to 31.

39. 39. The pharmaceutical product of claim 38, comprising a multispecific binding molecule comprising a first antibody or antigen-binding fragment thereof comprising a first antigen-binding domain that binds to human Dectin-1 and a second antibody or antigen-binding fragment thereof comprising a second antigen-binding domain that binds to a target of interest, wherein the target of interest is an antigen expressed on the surface of a disease-causing agent, and the disease-causing agent is a bacterial cell, a fungal cell, a virus, a senescent cell, a tumor cell, a protein aggregate, an LDL particle, a mast cell, an eosinophil, an ILC2 cell, or an inflammatory immune cell. Pharmaceuticals.

40. (a) 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, an eosinophil, an ILC2 cell, or an inflammatory immune cell; (b) the target of interest is a surface antigen of the virus; (c) the disease or disorder is cancer, a bacterial infection, a fungal infection, a viral infection, a mast cell disease or disorder, systemic mastocytosis, amyloidosis, or an age-related disease or disorder; or (d) the target of interest is an antigen expressed on the surface of a cancer cell; 40. The pharmaceutical product of claim 39.

41. The pharmaceutical product according to any one of claims 38 to 40, wherein the individual is a human.

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