Multi-specific binding proteins that bind detin-1 and CD20 and methods of use thereof

Abstract

The present disclosure relates to multispecific (e.g., bispecific) binding proteins that bind to human Dectin-1 and human CD20 and methods of use and production related thereto.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 327,281, filed on April 4, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (186542000840SEQLIST.xml; size: 33,783 bytes; and creation date: March 30, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure relates to multispecific (eg, bispecific) binding proteins that bind to human Dectin-1 and human CD20 and methods of use and production thereof. Background Art

[0006] Phagocytosis is the primary mechanism used to remove pathogens and cellular debris. Professional phagocytes, such as monocytes, macrophages, dendritic cells, and granulocytes, specialize in recognizing and engulfing abnormal or disease-causing host or foreign agents. Engulfed material is destroyed via the endolysosomal pathway in phagocytes. In addition, dendritic cells and macrophages can present antigens to cells of the adaptive immune system to further facilitate elimination of pathogens.

[0007] Dectin-1 is a C-type lectin receptor that recognizes β-glucan and promotes antifungal phagocytic activity. It is expressed on phagocytic cells and has been clearly shown to be sufficient to activate phagocytosis. Dectin-1 can be used to target phagocytosis and eliminate pathogens with antibodies.

[0008] Targeted removal and degradation of accumulated pathogens without increasing overall phagocytosis would be beneficial. The present disclosure provides solutions to these problems and describes other advantages.

[0009] All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety to the same extent as if each reference was specifically and individually indicated to be incorporated by reference. Summary of the invention

[0010] The present disclosure relates to multispecific (e.g., bispecific) binding molecules that bind to human Dectin-1 and methods of use and production thereof. Described herein is a method of targeting phagocytosis to remove a pathogen (e.g., CD20) after administering a multispecific (e.g., bispecific) binding molecule comprising a Dectin-1 binding arm and a second arm that specifically binds to a pathogen (including a host cell / host cell product, a microorganism or its product, etc.). The multispecific (e.g., bispecific) binding molecule allows phagocytes to engage with the target pathogen and form a synapse therebetween and promotes the aggregation of Dectin-1 on phagocytes. This stimulates phagocytosis of the target pathogen while stimulating phagocytes to secrete cytokines via the Dectin-1 / Syk / NfkB pathway. In addition, antigens from the engulfed material are presented on the surface of dendritic cells / macrophages to enhance adaptive immune responses against pathogens. Overall, it is believed that Dectin-1 agonistic, multispecific (eg, bispecific) binding molecules can promote immune stimulation, targeted phagocytosis, and neoantigen presentation / activation of the adaptive immune system to eliminate pathogens.

[0011] Thus, the present disclosure describes, among other things, the generation and functional characterization of an agonistic anti-human Dectin-1 antibody that exhibits high affinity binding to Dectin-1 and can promote immune stimulation. Further described is the generation of a bispecific antibody format comprising an anti-human Dectin-1 antibody and an antibody targeting an antigen on a pathogen, with data supporting target engagement, immune stimulation, phagocytosis, and antigen presentation.

[0012] In some embodiments, provided herein is a multispecific binding protein comprising: a first polypeptide chain comprising the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTL TVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ IDNO: 31) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35); a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence QVQLQQPGAELVKP GASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ IDNO:32) or QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is a composition comprising a mixture of multispecific binding protein species, wherein each species comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 35, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36, a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37, a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 38, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 44, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NONO:32 or a second polypeptide chain having an amino acid sequence of SEQ ID NO:36 and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:33.

[0013] In some embodiments, provided herein is a multispecific binding protein comprising: a first polypeptide chain comprising the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTL TVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ IDNO: 37) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39); a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ IDNO:38) or QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is a composition comprising a mixture of multispecific binding protein species, wherein each species comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 39, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40, a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41, a fourth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 44, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 48, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 49, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NO: 50, a fifth polypeptide chain comprising the amino acid sequence of SEQ ID NONO:38 or a second polypeptide chain having an amino acid sequence of SEQ ID NO:40 and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:33.

[0014] In some embodiments, the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are associated in a multispecific binding protein comprising a first antigen binding domain that binds human Dectin-1 and a second antigen binding domain that binds human CD20.

[0015] In some embodiments, at least one of the first polypeptide chain and the second polypeptide chain is non-fucosylated. In some embodiments, both of the first polypeptide chain and the second polypeptide chain are non-fucosylated. 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 2nM; is capable of binding to human or cynomolgus monkey 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 human CD20 expressed on the surface of B cells.

[0016] In some embodiments, a polynucleotide is provided herein, the polynucleotide encoding the multispecific binding protein of any one of the above embodiments. In some embodiments, a vector (e.g., an expression vector) is provided herein, the vector comprising the polynucleotide of any one of the above embodiments.

[0017] In some embodiments, a host cell (e.g., an isolated host cell or cell line) is provided herein, comprising a polynucleotide or vector of any one of the above embodiments. In some embodiments, the host cell is a yeast, insect, plant, or prokaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell comprises α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) knockout. In some embodiments, the host cell overexpresses β1,4-N-acetylglucosamine transferase III (GnT-III). In some embodiments, the host cell also overexpresses Golgi μ-mannosidase II (ManII).

[0018] In some embodiments, provided herein is a method of producing a multispecific binding protein, the method comprising culturing a host cell of any one of the above embodiments under conditions suitable for producing the multispecific binding protein. In some embodiments, the method further comprises recovering the multispecific binding protein. In some embodiments, prior to producing the antibody or multispecific binding protein, the host cell is treated with kifunensine.

[0019] In some embodiments, provided herein is a pharmaceutical composition comprising a multispecific binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a mixture of multispecific binding protein species, wherein each species comprises a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 35, a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 36, and a third polypeptide chain comprising an amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 31, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 32, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises an amino acid sequence of SEQ ID NO: 35, the second polypeptide chain comprises an amino acid sequence of SEQ ID NO: 36, and the third polypeptide chain comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, the composition comprises a mixture of multispecific binding protein species, wherein each species comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 39, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33.

[0020] In some embodiments, provided herein is a method for treating a disease or condition, the method comprising administering to an individual in need thereof an effective amount of a multispecific binding protein or composition of any one of the above embodiments. In some embodiments, the disease or condition is a B cell-mediated disease or condition. In some embodiments, the individual suffers from or has been diagnosed with a B cell-mediated disease or condition. In some embodiments, the B cell-mediated disease or condition is cancer. In some embodiments, the cancer is a B cell-mediated cancer or is characterized by a B cell malignancy. In some embodiments, the cancer is non-Hodgkin's lymphoma or chronic lymphocytic leukemia. In some embodiments, the B cell-mediated disease or condition is an autoimmune disease or condition. In some embodiments, the autoimmune disease or condition is rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and Wegener's granulomatosis. In some embodiments, the individual is a human.

[0021] It should be understood that one, some or all of the properties of the various embodiments described herein can be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figures 1A-1C Shown is the binding analysis of anti-human Dectin-1 antibody (clone 2M24) in human and monkey monocytes derived from peripheral blood mononuclear cells (PBMC) by flow cytometry. Single cells, live cells and CD14+ cells were gated to identify monocytes. Cells were incubated with 2M24 anti-Dectin-1 primary antibody or mIgG1 isotype control antibody, and then incubated with fluorescent anti-mouse secondary antibody. Primary antibody was used for continuous dose titration. Figure 1A Binding analysis of anti-human Dectin-1 clone 2M24 in human monocytes is shown. Figure 1B Binding analysis of anti-human Dectin-1 clone 2M24 antibody in cynomolgus monkey monocytes is shown. Figure 1C Comparison of binding between the 2M24 clone and other Dectin-1 antibodies identified from immunization of ATX-Gx Alloy transgenic mice and commercial anti-Dectin-1 antibodies to human monocytes, HEK cells overexpressing human Dectin-1, and cynomolgus monkey monocytes is depicted. The anti-human Dectin-1 clone 2M24 antibody exhibits high affinity for human and cynomolgus monkey Dectin-1 expressed in monocytes and exhibits higher affinity than other anti-Dectin-1 antibodies, including commercial antibodies.

[0023] Figure 2A-2B Phagocytosis of pHrodo-labeled polystyrene anti-mouse Fc IgG beads conjugated with anti-Dectin-1 antibody 2M24 or isotype control antibodies by HEK-Blue hDectin-1a cells and human monocytes is shown. Polystyrene anti-mouse Fc IgG beads (approximately 3.4 μm) were labeled with a pH-sensitive fluorescent dye (pHrodo red) and conjugated with Dectin-1 antibody 2M24 or isotype control. The beads were then incubated with cultured HEK-Blue hDectin-1a cells or human monocytes at a ratio of 1:2 (cell:bead). HEK-Blue hDectin-1a cells were labeled with the cell permeable dye Calcein AM. Phagocytosis of beads was monitored by IncuCyte live cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the overlap of red object counts (pHrodo) with calcein-positive cells. Figure 2A Shown are phagocytosis of beads in HEK-Blue hDectin-1a cells over 2.5 hours (top) and representative images of pHrodo-positive cells at 2.5 hours of phagocytosis (bottom). Figure 2B Shown are phagocytosis of beads in human monocytes over 4 hours (top), and representative images of pHrodo-positive cells at 2.5 hours of phagocytosis (bottom). In the representative images, engulfed beads fluoresce brightly in the phagosome.

[0024] Figure 3A-3B Binding of fully human 2M24 anti-Dectin-1 antibody (hIgG4) or isotype control antibody in HEK-BluehDectin-1a cells and primary human monocytes is shown. Figure 3A Binding analysis of the fully human 2M24 anti-Dectin-1 antibody to HEK cells is shown, while Figure 3B Binding to primary human monocytes is shown. Primary antibodies were used for serial dose titration followed by fluorescent secondary antibodies directed against the primary antibodies. The fully human 2M24 anti-Dectin-1 hIgG4 antibody binds to cells expressing Dectin-1 with high affinity.

[0025] Figure 4Targeted phagocytosis of Dectin-1-expressing cells to pHrodo-labeled polystyrene biotin beads conjugated to fully human 2M24 anti-Dectin-1 antibody (hIgG4) or isotype control antibodies is shown. Polystyrene biotin beads were labeled with pHrodo red and conjugated to anti-Dectin-1 antibody 2M24 or isotype control via streptavidin. The conjugated beads were mixed with cells at a ratio of 1:3 and the phagocytosis of the beads was monitored by IncuCyte live cell imaging. Phagocytosis of phrodo-biotin beads conjugated to streptavidin 2M24 anti-Dectin-1hIgG4 antibodies by HEK-Blue hDectin-1a cells (upper left), human monocytes (upper right) and human macrophages (lower) is shown. Fully human 2M24 anti-Dectin-1 antibody (hIgG4) promotes phagocytosis of cells expressing Dectin-1.

[0026] Figure 5A-5B Shown are the results of a secreted alkaline phosphatase reporter assay for Dectin-1 in HEK-Blue hDectin-1a cells. Figure 5A Results of a secreted alkaline phosphatase assay using immobilized fully human 2M24 anti-Dectin-1 antibody are shown. Fully human 2M24 (hIgG4) anti-Dectin-1 antibody or isotype control antibody was immobilized overnight in U-bottom polypropylene microtiter plates at a range of 0.1-10 μg per well, HEK-Blue hDectin-1a cells were then cultured for 22 hours, and alkaline phosphatase secretion in the supernatant was assessed at OD 630nm. Figure 5B Results of a secretory alkaline phosphatase assay using bead-conjugated fully human 2M24 anti-Dectin-1 antibody are shown. Biotin beads of size 3 μm, 10 μm, and 16.5 μm were conjugated to streptavidin 2M24 (hIgG4) anti-Dectin-1 antibody. Antibody-conjugated beads were mixed with HEK-Blue hDectin-1a cells for 22 hours, and supernatants were assessed for alkaline phosphatase secretion at OD 630nm. Bars represent mean ± sd; n = 2 replicates. 2M24 (hIgG4) anti-Dectin-1 antibody in immobilized and bead-conjugated form induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells.

[0027] Figure 6A-6BCytokine secretion of human primary macrophages stimulated with anti-Dectin-1 (15E2) antibody in solution is shown. Primary human macrophages and primary monocytes were stimulated with 10 μg / ml 15E2 anti-Dectin-1 antibody or isotype antibody in solution for 24 hours, and secretion of TNFα and IL6 was assessed by ELISA analysis of the supernatant. Zymosan was used as a positive control for cytokine secretion. Bars represent mean ± sd; n = 2 replicates. Fig. 6A Results are shown for primary human monocytes stimulated with soluble 15E2 anti-Dectin-1 antibody, whereas Figure 6B Results for stimulated primary human macrophages are shown.Soluble 15E2 anti-Dectin-1 antibody did not induce cytokine secretion in primary human monocytes and macrophages.

[0028] Figure 7A-7B Cytokine secretion of human primary monocytes and PBMCs stimulated with immobilized 2M24 or 15E2 anti-Dectin-1 antibodies is shown. Anti-Dectin-1 antibodies or isotype control antibodies were immobilized at 10 μg per well in U-bottom polypropylene microtiter plates overnight, and then human monocytes or human PBMCs were cultured for 24 hours. Secretion of TNFα, IL6, and IFNg was assessed by ELISA analysis of the supernatant. Fig. 7A shows cytokine secretion by human monocytes after stimulation with immobilized anti-Dectin-1 antibody. Figure 7B Cytokine secretion of cultured human PBMCs after stimulation is shown. Bars represent mean ± sd; n = 2 replicates. 2M24 anti-Dectin-1 antibody induced cytokine secretion in primary human monocytes and PBMCs and exhibited superior immunostimulation to the 15E2 Dectin-1 agonistic antibody.

[0029] Figure 8 Results of competition assays using 12M4 anti-Dectin-1 antibody clones and the natural ligands of Dectin-1 are shown. HEK-Blue hDectin-1a cells were incubated on ice for 30 minutes in 1 / 3 serial dose titrations starting at 300 nM with 2M24 (hIgG4) anti-Dectin-1 antibody or 15E2, 259931, GE2 anti-Dectin-1 commercial antibodies and in the presence of 8 ug / ml biotin-laminarin. 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.

[0030] Fig. 9A summary of the functional characterization of the 2M24 and 15E2 anti-Dectin-1 antibodies is depicted.

[0031] Figures 10A-10B A schematic diagram of the generation of bispecific antibodies by click chemistry is shown. Fig. 10A Differential labeling of antibodies with MTA or FOL reagents is depicted. Fig. 10B Covalent cross-linking of antibodies via specific MTA-FOL interactions is depicted.

[0032] Fig.11 Potential modes of activity employed by anti-Dectin-1 agonistic bispecific antibodies to eliminate target cancer cells are shown. These include immune stimulation, phagocytosis, neoantigen presentation, and activation of T and B lymphocytes of the adaptive immune system.

[0033] Figures 12A-12B Characterization of a click-chemistry conjugated bispecific antibody comprising anti-Dectin-1 (clone 2M24) and anti-hCD70 arms is shown. Fig. 12A Shown is SDS-PAGE analysis of covalently conjugated antibody pairs (2M24 / anti-hCD20, 2M24 / anti-hCD70, and isotype control) under non-reducing and reducing conditions. Fig. 12B Shown are flow cytometry-based characterization of the binding of bispecific antibodies (2M24 / anti-hCD70 or isotype control) to HEK293 cells expressing Dectin-1 (upper left) and two renal cancer cell lines - A498 (upper right) and 786-0 (lower left). Fig. 12B EC50 concentrations (nM) based on nonlinear regression fits are also depicted (bottom right).The anti-Dectin-1 / anti-hCD70 bispecific antibody binds to cells expressing Dectin-1 or CD70 with an affinity of 1.8 nM or 12.34 nM, respectively.

[0034] Fig.13 The coupling of the HEK293 cell line expressing Dectin-1 and the A498 renal cancer cell line induced by the 2M24 / anti-hCD70 bispecific antibody is shown. Flow cytometric analysis of co-cultures of HEK293 cells (labeled with calcein green) and A498 cells (labeled with calcein red) in the presence of 2M24 / anti-hCD70 bispecific antibodies or isotype controls (left) is shown. Double positive signals (green + red +, squares) indicate the coupling of HEK293 and A498 cells. The coupling efficiency is also shown, which is quantified as the percentage of cells that form doublets with HEK293 cells relative to the total target cells (A498) (right). The bars represent the mean ± sd; n = 3 replicates. The 2M24 / anti-hCD70 bispecific antibody induces the coupling of the HEK293 cell line expressing Dectin-1 to the A498 renal cancer cell line.

[0035] Figures 14A-14B The coupling of Dectin-1 expressing cells and B cells induced by the anti-Dectin-1 / anti-hCD20 bispecific antibody is shown. Fig.14A The coupling of HEK293 cells expressing Dectin-1 and B cells induced by anti-Dectin-1 / anti-hCD20 bispecific antibodies is shown. Flow cytometric analysis of co-cultures of HEK293 cells (labeled with calcein green) and Raji cells (labeled with calcein red) in the presence of 2M24 / anti-hCD70 bispecific antibodies or isotype controls (left) is shown. Double positive signals (green + red +; boxes) indicate the coupling of HEK293 and Raji cells. The coupling efficiency is also shown, which is quantified as the percentage of cells that form doublets with HEK293 cells relative to total target cells (Raji) (right). Bars represent mean ± sd; n = 2 replicates. Fig. 14B Results of similar experiments performed to assess coupling of human M0 macrophages and Raji cells induced by anti-Dectin-1 / anti-hCD20 bispecific antibodies are shown. Bars represent mean ± sd; n = 2 replicates. 2M24 / anti-hCD20 bispecific induces coupling of Dectin-1 expressing cells and CDC20 positive B cells (Raji cells).

[0036] Fig.15 The results of a secretory alkaline phosphatase reporter assay of Dectin-1 performed in HEK-Blue hDectin-1a cells using an anti-Dectin-1 / anti-CD20 bispecific antibody in the presence of Raji cells are shown. 2M24 (hIgG4) / anti-CD20 bispecific antibody was incubated with Raji cells and then washed twice to remove unbound bispecific antibody. 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 in the supernatant was assessed at OD 630nm. Bars represent mean ± sd; n = 2 replicates. Raji cells coated with anti-Dectin-1 / anti-CD20 bispecific antibodies induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells.

[0037] Fig.16Induction of phagocytosis of Raji cells by HEK 293 cells expressing Dectin-1 by anti-Dectin-1 / anti-hCD20 bispecific antibodies is shown. Representative Incucyte images (left) illustrating phagocytosis of Raji cells by HEK cells (arrows) at 16 hours versus 0 hours are shown. Yellow fluorescence indicates colocalization. The reduction of the calcein red signal of Raji cells at 16 hours indicates phagocytosis-mediated cell death. Quantification of the overlap or colocalization of HEK (calcein green) and Raji (calcein red) in different treatment groups is shown (right). Pre-incubation of HEK cells with the ADCP inhibitor Latrunculin A can block phagocytosis mediated by 15E2 / anti-hCD20 bispecific antibodies. (n=2 replicates).

[0038] Fig.17 The coupling of Dectin-1-expressing cells and HER2-expressing cells induced by anti-Dectin-1 / anti-hHER2 bispecific antibodies is shown. Flow cytometric analysis of co-cultures of HEK 293 cells expressing Dectin-1 (labeled with calcein green) and HER2-expressing SKBR3 cells (labeled with pHrodo red) in the presence of 15E2 / anti-hHER2 bispecific antibodies or isotype controls is shown (left). Double positive signals (green+red+, boxes) indicate coupling of HEK293 and SKBR3 cells. Also shown is the coupling efficiency, which is quantified as the percentage of cells that form doublets with cells expressing Dectin-1 relative to total target cells (SKBR3) (right). Bars represent mean ± sd; n = 2 replicates. Anti-Dectin-1 / anti-hHER2 bispecific antibodies induce coupling of Dectin-1 and HER2-positive cancer cells.

[0039] Fig.18 Coupling of HEK293 cells expressing Dectin-1 and BaF3 cells expressing CD94 induced by anti-Dectin-1 / anti-hCD94 bispecific antibodies is shown. Flow cytometric analysis of co-cultures of HEK293 cells (labeled with calcein green) and BaF3 cells (labeled with pHrodo red) in the presence of 2M24 / anti-hCD94 bispecific antibodies or isotype controls is shown (left). Double positive signals (green+red+; boxes) indicate coupling of HEK293 and BaF3 cells. Coupling efficiency is also shown, which is quantified as the percentage of cells that form doublets with HEK293 cells relative to total target cells (BaF3) (right). Bars represent mean ± sd; n = 2 replicates. Anti-Dectin-1 / anti-hCD94 bispecific antibodies induce coupling of cells expressing Dectin-1 and CD94.

[0040] Figures 19A-19B Schematic representation of Fab 2M24-mSA or full-length 2M24-mSA bound to a biotinylated target antibody is shown. Fig.19A Chimeric fusions of monomeric streptavidin (mSA) and Fab 2M24 or full-length 2M24 are shown. mSA is genetically fused to Fab 2M24 or full-length 2M24. Fig.19B Coupling of Fab 2M24-mSA or 2M24-mSA to a biotinylated target antibody is shown. The chimeric fusion was incubated with a biotinylated target antibody to generate a bispecific antibody comprising a Dectin-1 binding arm and a second arm that binds a target receptor or protein of interest.

[0041] Figures 20A-20C Biochemical and functional characterization of the Fab 2M24-mSA fusion protein is shown. Fig. 20A HPLC characterization of the recombinant Fab 2M24-mSA is shown. Fig. 20B Shown is SDS-PAGE analysis of Fab 2M24-mSA purified under reducing conditions. Fig. 20C The Fab 2M24-mSA was shown to be stable overexpressing human Dectin-1 (EC 50 =1.45 nM). Fab 2M24 was fused to monomeric streptavidin and bound to cells expressing Dectin-1 with an affinity of 1.45 nM.

[0042] Figures 21A-21B Phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated to Fab-2M24 anti-Dectin-1 antibody labeled with monomeric streptavidin (Fab-2M24-mSA) is shown. Fig.21A Shown are dyad formation by HEK-Blue hDectin-1a cells with Fab-2M24-mSA conjugated to biotin beads and phagocytosis of the beads, assessed by flow cytometry. Fig. 21B Phagocytosis of pHrodo-biotin beads (approximately 3 μm) conjugated to Fab-2M24-mSA assessed by IncuCyte real-time imaging (top), and representative images of pHrodo-positive cells (engulped beads fluoresce brightly red in phagosomes) versus no-bead controls at 3 hours of phagocytosis (bottom). Fab2M24-mSA fusion induces binding and phagocytosis of beads by HEK 293 cells expressing Dectin-1.

[0043] Figures 22A-22DBispecific complexes containing Fab 2M24-mSA and target biotinylated antibody are shown. Fig.22A ), biotinylated anti-hCD19 ( Fig. 22B ), biotinylated anti-hCD70 ( Fig. 22C ) or biotinylated anti-β-amyloid 1-42 ( Fig.22D ) HPLC analysis of Fab 2M24-mSA in complex. Each graph contains an overlay of the A280 traces for Fab 2M24-mSA alone, target biotinylated antibody alone, and Fab 2M24-mSA in complex with biotinylated target antibody.

[0044] Fig.23 The coupling of HEK293 cells expressing Dectin-1 and Raji cells expressing CD20 induced by Fab 2M24-mSA / biotin anti-hCD20 bispecific antibodies is shown. Flow cytometric analysis of co-cultures of HEK293 (labeled with calcein green) and Raji (labeled with calcein red) in the presence of Fab 2M24-mSA / biotin anti-hCD20 bispecific antibodies or isotype bispecific controls is shown (left). The co-cultures were incubated at 4°C or 37°C. Double positive signals (green + red +; dotted squares) indicate the coupling of HEK293 and Raji cells. Coupling efficiency is also shown, which is quantified as the percentage of cells forming doublets relative to total target cells (Raji) (right). Bars represent mean ± sd; n = 4 replicates. Fab 2M24-mSA / biotin anti-hCD20 bispecific antibody induced coupling of HEK293 cells expressing Dectin-1 and Raji cells.

[0045] Fig.24A and 24B Bispecific antibody designs of human bispecific antibodies (eg, human IgG1 bispecific antibodies) targeting Dectin-1 and a disease target or antigen are shown. Fig.24A Design diagram is provided. One arm (2M24A.X) with VH domain A and VL domain B targets human Dectin-1, while the other arm (2M24B.X) with VH domain C and VL domain D targets a disease target or antigen. Fig. 24B An exemplary mechanism of action diagram of an anti-Dectin-1 bispecific antibody with an active Fc domain is provided, which targets hDectin-1 on myeloid cells (via the first arm), antigens on target cells / pathogens (via the second arm), and Fc receptors on myeloid and NK cells, triggering strong immune stimulation and phagocytosis.

[0046] Fig.25Aand 25B Bispecific antibodies with one arm targeting hDectin-1 and the other targeting hCD20 (using the variable domain of rituximab) are shown to bind to cells expressing either human Dectin-1 or human CD20. Fig.25A (Top) Shows the binding of bispecific antibodies targeting hDectin-1 and hCD20 (2M24 / CD20) or hDectin-1 and RSV (2M24 / RSV) to HEK293 cells stably expressing human Dectin-1 assessed by flow cytometry. Fig.25A (Bottom) shows the binding of bispecific antibody 2M24 / RSV hIgG1-FITC conjugate and 2M24 bivalent hIgG1-FITC conjugate to PBMCs assessed by flow cytometry. Fig.25B Binding of rituximab (human IgG1), 2M24 / CD20 with active human IgG1 Fc, 2M24 / CD20 with inert human IgG1 Fc, 2M24 / RSV with active human IgG1 Fc, or 2M24 / RSV with inert human IgG1 Fc to the CD20-expressing B-cell lymphoma Raji cell line is shown.

[0047] Fig.26A and 26B It is shown that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) induces the coupling of cells expressing Dectin-1 and CD20. Fig.26A : To evaluate the coupling of HEK293 cells expressing Dectin-1 (effector cells) and Raji cells expressing CD20 (target cells), cells were differentially labeled with calcein green (effector cells) or calcein red (target cells) dyes. Labeled cells were co-cultured and treated with hIgG1 inert 2M24 / CD20 or 2M24 / RSV (control) bispecific antibodies to induce effector cell: target cell coupling. Double positive staining (calcein green +, calcein red +, squares) indicates successful coupling of effector cell: target cell. Fig.26B : Dose titration of bispecific antibodies in effector cell:target cell co-cultures. Coupling efficiency was quantified as the percentage of cells bound or coupled to effector cells relative to total target cells.

[0048] Fig.27A and 27BIt was shown that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with active hIgG1 Fc did not induce monocyte depletion through antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). Fig.27A ) and donor 77( Fig.27B ) were treated with increasing concentrations of 2M24 / CD20 bispecific antibody (hIgG1 active or inert isotype) and rituximab for 24 h and subsequently analyzed by flow cytometry to quantify the levels of remaining viable CD14+ monocytes (as a percentage of isotype control).

[0049] Fig.28A and 28B It was shown that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with active hIgG1 Fc caused better B cell depletion than rituximab. Fig.28A ) and 84( Fig.28B ) were treated with increasing concentrations of the indicated antibodies for 24 h and subsequently analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as the percentage of B cells in isotype control-treated PBMCs).

[0050] Fig.29A and 29B Rituximab induced higher B cell tailoring (CD19 downregulation) compared to 2M24 / CD20 active IgG1 bispecific antibody. After 24 hours of incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab or isotype control, B cells from two healthy donors - Donor 83 ( Fig.29A ) and donor 84( Fig.29B ) on B cells. The effect of 2M24 / CD20 bispecific antibody and rituximab on CD19 expression on B cells was evaluated by the mean fluorescence intensity (MFI) of CD19 staining using anti-CD19 (BV605 conjugated). EC50 values ​​were calculated based on nonlinear regression analysis.

[0051] Fig.30Shown the difference cytokine release induced by 2M24 / CD20 active IgG1 bispecific antibody compared with rituximab.In the supernatant separated from the healthy donor PBMC with 2M24 / CD20 active hIgG1 bispecific antibody, rituximab or isotype control, the cytokine quantification based on ELISA (mesoscale discovery) is carried out.PBMC stimulates overnight with antibody, then analyzes supernatant with MSD.The cytokine tested is IFNγ, IL-12p70, IL-6, TNFα, IL-1β, IL-4, IL-13, IL-10 and IL-8.Each figure shows that cytokine secretion (in pg / mL) changes with the antibody for processing (from left to right: 2M24 / CD20 hIgG1 bispecific antibody, 2M24 / RSV hIgG1 bispecific antibody, rituximab hIgG1 and isotype control hIgG1).

[0052] Fig.31A and 31B It was shown that in co-cultures of human macrophages and GFP-expressing Raji B cells, the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody induced better B cell depletion and lower CD19 trimming compared to rituximab. Fig.31A : Flow cytometric analysis of co-cultures of human macrophages and Raji-GFP cells (3:1 ratio) in the presence of 2M24 / CD20 hIgG1 (active isotype) bispecific antibodies, 2M24 / RSV controls, fucosylated rituximab or isotype hIgG1 controls. The co-cultures were incubated at 37°C for 24 hours and then stained with PE a-CD206 Ab to mark macrophages and with BV-605a-CD19 antibody to mark Raji cells. The number of remaining live / Raji-GFP+ cells was assessed at the end of the experiment. Primary antibodies were used for serial dose titration. Fig.31B : Evaluation of CD19 on Raji-GFP cells after 24 hours. B cell receptor trimming is shown as a decrease in CD19 MFI in the presence of anti-Dectin-1 / anti-hCD20 bispecific antibody or rituximab.

[0053] Figures 32A-32CIt was shown that the 2M24 / CD20 active IgG1 bispecific antibody induced better tissue B cell depletion in single cell suspensions of renal cancer biopsies compared to rituximab. Single cell suspensions from two renal cancer tissue biopsies were analyzed by flow cytometry in the presence of 2M24 / CD20 hIgG1 (active or inert) bispecific antibody, 2M24 / RSV hIgG1 control, fucosylated rituximab and corresponding isotype controls. Renal cancer tissue biopsies were dissociated into single cell suspensions and treated with primary antibodies (2μg / ml) for 24 hours at 37°C. Immune cell populations were analyzed by flow cytometry. Cells were initially gated for live cells and further divided into CD45+ cells (immune cells) and CD45- cells (non-immune cells), and then CD19+ (B cells) and CD3+ (T cells) cells were identified within the CD45+ cell population ( Fig.32A and 32B The number of remaining B cells was assessed using anti-CD19 antibody and expressed as a percentage relative to the CD45+ immune cell population ( Fig.32C ).

[0054] Figures 33A-33C Anti-Dectin 1 antibody (clone 2M24) was shown to induce Dectin 1 aggregation and TNFα secretion by human macrophages. Cytokine secretion was tested in cultured macrophages and single cell suspensions of renal cancer biopsies stimulated with immobilized anti-Dectin-1 antibody (clone 2M24) or 2M24 / CD20 bispecific antibody. Anti-Dectin-1 antibody (clone 2M24), isotype control or 2M24 / CD20 bispecific antibody was immobilized at 10 ug per well in U-bottom polypropylene microtiter plates overnight, and then human monocyte-derived macrophages ( Fig.33A and 33B ) or single cell suspensions from renal cancer biopsies ( Fig.33C ). The cells were cultured for 24 hours and the supernatant was evaluated for TNFα secretion by ELISA. As a positive control, the cells were stimulated with zymosan.

[0055] Fig.34Show that immobilized anti-Dectin 1 antibody (clone 2M24) promotes immune stimulation in single cell suspensions of renal cancer biopsies. Single cell suspensions from renal cancer biopsies were treated with immobilized anti-Dectin-1 antibody (clone 2M24) or isotype control hIgG4 antibodies for 24 hours. The release of various cytokines in the supernatant was analyzed by ELISA, including IFNγ, IL-6, TNFα, IL-23, IL-12p70, IL-10 and IL-13. Each figure shows that the amount of cytokines (pg / mL) changes with antibody treatment. Show the results of using renal cancer donor 3 (left) or donor 4 (right) using anti-Dectin-1 antibody (clone 2M24) or isotype control hIgG4 antibody treatment.

[0056] Fig.35 The effect of the 2M24 / CD20 bispecific antibody on CD16 expression in human NK cells compared to rituximab or an isotype control (RSV) is shown. The results show that CD16 antigen levels on NK cells are better maintained in PBMCs treated with the 2M24 / CD20 bispecific antibody compared to rituximab.

[0057] Fig.36 The effect of the 2M24 / CD20 bispecific antibody on CD19 expression in human B cells compared to rituximab or an isotype control (2M24 / RSV bispecific antibody) is shown. The results show that CD19 antigen levels are better maintained on B cells treated with the 2M24 / CD20 bispecific antibody compared to rituximab.

[0058] Fig.37 Depletion of human B cells by 2M24 / CD20 bispecific antibodies derived from rituximab or 2M24 / CD20 bispecific antibodies derived from obinutuzumab was shown. The results showed that the 2M24 / CD20 bispecific antibody derived from the rituximab arm depleted B cells better than the bispecific antibody derived from obinutuzumab.

[0059] Fig.38 Shown is the design of an exploratory study of the safety and efficacy of the 2M24 / CD20 bispecific antibody in non-human primates.

[0060] Fig.39 and 40 Depletion of circulating B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody produced in cells treated with kifunine (KIF) is shown. Fig.39: B cell depletion in monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF (top) or 2M24 / CD20 hIgG1 inert (bottom). Fig.40 : B cell depletion in monkeys treated with 5 mg / kg rituximab hIgG1 KI F.

[0061] Fig.41A and 41B Depletion of tissue-resident B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody produced in cells treated with kifunine (KIF) is shown. Fig.41A : B cell depletion in the bone marrow of monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF. Fig.41B : B cell depletion in lymph nodes of monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF.

[0062] Fig.42 Ex vivo depletion of B cells from cynomolgus monkey PBMCs is shown.

[0063] Fig.43 The format of a bispecific molecule is shown that pairs an anti-CD20 conventional half antibody with an anti-Dectin-1 single chain variable fragment (scFv) Fc fusion arm (2M24 scFv / CD20) using the knobs-in-holes technology. H: 2M24 VH domain; L: 2M24 VL domain.

[0064] Figures 44A-44C The purification and functional characterization of the 2M24 / CD20 bispecific antibody is shown. Fig.44A Purification of the molecules by size exclusion chromatography (SEC) is shown. Fig.44B It was shown that the purified bispecific antibodies promoted targeted immune stimulation as assessed in a NFκB reporter assay. Fig.44C Depletion of human B cells by the 2M24 scFv / CD20 bispecific antibody is shown.

[0065] Fig.45Depletion of B cells in healthy donor PBMCs by antibodies targeting CD20, including rituximab, CD3×CD20 bispecific T cell engagers, and bispecific binding molecules comprising an anti-CD20 conventional half antibody arm and an anti-Dectin-1 single chain variable fragment (scFv) arm (2M24 scFv / CD20) in a non-fucosylated hIgG1 Fc format are shown. PBMCs isolated from healthy donors were incubated with antibodies targeting CD20 for 24 hours and subsequently stained with anti-CD19 (B cell specific marker) antibodies to characterize the remaining B cells by flow cytometry. Data are presented as the percentage of remaining B cells relative to the untreated control group. NF: non-fucosylated.

[0066] Fig.46 Depletion of B cells in prostate cancer tumor biopsy specimens by rituximab, a bispecific binding molecule comprising an anti-CD20 conventional half antibody arm and an anti-Dectin-1 single chain variable fragment (scFv) arm (2M24scFv / CD20) in a non-fucosylated hIgG1 Fc format, a 2M24×CD20 hIgG1 bispecific antibody (DuetMab format), or an isotype control is shown. Single cell suspensions generated from prostate cancer biopsies were treated with antibodies targeting CD20 for 24 hours. Cells were subsequently stained with antibodies against CD45, CD3, CD11b, CD16, CD163, and CD19. B cells were identified as CD45+CD3-CD19+. The data show the percentage of remaining B cells relative to the number of B cells in the isotype control group (RSV hIgG1).

[0067] Fig.47A The effect of 2M24×CD20 bispecific binding protein (DuetMab format) treatment on CD16 expression on NK cells in healthy human PBMCs compared to rituximab or control antibody is shown. Compared to rituximab, the 2M24×CD20 bispecific antibody better maintained CD16 levels on NK cells.

[0068] Fig.47B The effect of 2M24×CD20 bispecific binding protein treatment on CD19 expression on B cells in healthy human PBMCs compared to rituximab or control antibodies is shown. Results from 3 donors are shown; for each donor, the order of data points is: 2M24×CD20 bispecific antibody, rituximab, control (from left to right). Compared with rituximab, the 2M24 / CD20 bispecific antibody better maintains CD19 levels on B cells.

[0069] Fig.47CThe effect of 2M24×CD20 bispecific binding protein treatment on B cell depletion in healthy human PBMCs compared to rituximab, obinutuzumab or control antibodies is shown. Two forms of 2M24×CD20 bispecific binding proteins were tested: one with the variable domains from rituximab and the other with the variable domains from obinutuzumab. B cells were quantified relative to the untreated control (dashed line). The 2M24×CD20 bispecific binding protein with the anti-CD20 arm of rituximab showed better B cell depletion than the comparable 2M24 bispecific binding protein with the anti-CD20 arm of obinutuzumab.

[0070] Fig.47D The effect of 2M24×CD20 bispecific binding protein treatment on B cell depletion compared to 2M24 / RSV bispecific antibody, rituximab, isotype control antibody or no treatment is shown using single cell suspensions from renal cancer biopsies. 2M24×CD20 bispecific binding protein induced superior B cell depletion compared to rituximab.

[0071] Fig.48 The design of an exploratory study of the 2M24×CD20 bispecific binding protein in non-human primates (cynomolgus monkeys) is shown. Time points for whole blood collection, tissue collection, whole blood collection for chemistry and coagulation, and body weight measurement are indicated. CBC: complete blood count. PK: pharmacokinetics. BM: bone marrow. LN: lymph node.

[0072] Figures 49A-49C Depletion of B cells by the 2M24×CD20 bispecific binding protein in non-human primate studies is shown. Fig.49A Depletion of CD19+ B cells in blood by 2M24×CD20 hIgG1 bispecific binding protein treated with KIF (upper left), 2M24×CD20 bispecific binding protein with an inert hIgG1 Fc (lower left), or rituximab hIgG1 treated with KIF (upper right) is shown. Fig.49B Depletion of B cells (as a percentage of CD45+) in the bone marrow (top) or lymph nodes (bottom) by 2M24×CD20 hIgG1 bispecific binding protein treated with KIF, 2M24×CD20 bispecific binding protein with an inert hIgG1 Fc, or rituximab hIgG1 treated with KIF is shown, as indicated. Fig.49C Shown are ex vivo depletion of B cells in cynomolgus monkey PBMCs treated with serial dilutions of 2M24×CD20 hIgG1 bispecific binding protein treated with KIF, rituximab hIgG1 treated with KIF, or control antibody treated with KIF.

[0073] Fig.50A and 50B Shown are the results of mouse studies on B cell depletion by the anti-mouse Dectin-1 antibody 2A11×mCD20 bispecific binding protein. Fig.50A The design of the study is shown. Fig.50B Results are shown for treatment with 2A11×mCD20 bispecific mlgG1 binding protein, anti-mouse CD20 rat IgG2a antibody, or mlgG1 isotype control on CD19+ B cells (percentage of total CD45+ cells) from blood, peritoneum, bone marrow, and spleen. Differences between study groups were analyzed using ordinary one-way Anova and Tukey's test. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001.

[0074] Figures 51A-51E Shown are the results of ex vivo testing of various properties of the 2M24×CD20 bispecific binding protein. Fig.51A Activation of a reporter HEK cell line expressing Dectin-1 is shown as a function of antibody concentration. Results are shown for treatment with 2M24×CD20 bispecific binding protein (circles), rituximab (squares) or D-zymosan (triangles), an established ligand for Dectin-1 used as a positive control. Fig.51B Induction of B cell line phagocytosis (percentage relative to isotype control treated B cells) as a function of antibody concentration is shown. Results are shown for treatment with 2M24×CD20 bispecific binding protein (black circles), rituximab (triangles) or nonspecific RSV antibodies (grey circles). Fig.51C B cell depletion (percentage of B cells relative to untreated controls) is shown as a function of antibody concentration. Results are shown for treatment with 2M24×CD20 bispecific binding protein (black squares), rituximab (triangles), anti-CD20 / anti-CD3 bispecific conjugates (grey squares), or nonspecific RSV antibodies (grey circles). Fig.51D and 51E Ex vivo cytokine stimulation of human PBMCs by 2M24×CD20 bispecific binding protein or comparative anti-CD20 antibodies (pg / mL of the indicated cytokines) is shown. Results of treatment with 2M24×CD20 bispecific binding protein, rituximab, anti-CD20 / anti-CD3 bispecific conjugate, nonspecific RSV antibody, or untreated control are shown. The cytokines measured included IL-6 ( Fig.51D , upper left), TNF-α( Fig.51D , lower left), IFN-γ( Fig.51D , right), IL-12p70( Fig.51E, upper left), IL-1β( Fig.51E , lower left) and IL-2 ( Fig.51E ,right).

[0075] Figures 52A-52H Shown are the results of in vivo testing of alternative anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. Fig.52A Shown is the effect of treatment with alternative anti-mDectin-1 / anti-mCD20 mIgG2a bispecific antibodies (squares) or isotype control (circles) on tumor volume (mm) in an in vivo mouse xenograft model. 3 ) over time (days post-injection) using Ramos B-cell lymphoma xenografts in SCID mice (upper left), Daudi B-cell lymphoma xenografts in SCID mice (lower left), or Raji B-cell lymphoma xenografts in SCID-beige mice. Efficacy data are shown as mean + / - SEM. Fig.52B Shown are myeloid cell activation (myeloid cells expressed as a percentage of CD45+ cells) of the indicated cell populations in the spleen (left), lymph nodes (center), or tumors (right) of mice implanted with MC38 murine colon cancer cells expressing human CD20. For each cell population, cell numbers are given as a percentage of total CD45+ cells, with data for isotype control on the left and anti-mDectin-1 / anti-mCD20 on the right. GRN: granulocyte. DC: dendritic cell. Macs: macrophage. Two-way Anova was used to analyze differences between the study groups. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. Fig.52C Activation of CD3+ T cells, Ki67+ T cells, effector T cells, and splenic T cells expressing the indicated markers in the MC38 xenograft mouse model after treatment with anti-mDectin-1 / anti-mCD20 mIgG1 bispecific antibody (right side in all figures) or isotype control (left side in all figures) is shown. For each cell population, the number of cells is provided as a percentage of the indicated cell population. Two-way Anova was used to analyze the differences between the study groups. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. Fig.52DThe activation of intratumoral CD3+ T cells (percentage of tumor CD45+ cells; left), activation of intratumoral CD4+ or CD8+ T cells (percentage of tumor T cells; middle), and production of cytokines IL-2 and GmzB by intratumoral CD8+ T cells in the MC38 xenograft mouse model after treatment with anti-mDectin-1 / anti-mCD20mIgG1 bispecific antibody (right in all figures) or isotype control (left in all figures) are shown. Two-way Anova was used to analyze the differences between the study groups. *P≤0.05; **P≤0.01; ***P≤0.001; ****P≤0.0001. Fig.52E Shown is B cell depletion in blood (upper left), lymph nodes (upper right), spleen (lower left) and bone marrow (lower right) in naive C57BL / 6 mice after treatment with anti-mDectin-1 / anti-mCD20 mIgG2a bispecific antibody (grey circles) or isotype control (black circles). Data are depicted as B cells (percentage of B cells in isotype control) at the indicated days after injection. Fig.52F Shown is B cell depletion in lung (upper left), liver (upper right), brain (lower left) and heart (lower right) in naive C57BL / 6 mice after treatment with anti-mDectin-1 / anti-mCD20 mIgG2a bispecific antibody (grey circles) or isotype control (black circles). Data are depicted as B cells (percentage of B cells in isotype control) at the indicated days after injection. Figure 52G Comparison of tumor growth inhibition of anti-mDectin-1 / anti-mCD20 bispecific antibodies with active Fc (mIgG2a) and inert mIgG2a Fc versus isotype control in the Daudi xenograft model in SCID mice. Tumor volume (mm 3 ) Over time (days post-implantation). Data for isotype control (circles), anti-mDectin-1 / anti-mCD20 mIgG2a active (squares), and anti-mDectin-1 / anti-mCD20 mIgG2a inert (triangles) as indicated. Error bars represent SEM. Fig.52H Figure 2 shows the expression of resident monocytes (CD11b) in tumor tissues after treatment with mIgG2a isotype control (circles) or anti-mDectin-1 / anti-mCD20 bispecific antibody with mIgG2a inert Fc (triangles). hi MHCII-Ly6c-; upper left), inflammatory monocytes (CD11b hi MHCII-Ly6c+; upper right), macrophages (CD11b+F4 / 80+; lower left), and proportions of M1 and M2 macrophages (lower right) (all graphs are presented as percentage of CD45+, except the lower right graph, which shows the percentage of macrophages).

[0076] Figures 53A-53I Shown are the results of in vivo testing of the 2M24×CD20 bispecific binding protein in a cynomolgus monkey model. Fig.53A Shown are B cell levels (percentage relative to baseline) in peripheral blood over time after administration of 1 mg / kg (upper left), 10 mg / kg (lower left), or 100 mg / kg (right) of the 2M24×CD20 bispecific antibody. Fig.53B Shown are B cell levels in the lymphoid organs bone marrow (top) and lymph nodes (bottom) over time after administration of 1 mg / kg (left), 10 mg / kg (middle), or 100 mg / kg (right) of the 2M24×CD20 bispecific antibody. Fig.53C Shown are B cell levels (as a percentage of tissue CD45+ cells) in the indicated lymphoid tissues (left) and non-lymphoid tissues (right) at day 15 after treatment with vehicle control (for all tissues, on the left) or 100 mg / kg 2M24×CD20 bispecific antibody (for all tissues, on the right). Fig.53D and 53E The vehicle control ( Fig.53D Below: Fig.53E Left side) or 100 mg / kg 2M24×CD20 bispecific antibody ( Fig.53D Picture above; Fig.53E Right) Depletion of the indicated B cell subsets in spleen at day 15 after treatment. Fig.53F Shown are the levels of circulating classical dendritic cells (as a percentage of CD45+ cells) over time (days) after treatment with 2M24×CD20 bispecific antibody (black circles) or rituximab-like anti-CD20 mAb (grey circles). Figure 53G Shown are the levels of classical dendritic cells (as a percentage of CD45+ cells) in circulation over time (days) after treatment with 1 mg / kg (black circles) or 10 mg / kg (grey squares) of the 2M24×CD20 bispecific antibody. Fig.53H and 53I showed that after three weekly doses of 1 mg / kg ( Fig.53H )、10mg / kg( Fig.53H ) or 100 mg / kg( Fig.53I ) of 2M24×CD20 bispecific binding protein. DETAILED DESCRIPTION

[0077] Several aspects are described below in conjunction with example applications for illustration. It should be understood that many specific details, relationships, and methods are set forth to provide a complete understanding of the features described herein. However, those of ordinary skill in the relevant art will readily recognize that the features described herein can be practiced without one or more specific details or using other methods. The features described herein are not limited by the actions or event sequences shown, because some actions can occur in different orders and / or occur simultaneously with other actions or events. In addition, not all actions or events shown need to be implemented according to the methods of the features described herein.

[0078] Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an", and "the" are intended to include the plural forms as well. In addition, if the terms "including", "includes", "having", "has", "with" or variations thereof are used in the detailed description and / or claims, such terms are intended to be included in a manner similar to the term "comprising". As used herein, the term "comprising" is synonymous with "including" or "containing" and is inclusive or open-ended.

[0079] Unless otherwise indicated, any reference to "or" herein is intended to include "and / or". As used herein, the term "about" with respect to a number means that number plus or minus 10% of that number. The term "about" with respect to a range means that range minus 10% of its lower value and plus 10% of its upper value.

[0080] I. Multispecific Binding Proteins

[0081] In certain aspects, the present disclosure provides antigen binding domains that bind to human Dectin-1, and multispecific (e.g., bispecific) binding molecules comprising them. In certain aspects, the present disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments, which include a first antigen binding domain that binds to a first target of interest (i.e., Dectin-1) and a second antigen binding domain that binds to a second target of interest (i.e., CD20). In some embodiments, the present disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments, which include a first antigen binding domain that binds to human Dectin-1 and a second antigen binding domain that binds to CD20.

[0082] In some embodiments, the multispecific binding protein comprises a first polypeptide chain comprising the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPED FATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:31) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:35); The second polypeptide chain, the second polypeptide chain comprises the amino acid sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ IDNO: 32) or QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36); and a third polypeptide chain, the third polypeptide chain comprising the amino acid sequence QIVLSQSPAILSASPG EKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0083] In some embodiments, the multispecific binding protein comprises a first polypeptide chain comprising the amino acid sequence QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPED FATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:37) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:39); The second polypeptide chain, the second polypeptide chain comprises the amino acid sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:38) or QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSV FPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:40); and a third polypeptide chain comprising the amino acid sequence QIVLSQSPAILSASPG EKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:33).

[0084] As is known in the art, the C-terminal lysine of some antibody heavy chain species may be cleaved off in certain portions of the molecule. In some embodiments, one or both antibody Fc domains do not have a C-terminal lysine.

[0085] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is a composition comprising a mixture of multispecific binding protein species, wherein each species comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 35, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 36, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33.

[0086] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, provided herein is a composition comprising a mixture of multispecific binding protein species, wherein each species comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 39, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 40, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33.

[0087] In some embodiments, the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain are associated in a multispecific binding protein comprising a first antigen binding domain that binds human Dectin-1 and a second antigen binding domain that binds human CD20.

[0088] In some embodiments, the multispecific binding protein, antigen binding domain, antibody or antibody fragment binds to human Dectin-1. In some embodiments, the multispecific binding protein, 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 multispecific binding protein, antigen binding domain, antibody or antibody fragment binds to human Dectin-1 isoform A and / or human Dectin-1 isoform B. 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 MEY HPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO: 10). In some embodiments, the multispecific binding protein, 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 is capable of binding to human Dectin-1 and monkey Dectin-1, such as cynomolgus monkey Dectin-1.

[0089] In some embodiments, the multispecific binding protein, antigen binding domain, antibody or antibody fragment binds human CD20, also known as MS4A1, B1, S7, Bp35, FMC7, CVID5 and LEU-16. In some embodiments, human CD20 refers to the polypeptide encoded by NCBI Gene ID No.931. An exemplary and non-limiting human CD20 polypeptide is provided by NCBI Ref. Seq. NP_068769: MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP (SEQ ID NO: 34).

[0090] In some embodiments, antibody and immunoglobulin are used interchangeably and are used herein in the broadest sense and 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 (as described in more detail herein), so long as they exhibit the desired antigen-binding activity.

[0091] In some embodiments, antibody (immunoglobulin) refers to a protein having a structure substantially similar to that of a natural antibody, or a protein having a heavy chain and a light chain variable region having a structure substantially similar to that of a natural heavy chain and a light chain variable region. Natural antibodies refer to naturally occurring immunoglobulin molecules with different structures. For example, IgG class natural immunoglobulins are heterotetrameric glycoproteins of about 150,000 daltons consisting of two light chains and two heavy chains connected by disulfide bonds. From N-terminal to C-terminal, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also referred to as a heavy chain constant region. Similarly, from N-terminal to C-terminal, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain, also referred to as a light chain constant region. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known and are generally described in, for example, Abbas et al., 2000, Cellular and Mol and Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007). Depending on the amino acid sequence of the heavy chain constant domain, antibodies (immunoglobulins) can be designated as different classes. Antibodies are mainly divided into five categories: α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). Based on the amino acid sequence of its constant domain, the light chain of the antibody can be designated as one of two types (called κ and λ). Immunoglobulin is basically composed of two Fab molecules and an Fc domain, connected via the immunoglobulin hinge region.

[0092] In some embodiments, Fc, Fc region or Fc domain refers to the C-terminal region of the antibody heavy chain containing at least a portion of the constant region. The term includes native Fc region and variant Fc region. Fc may 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 at the N-terminus of these domains. For IgA and IgM, Fc may include a J chain. The IgG Fc region includes IgG CH2 and IgG CH3 domains, and in some cases, includes a hinge. Unless otherwise specified herein, the numbering of the amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991. Human IgG Fc domains are particularly useful in the present disclosure, and may be Fc domains from human IgG1, IgG2, or IgG4.

[0093] 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.

[0094] "Fab" (fragment antigen binding) is the part of an antibody which binds to the antigen and comprises the variable region and CH1 of the heavy chain connected to the light chain via interchain disulfide bonds.

[0095] In some embodiments, the multispecific binding protein or antibody of the present disclosure comprises an Fc region. Antibodies / multispecific binding proteins may belong to any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA and IgD. The immunoglobulin Fc region of the molecule causing targeted phagocytosis may play an important role in the process by binding to Fc receptors and inducing additional phagocytosis. In some embodiments, the molecule has a modified Fc region that has reduced ADCC activity compared to wild-type human IgG1 (e.g., comprising one or more mutations that reduce effector function as described herein).

[0096] In some embodiments, the multispecific binding protein or antibody of the present disclosure comprises an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, for example, at least one or two heavy chains of the antibody are non-fucosylated. In some embodiments, a composition is provided herein, comprising a multispecific binding protein or antibody of the present disclosure, the multispecific binding protein or antibody comprising an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, for example, at least one or two heavy chains of the antibody are non-fucosylated. In some embodiments, less than 50% of the N-glycoside-linked carbohydrate chains in the composition contain fucose residues. In some embodiments, substantially no N-glycoside-linked carbohydrate chains contain fucose residues. In some embodiments, the multispecific binding protein or antibody with reduced fucose or lack of fucose has improved ADCC function.

[0097] In other embodiments, the multispecific binding proteins (e.g., IgG1 antibodies) of the present disclosure or compositions comprising the multispecific binding proteins (e.g., IgG1 antibodies) of the present disclosure comprise wild-type glycosylation of the Fc region. In some embodiments, provided herein are fucosylated binding proteins (e.g., IgG1 antibodies) of the present disclosure or compositions comprising the fucosylated binding proteins (e.g., IgG1 antibodies) of the present disclosure.

[0098] Fucosylation or fucosylated binding protein may refer to the presence of fucose residues in oligosaccharides attached to the antibody peptide backbone. Specifically, fucosylated antibodies contain α(l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue in one or two N-linked oligosaccharides attached to the Fc region of the antibody, such as at position Asn 297 of the Fc region of human IgG1 (EU numbering of Fc region residues). Due to minor sequence variations in immunoglobulins, Asn297 may also be located about +3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Non-fucosylated or fucose-deficient antibodies have reduced fucose relative to the amount of fucose on the same antibody produced in a cell line. Antibody fucosylation can be measured, for example, by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOFMS) evaluation in an antibody composition treated with N-glycosidase F.

[0099] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate fucosylation, such as a substitution at Asn 297 (EU numbering of Fc region residues) of the human IgG1 Fc region. Optionally, the Fc region further comprises one or more amino acid substitutions therein that further improve ADCC, such as a substitution at positions 298, 333, and / or 334 (Eu numbering of residues) of the Fc region. Examples of publications relating to "defucosylated" or "fucose-deficient" antibodies include: US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004).

[0100] In some embodiments, the afucosylated or non-fucosylated binding protein is produced in a cell line with a genetic modification that results in an afucosylated or non-fucosylated antibody. Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells that are defective in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., particularly Example 11), and knockout cell lines, such as CHO cells with the α-1,6-fucosyltransferase gene FUT8 knocked out (Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004)), cells overexpressing β1,4-N-acetylglucosamine transferase III (GnT-III) and Golgi μ-mannosidase II (ManII), and cells with mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase knocked out (MGAT1; see Byrne, G. et al. (2018) PLoS Biol. 16:e2005817).

[0101] In some embodiments, the afucosylated or non-fucosylated binding protein is produced in a cell line treated with a carbohydrate processing enzyme inhibitor, such as kifunine, which is a mannosidase I inhibitor (see, e.g., Elbein, AD et al. (1990) J. Biol. Chem. 265: 15599-15605). For example, the cells can be centrifuged and resuspended in a growth medium containing kifunine (e.g., 250 μg / mL), then cultured and used to produce antibodies.

[0102] In some embodiments, one or more 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.

[0103] In some embodiments, a multispecific (e.g., bispecific) binding molecule comprises: a first antibody arm 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 Fc region; and a second antibody arm comprising an antibody heavy chain comprising a VH domain, the antibody heavy chain 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 Dectin-1, and a conventional antibody arm having VH and VL domains on separate polypeptides binds to a target of interest, such as described herein, such as a pathogen. In some embodiments, the first Fc region comprises one or more mutations forming a knob, and the second Fc region comprises one or more homologous mutations forming a hole, or wherein the second Fc region comprises one or more mutations forming a knob, and the first Fc region comprises one or more homologous mutations forming a hole. Fig.43 A non-limiting example of this format is shown in .

[0104] In some embodiments, the pathogen is a B cell, a tumor or a cancer cell, such as a malignant B cell. In some embodiments, CD20 is expressed on the surface of a B cell, such as a malignant B cell. CD20 is expressed on most B cells from the late pre-B lymphocyte stage. Therefore, the therapy of depleting B cells has been used to treat a variety of indications for CD20, such as cancer (e.g., non-Hodgkin's lymphoma or chronic lymphocytic leukemia) and autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis and Wegener's granulomatosis).

[0105] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising: a first antibody arm, the first antibody arm comprising a single chain variable fragment (scFv) comprising VH and VL domains of the present disclosure that binds to human Dectin-1 and a first Fc region; and a second antibody arm, the second antibody arm comprising an antibody heavy chain comprising a VH domain, the antibody heavy chain associated with an antibody light chain comprising a VL domain and a second Fc region connected to the VH domain, wherein the VH domain and the VL domain of the second antibody arm form an antigen binding domain that binds to a target of interest (e.g., a pathogen of the present disclosure). In some embodiments, the first Fc region comprises one or more mutations that form a knob, and the second Fc region comprises one or more homologous mutations that form a hole, or the second Fc region comprises one or more mutations that form a knob, and the first Fc region comprises one or more homologous mutations that form a hole. 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 the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:31) or QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:37).

[0106] In some embodiments, a multispecific (e.g., bispecific) binding molecule is provided herein, comprising: a first antibody arm, the first antibody arm comprising a single chain variable fragment (scFv) comprising VH and VL domains of the present disclosure that binds to human Dectin-1 and a first Fc region; and a second antibody arm, the second antibody arm comprising an antibody heavy chain comprising a VH domain, the antibody heavy chain associated with an antibody light chain comprising a VL domain and a second Fc region connected to the VH domain, wherein the VH domain and the 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 mutations that form a knob, and the second Fc region comprises one or more homologous mutations that form a hole, or the second Fc region comprises one or more mutations that form a knob, and the first Fc region comprises one or more homologous mutations that form a hole. 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 QVQLVQSG AEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDI EEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:31) amino acid sequence.In some embodiments, the second antibody arm comprises a polypeptide comprising QVQLQQPGAELVKPGASV KMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ (SEQ ID NO: 32) and a third polypeptide comprising the amino acid sequence of QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).In some embodiments, the first antibody arm comprises the amino acid sequence of QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:37).In some embodiments, the second antibody arm comprises a polypeptide comprising QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ (SEQ ID NO: 38) and a third polypeptide comprising the amino acid sequence of QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0107] Multispecific antibodies have binding specificity for at least two epitopes, usually from different antigens. Multispecific or bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2 bispecific antibodies).

[0108] In order to enable targeted removal of pathogens via phagocytosis, the antigen binding domains of the present disclosure can be selected from IgG, intracellular antibodies, peptide antibodies, nanobodies, single domain antibodies, SMTP and multispecific antibodies (e.g., bispecific antibodies, double-chain antibodies, three-chain antibodies, four-chain antibodies, tandem two-scFV, tandem three-scFv, ADAPTIR).

[0109] Methods for preparing bispecific antibodies are known in the art. One recognized method for making bispecific antibodies is the "knob into hole" or "protrusion into cavity" method. See, for example, U.S. Patent No. 5,731,168. Two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each contain an interface; the interface of one immunoglobulin polypeptide interacts with a corresponding or homologous interface on another immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. In some embodiments, the interface can be engineered so that a "knob" or "protrusion" located in the interface of one immunoglobulin polypeptide corresponds to a homologous "hole" or "cavity" located in the interface of another immunoglobulin polypeptide. In some embodiments, a knob can be constructed by replacing a smaller amino acid side chain with a larger side chain. In some embodiments, a hole can be constructed by replacing a larger amino acid side chain with a smaller side chain. The knob or hole may be present in the original interface, or they may be introduced synthetically. Polynucleotides encoding modified immunoglobulin polypeptides having one or more corresponding mutations that form a knob or hole can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; 8,679,785; 8,844,834; U.S. Publication No. 2013 / 0089553; Spiess et al., Nature Biotechnology 31:753-758, 2013; and Ridgway and Carter (1996) Protein Eng. 9:617-621. The modified immunoglobulin polypeptides can be produced using prokaryotic host cells such as E. coli or eukaryotic host cells such as mammalian cells (e.g., CHO cells) or yeast cells. The corresponding immunoglobulin polypeptides with knobs and holes can be expressed in co-cultured host cells and purified together as heteromultimers, or they can be expressed in a single culture, purified separately, and assembled in vitro. Exemplary homologous knob and hole mutations (numbered according to the EU index) are provided below. EU numbering as used herein is known in the art; see, e.g., IMGT resources www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html and www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html. As used herein, "antibody arm" may refer to the pairing between an antibody heavy chain and an antibody light chain, wherein the variable domains of the heavy and light chains form an antigen binding site that binds to a target antigen.

[0110]

[0111] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences.

[0112] In some embodiments, multispecific (e.g., bispecific) antibodies also include one or more mutations that improve heavy chain / light chain pairing only on one antibody arm. For example, amino acid substitutions can be used to replace the natural disulfide bonds in the CH1-CL interface of an antibody arm with engineered disulfide bonds. See, for example, Mazor, Y. et al. (2015) MAbs 7:377-389 and EP3452089A2. In some embodiments, multispecific or bispecific antibodies include two antibody light chains and two antibody heavy chains, wherein only one antibody heavy chain includes amino acid substitutions F126C and C220V according to EU numbering, and only corresponding or homologous light chains include amino acid substitutions S121C and C214V.

[0113] Multispecific (e.g., bispecific) antibodies also include crosslinked or "heterologous coupling" antibodies. The technology of producing bispecific antibodies from antibody fragments has also been described in the literature. For example, chemical connections can be used to prepare bispecific antibodies. In some embodiments, the bispecific antibody comprises a first IgG antibody comprising a first antigen-binding domain that is covalently connected to a second IgG antibody comprising a second antigen-binding domain.

[0114] In some embodiments, the multispecific (e.g., bispecific) antibody further comprises one or more mutations on only one antibody arm that 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 antibody heavy chain comprises the amino acid substitutions H435R and Y436F according to EU numbering.

[0115] In some embodiments, the monospecific or multispecific (e.g., bispecific) antibody further comprises one or more mutations that reduce effector functions, such as reducing or eliminating the binding of the Fc region to an Fc receptor. In some embodiments, the antibody comprises two antibody Fc regions, wherein the antibody Fc region comprises 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 region comprises L234A, L235E, and G237A substitutions according to EU numbering.

[0116] In some embodiments, a monospecific or multispecific (e.g., bispecific) antibody comprises two antibody heavy chains and two antibody light chains, wherein the VH domain of the first antibody heavy chain forms an antigen binding domain with the VL domain of the first antibody light chain, wherein the VH domain of the second antibody heavy chain forms an antigen binding domain with the VL domain of the second antibody light chain, wherein according to EU numbering, the first antibody heavy chain comprises F126C, C220V and T366W substitutions, wherein the first antibody light chain comprises S121C and C214V substitutions, and wherein 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 human IgG1 Fc domains.

[0117] In some embodiments, a polynucleotide is provided herein, the polynucleotide encoding the antibody or multi-specific binding protein of any one of the above embodiments. In some embodiments, a vector (e.g., an expression vector) is provided herein, the vector comprising the polynucleotide of any one of the above embodiments. In some embodiments, a host cell (e.g., an isolated host cell or cell line) is provided herein, the host cell comprising the polynucleotide or vector of any one of the above embodiments. In some embodiments, a pharmaceutical composition is provided herein, the pharmaceutical composition comprising the multi-specific binding protein of any one of the above embodiments and a pharmaceutically acceptable carrier. Any of these can be used for the production and / or treatment methods disclosed herein.

[0118] In some embodiments, provided herein is a method of producing a multispecific binding protein, the method comprising culturing a host cell of any of the above embodiments under conditions suitable for producing the multispecific binding protein. In some embodiments, the method further comprises recovering the multispecific binding protein. The multispecific binding protein can be produced using standard recombinant techniques as described herein and / or as exemplified below.

[0119] Recombinant methods can be used to produce antibodies and antibody fragments. For example, nucleic acids encoding antibodies / fragments can be isolated and inserted into replicable vectors for further cloning or expression. The DNA encoding antibodies / fragments can be easily separated and sequenced using conventional procedures (e.g., via the use of oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibodies / fragments). Many vectors are known in the art; vector components generally include, but are not limited to, one or more of the following: signal sequences, replication origins, one or more marker genes, enhancer elements, promoters, and transcription termination sequences. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryotic cells, yeast cells, or higher eukaryotic cells. When recombinant techniques are used, antibodies / fragments can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody / fragment is produced intracellularly, the particle debris, host cells, or dissolved fragments are removed by, for example, centrifugation or ultrafiltration. When the antibody / fragment is secreted into the culture medium, the supernatant from this type of expression system is usually first concentrated using a commercially available protein concentration filter.

[0120] In some embodiments, the multispecific binding proteins of the present disclosure are part of a pharmaceutical composition, for example, comprising an antibody and one or more pharmaceutically acceptable carriers. Pharmaceutical compositions and formulations as described herein can be prepared by mixing an active ingredient (e.g., a fusion protein) having a desired purity with one or more optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution (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 such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0121] Certain aspects of the present disclosure relate to kits or articles of manufacture comprising any of the multispecific binding proteins 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 multispecific binding protein according to any of the methods disclosed herein, for example, to treat a disease or condition such as cancer.

[0122] Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed of a variety of materials, such as glass or plastic. The container contains a composition effective for treating the disorder and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a multispecific binding protein as described herein. The label or package insert indicates that the composition is used to treat a specific disorder. The label or package insert will also include instructions for administering the multispecific binding protein composition to a subject. Articles and kits containing the combination therapies described herein are also contemplated.

[0123] II. How to use

[0124] In certain aspects, the disclosure provides methods for treating a disease or condition, the methods 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. In some embodiments, the individual suffers from or has been diagnosed with cancer (e.g., non-Hodgkin's lymphoma or chronic lymphocytic leukemia) or an autoimmune disorder associated with B cells (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and Wegener's granulomatosis).

[0125] In some embodiments, the method comprises 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 pathogen, such as CD20 or hCD20. In some embodiments, the pathogen is a B cell, a tumor or a cancer cell, such as a malignant B cell. In some embodiments, CD20 is expressed on the surface of a B cell, such as a malignant B cell. The binding of a molecule that mediates targeted removal of a pathogen via phagocytosis may or may not have avidity, i.e., induce or not induce dimerization of a phagocytosis receptor such as Dectin-1 or a target antigen present on a pathogen.

[0126] In addition to beneficially removing pathogens via phagocytosis, molecules can also induce the production of inflammatory mediators to change disease microenvironments, such as tumors, cancers, and lymphomas. Without wishing to be bound by theory, it is believed that molecules that carry out targeted phagocytosis may show obvious benefits to patients such as cancer, inflammation or immune diseases (such as autoimmune diseases, inflammatory bowel disease, multiple sclerosis), degenerative diseases (such as joints and cartilage) rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD, etc. In addition, compared to ADCC that depends on NK cells, targeted phagocytosis antibody therapy may have better activity of cells in depletion tissues. Compared with the therapy that targets bone marrow cells and improves phagocytosis in general, the treatment may have the activity of selectively removing specific pathogens. For example, targets of interest for treating cancer include CD20.

[0127] The following description is presented to enable one of ordinary skill in the art to make and use various embodiments. The description of specific devices, methods, and applications is provided as an example only. Various modifications to the embodiments described herein will be apparent to one of ordinary skill in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the embodiments described and shown herein, but to conform to the scope consistent with the claims.

[0128] Example

[0129] Example 1: Functional Characterization of 2M24 Anti-Dectin-1 Antibody

[0130] This example describes the generation of monoclonal antibodies specific for human Dectin-1. This example also describes the characterization of novel anti-human Dectin-1 antibodies.

[0131] Materials and methods

[0132] Generation of anti-Dectin-1 antibodies

[0133] Four-week-old ATX-Gx transgenic mice were subcutaneously immunized with recombinant human Dectin-1 isotype B for five weeks, with weekly boosts of antigen. Antibody titers in mouse sera were assessed by ELISA and flow cytometry before and after boosts. Mice with the highest serum antibody titers were selected to supply B cells for hybridoma generation.

[0134] Prior to cell fusion, mice were administered an additional boost of recombinant human Dectin-1 isoform B. Mice were sacrificed and spleens were harvested. Splenocytes and SP2 / 0-Ag14 myeloma cells were mixed and then fused by incubation at 37°C and in the presence of polyethylene glycol (PEG) or electroporation. Cells were then harvested and plated in 96-well plates, with limiting dilution to achieve one cell per well. Cells were subsequently treated with hypoxanthine, aminopterin and thymidine (HAT) medium and selected for culture for more than 2 weeks.

[0135] To identify candidate antibodies specific for Dectin-1, hybridoma supernatants were screened by flow cytometry on cells overexpressing Dectin-1 and human primary monocytes. Cynomolgus monkey Dectin-1 cross-reactivity was assessed by antibody binding to cynomolgus monkey primary monocytes using flow cytometry.

[0136] Healthy donor samples

[0137] Fresh healthy donor buffy coats were obtained from Stanford Blood Center. Peripheral blood mononuclear cells were separated and cryopreserved in Bambanker cell freezing medium (Bulldog Bio, Portsmouth, NH) via ficoll paque (GE Healthcare, Chicago, IL). Briefly, the buffy coat was diluted in phosphate buffered saline (1: 1 ratio), and the diluted buffy coat was then layered in ficoll and centrifuged at 760g. Before downstream analysis, the PBMC layer was separated and washed in PBS. Peripheral blood leukocytes were separated by erythrocyte lysis. Cryopreserved cynomolgus monkey PBMCs were obtained from Human Cells Biosciences.

[0138] Primary cells and cell culture

[0139] According to the manufacturer's instructions of pan-monocyte separation kit (Miltenyi Biotec company, Auburn, CA), human monocytes were isolated from healthy donor PBMC. For the differentiation of macrophages and dendritic cells, monocytes were cultured for 6 days in the presence of 50ng / ml MCSF (Peprotech, Rocky Hill, NJ) in RPMI containing 10 human serum (Millipore Sigma) to be completely differentiated into macrophages, or cultured for 6 days in the presence of 50ng / ml GMCSF and 50ng / ml IL-4 (Peprotech, Rocky Hill, NJ) to be completely differentiated into dendritic cells. The culture medium containing cytokines was changed every 3 days.

[0140] HEK Blue hDectin-1-a cells 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 recommendations (Thermo Fisher, Waltham, MA). Briefly, viable cell density and percent viability were determined. Cells were diluted to 11 × 10 6 4 viable cells / mL. Dilute the Freestyle Max reagent with OptiPro SFM medium, mix, and incubate at room temperature for 5 minutes. Add the diluted Freestyle Max reagent to the plasmid DNA, diluted with OptiPro SFM medium and mix. Incubate the Freestyle Max reagent / plasmid DNA complex at room temperature for 10-20 minutes. Slowly transfer the complex to the cells, gently swirling the flask during the addition, and then incubate the cells in an incubator at 37°C with 80% relative humidity and 8% CO2 on an orbital shaker.

[0141] Binding of Dectin-1 Antibodies to Dectin-1-Expressing Cells

[0142] Dectin-1-expressing cells (HEK Blue hDectin-1-a, HEK Blue hDectin-1-b, HEK293FhDectin-1a FL, human monocytes, or cynomolgus monkey monocytes) were plated at 1 × 10 cells per well. 5 -2×10 5 Cells were plated in non-tissue culture treated 96-well V-bottom plates. In addition, human monocytes were incubated in human FcgR blocking antibodies (Biolegend, San Diego, CA) for 10 minutes at room temperature to reduce the binding of antibodies to Fc receptors. The cells were then stained on ice for 30 minutes with eFluor 506 viability dye (ThermoFisher, Waltham, MA) at a dilution of 1:1000, followed by a wash step in FACS buffer (PBS containing 2% fetal bovine serum). Primary Dectin-1 antibodies or isotypes were used at titers of 300nM, 100nM, 33.3nM, 11.1nM, 3.7nM, 1.23nM, 0.41nM, and 0.14nM and incubated on ice for 30 minutes, followed by another wash step in FACS buffer.

[0143] To detect mouse primary antibodies, cells were incubated with fluorescently labeled AF647 anti-mouse Fc-specific secondary antibodies (Jackson Immuno). To detect human IgG4 primary antibodies, cells were incubated on ice for 30 minutes with Alexa Fluor 647 anti-human Fc-specific secondary antibodies (Jackson Immuno) (detection in HEK cells) or FITC anti-human IgG4 antibodies (Sigma) (detection in primary monocytes). Data were collected using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA) and analyzed using Graphpad Prism 8.4.

[0144] Dectin-1 antibody blocks laminarin

[0145] HEK Blue hDectin-1a cells were cultured at 1 × 10 5 Cells were plated in non-tissue culture treated 96-well V-bottom plates. Primary anti-Dectin-1 antibodies were used at titers of 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.14 nM, 0.05 nM, 0.015 nM, and 0.005 nM and incubated on ice for 30 minutes in the presence of 8 μg / ml biotin-laminarin. After a wash step in FACS buffer, binding of biotin-laminarin on HEK cells was detected using streptavidin-AF647 on ice for 30 minutes. For analysis, 4000 cell events were collected in a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA) and analyzed using Graphpad Prism 8.4.

[0146] Labeling polystyrene beads with pHrodo and conjugating with antibodies

[0147] Polystyrene beads of different sizes coated with goat anti-mouse IgG (or biotin) (Spherotech, Lake Forest, IL) were washed twice with PBS / Tween20 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 with shaking for 60 minutes. The beads were then washed with PBS / BSA 0.1% to remove excess pHrodo red.

[0148] After pHrodo labeling, antibodies were conjugated to the beads according to the manufacturer's recommendations. Briefly, a 5-fold excess of antibody was added to the beads, depending on the binding capacity of the beads to the antibody, and incubated for 60 minutes at room temperature with shaking. 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 evaluated by flow cytometry in low pH buffer. Antibody binding to the beads was assessed using fluorescently labeled AF647 anti-mouse Fc specific or FITC anti-human IgG4 antibody secondary antibodies.

[0149] Antibody-dependent targeted phagocytosis of phrodo-labeled beads

[0150] For phagocytosis experiments, 50,000 HEK cells or primary cells (macrophages or dendritic cells) overexpressing Dectin-1 were seeded in 96-well plates in RPMI containing ultra-low IgG FBS at 10 μl. 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 to beads, and the plates were briefly spun.

[0151] In some experiments, the cell tracking agent Calcein AM (Thermo Fisher, Waltham, MA) was added to label cells. Phagocytosis was monitored in an IncuCyte S3 real-time imaging system (Germany) by taking images at the desired time points 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.

[0152] SEAP reporter gene assay in HEK cells overexpressing Dectin-1 using anti-Dectin-1 antibody

[0153] Anti-Dectin-1 monoclonal antibody 2M24 (VH and VL domains contain SEQ ID NO: 7 and 8, respectively) or 15E2 and control isotypes were immobilized by coating on the well surface of untreated 96-well U-bottom polypropylene microtiter plates. For coating, 10 μg, 2 μg, 1 μg, 0.5 μg and 0.1 μg of anti-Dectin-1 antibody diluted in 50 μl sterile PBS were added to each well. The plate was placed in a Class II laminar flow cabinet overnight with the lid opened to allow the solution to evaporate. The coated plate was washed twice with 200 μl sterile PBS to remove salt crystals and unbound antibodies. HEK Blue hDectin-1-a cells were then cultured on culture plates in RPM1 containing 10% ultra-low IgG FBS (VWR) for 22 hours, and alkaline phosphatase levels in the supernatant were assessed at OD 630nm using QUANTI Blue solution (Invivogen, San Diego, CA) according to the manufacturer's instructions.

[0154] To determine the SEAP secretion of HEK cells induced by anti-Dectin-1 antibody-conjugated beads, 3 μm, 10 μm, and 16 μm sized biotinylated polystyrene beads (Spherotech, Lake Forest, IL) were conjugated with antibodies by incubating them with streptavidin-2M24 (hIgG4) for 30 min at room temperature and then washing twice with PBS to remove unbound antibodies. 5 HEK Blue hDectin-1-a cells were mixed at a 1:3 cell:bead ratio in RPM1 containing 10% ultra-low IgG FBS for 22 hours, and alkaline phosphatase secretion in the supernatant was assessed at OD 630nm as described above.

[0155] Cytokine secretion

[0156] Anti-Dectin-1 monoclonal antibody 2M24 or 15E2 clone and control isotype are fixed by coating 10ug onto the well surface of untreated 96-well U-shaped bottom polypropylene microtiter plates, as described above. Freshly isolated monocytes or peripheral blood mononuclear cells are then cultured at 200,000 cells / well for 24 hours on a plate with immobilized antibodies in RPM1 containing 10% ultra-low IgG FBS. In other wells, cells are treated with 10μg / ml of Dectin-1 antibody solution instead of immobilized antibodies. TNFa, IL-6 and IFNg in the supernatant are assessed using the U-PLEX assay platform (Meso Scale Discovery), and their levels are expressed as the fold change of Dectin-1 antibody-induced cytokine secretion relative to isotype controls. As a positive control, cells are stimulated with 25μg / ml yeast polysan.

[0157] result

[0158] To generate Dectin-1 antibodies, four-week-old ATX-Gx Alloy transgenic mice were subcutaneously immunized with recombinant Dectin-1 isoform B protein and boosted with antigen once a week. The antibodies generated by this immunization have human variable domains and mouse constant domains.

[0159] Among the 56 candidate anti-Dectin-1 antibody clones generated in this study, clone 2M24 was the only one that showed binding to both Dectin-1 isoforms A and B in HEK cells and monocytes. Figure 1A As shown, the 2M24 anti-Dectin-1 antibody clone exhibits high affinity for human monocytes expressing Dectin-1. In contrast, other clones bind only to Dectin-1 isoform A (e.g., 2M08, 2M12, 2M38) or not at all (2M49). In addition, the affinity of 2M24 for Dectin-1 is superior to that exhibited by other clones and commercial Dectin-1 antibodies (15E2, 259931, GE2). Figure 1C Comparison of binding to human monocytes and HEK cells overexpressing Dectin-1 between the 2M24 clone and other Dectin-1 clones identified from immunization of Alloy transgenic mice and commercial Dectin-1 clones is shown.

[0160] The cross-reactivity of the 2M24 antibody to cynomolgus monkey Dectin-1 was also evaluated. Binding was assessed by flow cytometric analysis of cynomolgus monkey monocytes derived from PBMC. Figure 1BAs shown, the anti-human Dectin-1 clone 2M24 antibody showed cross-reactivity and high affinity to cynomolgus monkey Dectin-1 expressed on monocytes. The 2M24 anti-Dectin-1 antibody outperformed the commercial antibodies tested in terms of affinity, showing an EC50 of 0.3 nM. The agonistic 15E2 and 255931 commercial antibodies showed EC50s of 14 nM and 16 nM, respectively, in cynomolgus monkey monocytes. Figure 1C Comparison of binding to cynomolgus monkey monocytes between the 2M24 clone and the commercial clones 15E2 and 259931 is shown.

[0161] To evaluate the function of 2M24 Dectin-1 antibody in promoting phagocytosis, polystyrene beads were coated with 2M24 antibody and mixed with HEK-Blue hDectin-1a cells or primary human monocytes. 2M24 antibody effectively induced phagocytosis of the beads. Figure 2A-2B As shown, 2M24 anti-Dectin-1 antibody coupled to polystyrene beads promoted phagocytosis in HEK-Blue hDectin-1a cells and human primary monocytes.

[0162] From the mIgG1 2M24 clone, a fully human 2M24 antibody of the IgG4 isotype was developed. This antibody has human constant and variable regions. The hIgG4 2M24 was then evaluated for binding to two Dectin-1 expressing cell types, HEK-Blue hDectin-1a cells and human monocytes. Figure 3A-3B As shown, 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).

[0163] Next, the ability of the hIgG4 2M24 antibody to promote bead phagocytosis in cells expressing Dectin-1 was tested. Figure 4 As shown, hIgG4 2M24 antibody exhibited efficient phagocytosis in HEK cells, human monocytes and human macrophages overexpressing Dectin-1. Therefore, fully human IgG4 2M24 antibody can promote the phagocytosis of cells expressing Dectin-1.

[0164] The fully human 2M24 (hIgG4) anti-Dectin-1 antibody was also tested for its ability to promote Dectin-1 signaling. Activation of Dectin-1 signaling by the antibody can be assessed by a secreted alkaline phosphatase assay using HEK-Blue hDectin-1a cells. HEK-Blue hDectin-1a cells are engineered to respond to stimulation with the Dectin-1 ligand by expressing Dectin-1 isoform A and genes involved in the Dectin-1 / NF-κB / SEAP signaling pathway, thereby expressing secreted alkaline phosphatase (SEAP). Figure 5A-5B As shown, 2M24 (hIgG4) anti-Dectin-1 antibodies in immobilized form and in a form conjugated to beads induce alkaline phosphatase secretion in HEK-Blue hDectin-1a cells. These observations support the view that the 2M24 (hIgG4) antibody promotes SEAP secretion by engaging Dectin-1 on the cell surface, indicating receptor aggregation and the agonistic activity of the antibody. In addition, beads conjugated to 2M24 (hIgG4) can promote effective aggregation signaling of Dectin-1. Larger beads can better induce signal transduction, reflecting better aggregation of receptors. This supports that the aggregation of Dectin-1 promoted by a bispecific antibody comprising an anti-Dectin-1 antibody targeting phagocytes and an antibody targeting another cell (e.g., cancer cells) can promote the aggregation and signal transduction of Dectin-1 on phagocytes.

[0165] The natural ligand of Dectin-1 causes the receptor to cluster downstream of Dectin-1 / Syk / NFkB and signaling to induce inflammatory gene expression. To evaluate whether the engagement of Dectin-1 antibody in solution would trigger cytokine secretion, monocytes or macrophages were treated with 10ug / ml of commercial anti-Dectin-1 antibody. Figure 6A-6B As shown, the 15E2 commercial anti-Dectin-1 antibody did not induce cytokine secretion in primary human macrophages and monocytes, indicating insufficient Dectin-1 receptor aggregation. This data confirms that free Dectin-1 antibody in solution does not induce immune stimulation due to the lack of sufficient Dectin-1 aggregation.

[0166] To evaluate whether the 2M24 (hIgG4) anti-Dectin-1 antibody can induce cytokine secretion, the antibody was immobilized on beads and incubated with monocytes or PBMCs. Figure 7A-7BAs shown, the 2M24 anti-Dectin-1 antibody induces cytokine secretion in primary human monocytes and PBMCs. Compared with the cytokine secretion promoted by the 15E2 anti-Dectin-1 agonistic antibody, the 2M24 antibody not only promotes cytokine secretion, but also exhibits a better immunostimulatory effect. Among the cytokines measured in this experiment, TNFa and IL6 are secreted by monocytes expressing Dectin-1. In contrast, IFNg is mainly secreted by T cells present in PBMCs. Since T cells do not express Dectin-1, they are not directly activated by anti-Dectin-1 antibodies, but are activated by cytokines secreted by monocytes in PBMCs stimulated with Dectin-1 antibodies. Therefore, the differential effect of Dectin-1 antibodies on IFNg is more prominent in PBMCs than in pure monocytes.

[0167] Finally, activation of Dectin-1 by natural ligands in the presence of anti-Dectin-1 antibodies was tested. HEK-BluehDectin-1a cells were incubated with 2M24 (hIgG4) Dectin-1 antibody or 15E2, 259931, GE2 anti-Dectin-1 commercial antibodies starting at 300 nM in a 1 / 3 continuous dose titration in the presence of 8 ug / ml biotinylated laminarin. Figure 8 As shown, binding of 2M24 (hIgG4) antibody to Dectin-1 does not block binding of laminarin, the natural ligand of Dectin-1. Therefore, engagement of Dectin-1 with 2M24 anti-Dectin-1 antibody does not prevent pathogen clearance and is unlikely to increase susceptibility to potential fungal infections.

[0168] In conclusion, the 2M24 anti-Dectin-1 antibody can induce phagocytosis of cells expressing Dectin-1 and can induce activation of Dectin-1 signaling without competing with the natural ligands of Dectin-1. The properties of the 2M24 and 15E2 antibodies are summarized in Fig. 9 middle.

[0169] Example 2: Bispecific anti-Dectin-1 antibody

[0170] This example describes the generation and characterization of a bispecific antibody comprising a Dectin-1 binding arm and a second arm that binds a specific tumor antigen.

[0171] Materials and methods

[0172] Generation of bispecific antibodies

[0173] Antibodies were differentially labeled with MTA or FOL reagents according to the manufacturer's guidelines (AAT Bioquest). Labeled antibodies were mixed and incubated to allow covalent assembly via MTA and FOL interactions. The following antibodies were used for biotin:streptavidin-induced bispecific antibodies:

[0174] Anti-Dectin-1 15E2 antibody heavy chain:mSA fusion

[0175] QWQLQQSGAELARPGASWKMSCKASGYTFTTYTMHWWKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAWYYCARERAVLVPYAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH(SEQID NO:18)

[0176] Anti-Dectin-1 15E2 Antibody Light Chain

[0177] QIVLTQSPAVMSASPGEKWTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVPTRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:19)

[0178] Avi-tagged anti-CD20 Fab heavy chain (CH1 domain based on hIgG4 sequence)

[0179] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE (SEQ ID NO:20) Anti-CD20 Fab light chain

[0180] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO:21)

[0181] Avi-tagged anti-HER2 Fab heavy chain (CH1 domain based on hIgG4 sequence)

[0182] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE (SEQ ID NO:22)

[0183] Anti-HER2 Fab light chain

[0184] DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:23)

[0185] Cell coupling assay

[0186] Cells expressing Dectin-1 are labeled with calcein green, and target cells are labeled with calcein red. Cells are incubated in the presence of bispecific antibodies or isotype control antibodies and then analyzed by flow cytometry. The coupling of cells is indicated by double positive signals (green + red +). The coupling efficiency is quantified as the percentage of cells that form doublets with cells expressing Dectin-1 relative to total target cells.

[0187] Five million effector cells (cells expressing Dectin-1) or target cells (cells expressing the target of interest, such as CD20-positive Raji cells or HER2-positive SKBR3 cells) were differentially labeled with calcein green (0.5 nM) or calcein red / pHrodo-red (0.5 nM). Cells were washed thoroughly with PBS and kept on ice. Effector and target cells were then co-cultured at a 3:1 ratio (effector cells: target cells) in the presence of 2M24 bispecific antibody or isotype control and incubated at 37°C for 30 minutes. 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. Coupling efficiency is 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.

[0188] SEAP reporter gene assay in HEK cells overexpressing Dectin-1 using anti-Dectin-1 antibody

[0189] To determine the SEAP secretion induced by Raji cells (expressing CD20), Raji cells were coated with 2M24 / anti-hCD20 or hIgG4 / anti-CD20 bispecific antibodies on ice for 30 min and then washed twice with PBS to remove unbound bispecific antibodies. 5HEK Blue hDectin-1-a cells were mixed at a 1:2 ratio (HEK cells: Raji cells) in RPM1 containing 10% ultra low IgG FBS. After 22 hours, alkaline phosphatase secretion in the supernatant was assessed at OD 630nm as described in Example 2.

[0190] result

[0191] Dectin-1 agonist bispecific antibodies can exploit multiple modes of activity (e.g., immune activation, phagocytosis, neoantigen presentation, and adaptive immune activation) to target and deplete cancer cells ( Fig.11 As a proof of concept for conjugating a Dectin-1 antibody (15E2 or 2M24) and a target antibody, a click chemistry approach was used to develop bispecific antibodies comprising an anti-Dectin-1 targeting arm and a second arm targeting a protein of interest. This approach was able to generate bispecific antibodies for a variety of assays. A schematic diagram of this approach is shown in Figures 10A-10B Since the binding of Dectin-1-specific antibodies to Dectin-1 can induce phagocytosis of targets (see Examples 1 and 2), the ability of bispecific antibodies to promote phagocytosis of specific target cells was evaluated. First, the ability of bispecific antibodies to eliminate cancer cells expressing CD70 by phagocytosis was evaluated. CD70 is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) superfamily. CD70 is expressed at low levels in normal tissues but is highly overexpressed in various diseases including acute myeloid leukemia (AML), renal cell carcinoma, rheumatoid arthritis, and lupus.

[0192] Using click chemistry, a bispecific molecule containing a Dectin-1 targeting arm (anti-Dectin-1; clone 2M24) and a CD70 targeting arm (anti-hCD70; clone 113-16) was generated. The purity of the bispecific (2M24 / anti-hCD70) antibody was assessed by SDS-PAGE analysis ( Fig. 12A ), while binding was assessed by flow cytometry analysis ( Fig. 12B ).like Fig. 12B As shown, binding studies on cells showed that 2M24 / anti-hCD70 bound to the HEK293 cell line expressing Dectin-1 with an EC50 of 1.8 nM and bound to the CD70-positive renal cancer cell line with an EC50 of 12.34 nM (A498 cells) or 11.62 nM (786-0 cells). The ability of the bispecific antibodies to induce cell binding was then evaluated. Fig.13As shown, the 2M24 / anti-hCD70 bispecific antibody induced the coupling of HEK293 cells expressing Dectin-1 and renal cancer cells expressing CD70, generating cell doublets of HEK293 cells (labeled with calcein green) and A498 cells (labeled with calcein red).

[0193] Next, we evaluated bispecific antibodies targeting cells expressing CD20. CD20 is a transmembrane protein that is present on nearly all B cells from the stage of B cell development until it is downregulated during differentiation into antibody-secreting plasma cells and is considered a pan-B cell antigen marker. Figures 14A-14B As shown, 2M24 / anti-hCD20 bispecific antibodies induce the coupling of Dectin-1-expressing cells (DECtin-1-expressing HEK293 cells and human M0 macrophages) to CD20-expressing B cells (Raji cell line). This cell-cell coupling mediated by the bispecific antibody may induce synapse formation between effector cells and target cells, which may alter cytokine signaling, activate phagocytosis and ultimately target antigen presentation.

[0194] To test the induction of signaling caused by stimulation with the bispecific antibody binding to Dectin-1, a secreted alkaline phosphatase assay was performed. Fig.15 As shown, Raji cells coated with anti-Dectin-1 / anti-CD20 bispecific antibodies induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells. Therefore, using bispecific antibodies to connect target cells to Dectin-1 expressing cells (such as phagocytes) can promote signaling by Dectin-1 expressing cells. In the case of phagocytes, signaling may lead to cytokine production and immune stimulation.

[0195] Previously, it has been demonstrated that expression of Dectin-1 in HEK 293 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, a bispecific antibody comprising a Dectin-1 targeting arm and a CD20 targeting arm was developed. In a co-culture assay of HEK 293 cells and CD20-expressing Raji cells, phagocytosis was observed in cells treated with the anti-Dectin-1 / anti-hCD20 bispecific antibody compared to the isotype control bispecific antibody ( Fig.16). In addition, preincubation of cells with erythromycin A, a phagocytosis inhibitor that blocks actin polymerization, blocked phagocytosis of cells treated with the anti-Dectin-1 / anti-hCD20 bispecific antibody. These findings suggest that expression of Dectin-1 is sufficient to induce phagocytosis and that co-targeting of Dectin-1 and a target of interest using a Dectin-1 agonistic bispecific antibody is sufficient to induce phagocytosis of target cells.

[0196] Proof-of-concept experiments were performed using an anti-Dectin-1 / anti-HER2 bispecific antibody to co-target Dectin-1 expressing cells and HER2 positive breast cancer cells. Approximately 20% to 25% of invasive breast cancers show overexpression of the human epidermal growth factor receptor HER2 tyrosine kinase receptor. Fig.17 As shown, the anti-Dectin-1 (15E2) / anti-HER2 bispecific antibody induces the coupling of cells expressing Dectin-1 and HER2. This interaction is thought to promote synapse formation between effector cells and target cells, because Dectin-1 aggregation induces cytokine secretion by effector cells, triggers phagocytosis of target cells, and causes neoantigen presentation and activation of adaptive immune cells (B cells and T cells).

[0197] Finally, an anti-Dectin-1 (2M24) / anti-hCD94 bispecific antibody was also evaluated. Large granular lymphocytic (LGL) leukemia is a rare chronic lymphoproliferative disease of the T-cell and natural killer (NK) cell lineages. CD94 / NKG2 is a family of C-type lectin receptors that are primarily expressed on the surface of NK cells and CD8+ T lymphocyte subsets. Fig.18 As shown, the anti-Dectin-1 (2M24) / anti-hCD94 bispecific antibody induced the coupling of Dectin-1-expressing cells and CD94-expressing cells. Therefore, the bispecific antibody binding to Dectin-1 can mediate the coupling of Dectin-1-expressing cells with a variety of target cells.

[0198] Example 3: Generation of bispecific anti-Dectin-1 antibodies using streptavidin-biotin

[0199] This example describes the biochemical and functional characterization of bispecific antibodies that bind Dectin-1, generated using streptavidin-biotin conjugation.

[0200] Materials and methods

[0201] Generation of bispecific antibodies

[0202] mSA is genetically fused to Fab 2M24 or full-length 2M24. The chimeric fusion is incubated with a biotinylated target antibody to generate a bispecific antibody comprising a Dectin-1 binding arm and a second arm that binds to a target receptor or protein of interest.

[0203] Full-length 2M24 sequence fused to mSA:

[0204] (SEQ ID NO:15)

[0205] Sequence of Fab 2M24 fused to mSA:

[0206] QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH(SEQ IDNO:17)

[0207] Antibody-dependent targeted phagocytosis of phrodo-labeled beads

[0208] 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 for 30 minutes on ice or at 37°C for 30 minutes, then washed twice with PBS. Phagocytosis was assessed by detecting activated Phrodo red in the HEK cell / bead pair population by flow cytometry in the PE channel using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA).

[0209] result

[0210] In order to enable efficient generation of bispecific antibodies, a new strategy was developed that exploits the high affinity interaction of streptavidin and biotin. Monomeric streptavidin (mSA) constructs were fused to the Fc domain of 2M24 or the CH1 domain of Fab 2M24. The recombinant fusion proteins were incubated with various biotinylated antibodies of interest to assemble bispecific antibodies. A schematic diagram of this strategy is shown in Figures 19A-19B middle.

[0211] This fusion technology enables high-throughput generation and screening of bispecific antibodies. To test this approach, Fab 2M24-mSA fusion protein was generated and purified. Figures 20A-20CAs shown, the Fab 2M24-mSA fusion exhibits high affinity binding to cells expressing Dectin-1 (EC50 = 1.45 nM). This Fab 2M24-mSA fusion protein can be combined with various biotinylated antibodies against targets of interest. In addition, the Fab 2M24-mSA fusion also induced binding and phagocytosis of beads by HEK 293 cells expressing Dectin-1 ( Figures 21A-21B ), indicating that the Fab format of the 2M24 antibody can effectively promote phagocytosis in cells expressing Dectin-1.

[0212] Using anti-Dectin-1-streptavidin fusion proteins, bispecific antibodies against various targets (e.g., CD20, CD19, CD70, amyloid B (1-42)) have been developed. Figures 22A-22D As shown, these bispecific antibodies showed high homogeneity based on HPLC analysis. These data demonstrate the robust feasibility of this technology for the generation of bispecific antibodies.

[0213] Next, the ability of the anti-Dectin-1 bispecific antibody generated using the Fab 2M24-mSA fusion protein to induce cell binding was evaluated. Fig.23 As shown, Fab 2M24-mSA / biotin anti-hCD20 bispecific antibody induces the coupling of HEK293 cells expressing Dectin-1 with B cells (Raji cell line) expressing CD20. This interaction can promote Dectin-1 aggregation, thereby inducing cytokine secretion from effector cells, triggering phagocytosis of target cells, and causing new antigen presentation and activation of adaptive immune cells (B cells and T cells).

[0214] Example 4: Bispecific design for development of human bispecific antibodies targeting Dectin-1 and disease targets or antigens

[0215] In order to be able to assemble and efficiently produce highly purified and active bispecific antibodies, design principles based on previously reported strategies were adopted, including "knobs into sockets" (Ridgway, 1996; patent US8679785B2), DuetMab (Mazor, 2015; patent EP3452089A2), single-step protein A and G affinity purification methods (Ollier, 2019; AU2018204314B2), and mutations that eliminate FcR binding (patent WO 2016 / 081746 A2). The assembly of a complete bispecific antibody involves the expression of four separate subunits, for example, cloned into an expression vector such as pFUSE. A diagram of an exemplary anti-Dectin-1 bispecific antibody is shown in Fig.24A middle.

[0216] As shown in Table 1, bispecific antibodies using this design were constructed for proof-of-concept studies. One arm of these bispecific antibodies targets hDectin-1 and the second arm targets hCD20. The bispecific antibodies described in Table 1 were generated by expressing all four chains and purified to 95% purity and homogeneity. All bispecific antibodies were found to bind to their respective targets.

[0217] Table 1. Bispecific antibodies targeting human Dectin-1 and antigens expressed on cancer cells / disease targets.

[0218]

[0219] The variable domains of the antibody arms corresponding to anti-Dectin-1 in Table 1 are as follows.

[0220] CD20 VH:

[0221] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSS LTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA(SEQ ID NO:24)

[0222] CD20 VL:

[0223] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSP KPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQ QWTSNPPTFGGGTKLEIK(SEQ ID NO:25)

[0224] These hDectin-1 bispecific antibodies bind to three targets: Dectin-1 on myeloid cells, CD20 on target cells or pathogens, and Fc receptors on myeloid cells and NK cells, triggering strong immune stimulation and phagocytosis ( Fig. 24B). In particular, bispecific antibodies with non-fucosylated, active hIgG1 Fc domains allow the bispecific antibodies to recruit myeloid cells (e.g., monocytes, macrophages, and dendritic cells) and natural killer (NK) cells to eliminate pathogenic target cells, such as tumor cells expressing specific antigens. In the context of cancer and without wishing to be bound by theory, dual engagement of myeloid cells and NK cells by Dectin-1 and Fcγ receptors is thought to elicit a strong immune response that ultimately eliminates cancer cells via the following actions: (1) bispecific antibody-induced cross-linking of Dectin-1 with Fcγ receptors leads to ITAM-dependent activation of downstream inflammatory pathways and release of immunomodulatory cytokines and cytotoxic proteins (proteases, perforins), thereby modulating the tumor microenvironment and potentially killing targeted cells directly; (2) bispecific antibody-induced aggregation of Dectin-1 and Fcγ receptors triggers phagocytosis and elimination of targeted cancer cells by monocytes, macrophages, and dendritic cells; and (3) macrophages and DCs present the engulfed antigens, a process that triggers a T cell immune response aimed at eliminating cancer cells.

[0225] The 2M24 / CD20 bispecific antibodies described in Table 1 were tested for binding to cells expressing human Dectin-1 or CD20. 2M24 / RSV was used as an isotype control for the target binding arm in all assays. The bispecific variants tested here contained mutations in the hIgG1 Fc domain (hIgG1 inert) that eliminated the binding of Fc to Fc receptors (according to EU numbering L234A, L235E and G237A). The binding of 2M24 / CD20 or 2M24 / RSV bispecific antibodies to HEK293 cells stably expressing human Dectin-1 was assessed by flow cytometry ( Fig.25A ). The 2M24 / CD20 hIgG1 inert bispecific antibody was able to bind to cells expressing human Dectin-1 with similar affinity (cell-based binding EC50 values ​​were 1.4 and 1.7 nM, respectively). Therefore, the 2M24 / CD20 bispecific antibody showed high affinity binding to HEK293 cells expressing Dectin-1.

[0226] Binding of rituximab, 2M24 / CD20 or 2M24 / RSV hIgG1 active or inert bispecific antibodies was also evaluated using the CD20-expressing B-cell lymphoma Raji cell line ( Fig.25B). The 2M24 / CD20 bispecific (active or inert hIgG1 isotype) antibody was able to bind to Raji cells expressing CD20, but with at least a 10-fold reduction in affinity compared to rituximab. Without wishing to be bound by theory, it is believed that the difference in CD20 binding affinity between the 2M24 / CD20 bispecific antibody and rituximab may be mediated by a loss of avidity in the bispecific antibody (monovalent vs. bivalent binding).

[0227] Next, the ability of the 2M24 / CD20 bispecific antibody to induce coupling of cells expressing hDectin-1 with cells expressing hCD20 was determined. HEK293 cells expressing Dectin-1 (effector cells) and Raji cells expressing CD20 (target cells) were differentially labeled with calcein green (effector cells) or calcein red (target cells) dyes. The labeled cells were co-cultured and treated with hIgG1 inert 2M24 / CD20 or 2M24 / RSV (control) bispecific antibodies to induce effector cell: target cell coupling. Double positive staining (calcein green +, calcein red +, squares; Fig.26A ) indicates successful coupling of effector cells:target cells. Coupling efficiency (the percentage of cells bound or coupled to effector cells relative to total target cells) was determined using dose titration of the bispecific antibody in co-cultures of effector cells:target cells ( Fig.26B ).

[0228] These results indicate that the 2M24 / CD20 bispecific antibody can couple Dectin-1-expressing "effector" cells and CD20-expressing "target" cells with an effective EC50 of 0.17 nM. Although the 2M24 / CD20 bispecific antibody has a low affinity for binding to CD20 on Raji cells ( Fig.25B ), but the coupling efficiency of 2M24 / CD20 is very high. These findings suggest that high expression of Dectin-1 or CD20 on effector cells and target cells enhances 2M24 / CD20 binding affinity (avidity), thereby promoting efficient coupling of the two cells. Based on these findings, it is believed that the 2M24 / CD20 bispecific antibody can effectively engage monocytes, macrophages or dendritic cells expressing Dectin-1 with target disease cells (e.g., B cell lymphomas expressing high levels of CD20). Effector cell: target cell engagement is the first step of the 2M24 bispecific antibody MOA.

[0229] The human IgG1 active isotype binds to Fcγ receptors on NK cells or monocytes. Therefore, it was evaluated whether the hIgG1 active isotype of 2M24 / CD20 can trigger NK cells (via antibody-dependent cellular cytotoxicity, ADCC) or other monocytes (fratricidal or antibody-dependent cellular phagocytosis, ADCP) to kill monocytes. In this case, the active hIgG1 domain of 2M24 / CD20 engages with Fcγ receptors on NK cells or monocytes and Dectin-1 receptors on monocytes, thereby inducing Fcγ-mediated target activation and depletion. From two healthy donors - donor 76 ( Fig.27A ) and donor 77( Fig.27B ) were treated with increasing concentrations of 2M24 / CD20 bispecific antibody (hIgG1 active or inert isotype) and rituximab for 24 hours and subsequently analyzed by flow cytometry to quantify the remaining live CD14+ monocyte levels (as a percentage of isotype control). No reduction in the number of monocytes was found in either donor, indicating that 2M24 / CD20 active IgG1 did not induce monocyte depletion. Without wishing to be bound by theory, it is believed that 2M24 / CD20 hIgG1 (active isotype) should not affect monocyte levels, so the risk of infection is minimal.

[0230] Based on the proposed MOA of 2M24 / CD20 bispecific antibody ( Fig. 24B ), B cell depletion by 2M24 / CD20hIgG1 (active isotype) bispecific antibody or rituximab was evaluated to compare B cell depletion. Fig.28A ) and 84( Fig.28B ) were treated with increasing concentrations of the indicated antibodies for 24 h and subsequently analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as the percentage of B cells in isotype control-treated PBMCs). Thus, in two healthy donors, high concentrations of the 2M24 / CD20 bispecific antibody induced better B cell depletion (about 80% reduction) compared with rituximab (about 40% reduction), despite the bivalent binding properties of rituximab and an approximately 10-fold difference in binding affinity (e.g., Fig.25B Involves binding to Dectin-1 on myeloid cells, Fcγ receptors on NK cells and monocytes, and CD20 on target B cells ( Fig. 24B The unique mechanism of action of 2M24 / CD20 active IgG1 of Dectin-1 induces B cell depletion that is superior to rituximab overall. These data support the concept that Dectin-1 induces immune stimulation through 2M24 / CD20 bispecific antibodies and enhances target cell depletion.

[0231] The ability of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody or rituximab (hIgG1) to downregulate CD19 expression on B cells in a process called trimming or endocytosis was evaluated. CD19 expression in B cells from two healthy donors - Donor 83 ( Fig.29A ) and donor 84( Fig.29B ) on B cells. The effect of 2M24 / CD20 bispecific antibody and rituximab on CD19 expression on B cells was assessed using the mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugated). In PBMCs from donor 83, the EC50 for CD19 expression was 0.014 nM for rituximab and 0.080 nM for 2M24 / CD20 hIgG1 bispecific antibody ( Fig.29A 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 antibody ( Fig.29B ). Both 2M24 / CD20 active IgG1 bispecific antibody and rituximab caused downregulation of CD19 expression on B cells. Interestingly, rituximab had at least five times higher trimming potential than 2M24 / CD20 bispecific antibody. Downregulation of the target CD20 on B cells has previously been reported as a mechanism for malignant B cells to escape rituximab-mediated depletion (Beum, PV et al. (2006) J. Immunol. 176: 2600-2609). Therefore, these findings suggest that 2M24 / CD20 active IgG1 bispecific antibody may be superior to rituximab in depleting B cells due to reduced trimming potential.

[0232] Compared with rituximab, immune stimulation triggered by the 2M24 / CD20 active IgG1 bispecific antibody resulted in the secretion of a unique cytokine pool ( Fig.30). ELISA (mesoscale discovery)-based cytokine quantification was performed in supernatants isolated from healthy donor PBMCs treated with 2M24 / CD20 active hIgG1 bispecific antibody, rituximab, or isotype control. PBMCs were stimulated with antibodies overnight 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 show that 2M24 / CD20 active IgG1 triggers higher levels and different cytokine activation in PBMCs compared to rituximab. In addition, binding of the 2M24 / RSV bispecific antibody to Dectin-1 and Fc receptors alone did not induce cytokine release, ruling out the possibility of systemic cytokine activation. These findings highlight a unique MOA that distinguishes the 2M24 / CD20 active IgG1 bispecific antibody from rituximab. Without wishing to be bound by theory, these findings further suggest that 2M24 / CD20 can trigger the release of Th1 and Th2 type responses and promote immune stimulation of the tumor microenvironment.

[0233] It was also found that in co-cultures of human macrophages and GFP-expressing Raji B cells, 2M24 / CD20 hIgG1 (active isotype) bispecific antibody induced better B cell depletion and lower CD19 trimming compared to rituximab. Co-cultures of human macrophages and Raji-GFP cells (3:1 ratio) in the presence of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, 2M24 / RSV control, fucosylated rituximab, or isotype hIgG1 control were analyzed by flow cytometry ( Fig.31A The co-cultures were incubated at 37°C for 24 h and then stained with PE a-CD206 Ab to label macrophages and BV-605a-CD19 Ab to label Raji cells. The number of remaining live / Raji-GFP+ cells was assessed at the end of the experiment. Primary antibodies were used for serial dose titration. Assessment of CD19 on Raji-GFP cells after 24 h ( Fig.31B), where the B cell receptor is shown as a decrease in CD19 MFI in the presence of anti-Dectin-1 / anti-hCD20 bispecific antibodies or rituximab. The EC50 for CD19 expression is 0.020 nM for rituximab and 0.95 nM for the 2M24 / CD20 hIgG1 bispecific antibody. These results indicate that the 2M24 / CD20 bispecific antibody enhances B cell depletion (Fcγ receptor mediated) compared to rituximab. Rituximab is more effective in reducing B cell receptor CD19 surface levels than the anti-Dectin-1 / anti-hCD20 bispecific antibody. Similarly, rituximab was observed to trim the B cell receptor for CD20, and the reduction of CD20 limits the effectiveness of rituximab to deplete B cells. Without wishing to be bound by theory, it is believed that these data indicate that the advantage of the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody to deplete B cells is due to less trimming of the B cell receptor compared to rituximab. This highlights the differential mechanism of cell depletion by the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody.

[0234] B cell depletion was also analyzed in single cell suspensions from renal cancer tissue biopsies. Single cell suspensions from two renal cancer tissue biopsies were analyzed by flow cytometry in the presence of 2M24 / CD20 hIgG1 (active or inert) bispecific antibodies, 2M24 / RSV hIgG1 control, fucosylated rituximab, and corresponding isotype controls. Renal cancer tissue biopsies were dissociated into single cell suspensions and treated with primary antibodies (2 μg / ml) for 24 hours at 37°C. Immune cell populations were analyzed by flow cytometry ( Fig.32A and 32B The number of remaining live B cells was assessed using anti-CD19 antibody and expressed as a percentage relative to the CD45+ immune cell population ( Fig.32C ). The 2M24 / CD20 active IgG1 bispecific antibody induced better tissue B cell depletion in single-cell suspensions of renal cancer biopsies than rituximab. The 2M24 / CD20hIgG1 (active isotype) bispecific antibody reduced B cells by 44% and 46% (respectively) in two renal cancer donor biopsies, whereas rituximab induced a 33% and 18% reduction in B cells, respectively ( Fig.32C ). The data support that the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody depletes the function of cells in cancer tissues via Dectin-1-induced immune stimulation and Fcγ receptor engagement. Without wishing to be bound by theory, it is believed that since Dectin-1 is primarily expressed on tumor-associated macrophages (TAMs) in the above biopsies, the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody may engage with TAMs to enhance target cell depletion.

[0235] Cytokine secretion was tested in cultured macrophages and single cell suspensions of renal cancer biopsies stimulated with immobilized anti-Dectin-1 antibody (clone 2M24) or 2M24 / CD20 bispecific antibody. Human monocyte-derived macrophages ( Fig.33A and 33B ) or single cell suspensions from renal cancer biopsies ( Fig.33C ). The cells were cultured for 24 hours and the assessment of TNFα secretion in the supernatant was evaluated by ELISA. As a positive control, the cells were stimulated with zymosan. The anti-Dectin1 antibody (clone 2M24) was found to induce Dectin-1 aggregation 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 as well as in single-cell homogenates of cancer biopsies. Since Dectin-1 is expressed in myeloid cells, tumor-associated macrophages in cancer biopsies are expected to produce cytokines in response to anti-Dectin-1 antibody stimulation. This promotes the transition of tumor-associated macrophages from anti-inflammatory to proinflammatory, with a strong anti-tumor effect. In addition, the monovalent binding of the 2M24 / CD20 bispecific antibody to Dectin-1 is sufficient to promote Dectin-1 aggregation and immune stimulation of macrophages.

[0236] Immunostimulation by immobilized anti-Dectin-1 antibodies in single cell suspensions from renal cancer biopsies was also analyzed ( Fig.34 ). Single cell suspensions from renal cancer biopsies were treated with immobilized anti-Dectin-1 antibodies (clone 2M24) or isotype control hIgG4 antibodies for 24 hours. The release of various cytokines in the supernatant was analyzed by ELISA, including IFNγ, IL-6, TNFα, IL-23, IL-12p70, IL-10 and IL-13. These results indicate that activation of Dectin-1 on bone marrow cells (in this example, Dectin-1 is primarily expressed by tumor-associated macrophages TAMs) triggers the release of a specific cytokine pool located directly downstream of the Dectin-1 signaling pathway, or indirectly by activating other immune cells. Without wishing to be bound by theory, it is believed that the engagement of the 2M24 bispecific antibody with Dectin-1 promotes immune stimulation and can modulate the tumor microenvironment to support the elimination of cancer cells expressing the target.

[0237] Example 5: Characterization of bispecific antibodies targeting Dectin-1 and CD20

[0238] This example describes further characterization of a bispecific antibody targeting human Dectin-1 and human CD20. The anti-Dectin-1 arm includes the variable domain of 2M24, and the anti-CD20 arm includes the variable domain of rituximab (see SEQ ID Nos: 24 and 25 for the VH and VL domains, respectively).

[0239] Materials and methods

[0240] CD16 expression on NK cells

[0241] Human PBMCs from healthy donors were treated with serial dilutions of 2M24 / CD20 hIgG1 KIF, rituximab KIF, and isotype control RSV hIgG1 KIF antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. CD16 expression on CD56+ NK cells was quantified and compared to expression levels in the isotype control treated group.

[0242] CD19 expression on B cells

[0243] Human PBMCs from healthy donors were treated with 0.1 nM 2M24 / CD20 hIgG1 KIF, Rituximab KIF, and isotype control RSV hIgG1 KIF antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. CD19 expression on B cells was quantified (MFI).

[0244] B cell depletion in PBMCs

[0245] Human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. Fig.37 The dashed line in the figure indicates quantification of B cells.

[0246] B cell depletion in renal cancer biopsies

[0247] Single cell suspensions were generated from renal cancer biopsies and cells were treated with 2M24 / CD20 hIgG1, 2M24 / RSV hIgG1, rituximab hIgG1, and isotype control RSV hIgG1 antibodies. After 24 hours of treatment, cells were stained with antibodies against lineage-specific markers for flow cytometric analysis. B cells were quantified as the percentage of CD19+ cells in the CD45+ immune cell population.

[0248] result

[0249] First, the effect of the 2M24 / CD20 bispecific antibody on CD16 expression was examined in human NK cells. CD16 is required for the ADCC activity of NK cells, so the loss of CD16 expression reduces the cytotoxic potential of NK cells. Compared with 2M24 / CD20hIgG1 KIF, rituximab induced efficient and strong shedding of CD16 on NK cells ( Fig.35 ). In contrast, CD16 levels on NK cells were better maintained after 2M24 / CD20 bispecific antibody treatment compared to rituximab treatment. Without wishing to be bound by theory, it is believed that 2M24 / CD20 bispecific antibodies have the potential to better preserve the cytotoxic potential of NK cells.

[0250] Next, the effect of the 2M24 / CD20 bispecific antibody on CD19 expression was examined in human B cells. Preservation of target antigen expression is critical 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 via trimming / shedding after binding to anti-CD19 antibodies. With CD20-targeting antibodies, a bystander effect was observed, where CD19 expression was reduced after treatment with rituximab, but not with treatment with the 2M24 / CD20hIgG1 KIF bispecific antibody ( Fig.36 ). Compared with rituximab, 2M24 / CD20 bispecific antibody can better maintain CD19 levels on B cells. Without wishing to be bound by theory, it is believed that in therapy, 2M24 / CD20 bispecific antibody may exhibit prolonged activity due to minimal effect on target antigen expression.

[0251] To compare rituximab with the anti-CD20 antibody obinutuzumab, the 2M24 bispecific antibody against CD20 was generated using the variable domain sequences from rituximab or obinutuzumab. The obinutuzumab variable domain sequences are as follows. VH: QVQLVQSGAE VK KPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFP GDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCA RNVFDGYWLVYWGQGTLVTVSS (SEQ ID NO: 46); VL: DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQL LIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQN LELPYTFGGGTKVEIK (SEQ ID NO: 47).

[0252] In ADCC / ADCP assays, 2M24 / CD20 (derived from the rituximab sequence) demonstrated nearly complete depletion of B cells, superior to 2M24 / CD20 (derived from obinutuzumab) or the parental bivalent antibody and isotype control ( Fig.37 ). These data support the development of 2M24 / CD20 bispecific antibodies using the rituximab sequence.

[0253] Example 6: Characterization of bispecific antibodies targeting Dectin-1 and CD20 in exploratory studies in non-human primates

[0254] This example describes the results of an exploratory study of the safety and efficacy of the bispecific antibody targeting human Dectin-1 and human CD20 described in Example 9 in cynomolgus monkeys.

[0255] Materials and methods

[0256] Three groups of cynomolgus monkeys (1 male and 1 female per group) were treated with a single dose (5 mg / kg) of the test articles: A) 2M24 / CD20 hIgG1 KIF; B) 2M24 / CD20 hIgG1 Inert; and C) Rituximab hIgG1 KIF. Blood was collected at the indicated time points. Abbreviations of the test articles (2M24 / CD20 KIF, 2M24 / CD20 Inert, RTX KIF).

[0257] B cell levels were assessed by flow cytometry. Depletion was quantified by the number of CD19+B cells remaining in the post-dose sample compared to the level before the test article was administered. 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 post-dose sample (Day 7) compared to the level before the test article was administered (Day -7).

[0258] For PBMC assays, PBMCs from healthy cynomolgus monkeys were treated with serial dilutions of 2M24 / CD20 hIgG1 KIF, rituximab KIF, and isotype control RSV hIgG1 KIF antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. B cell depletion was quantified relative to isotype controls.

[0259] result

[0260] This exploratory study was designed to examine the safety and efficacy of the 2M24 / CD20 bispecific antibody in nonhuman primates. Fig.38As shown. Cynomolgus monkeys were divided into three treatment groups, each containing 2 animals (1 male, 1 female). Each group was administered a single dose of 5 mg / kg with a specific test article. The test articles included: 1) 2M24 / CD20 hIgG1KIF; 2) 2M24 / CD20 hIgG1 inert; and 3) rituximab hIgG1 KIF. Animals were monitored daily and samples such as whole blood, bone marrow, lymph nodes, and colorectal tissue were collected as indicated. The study was planned to last for 8 weeks.

[0261] like Fig.39 As shown in (top), the 2M24 / CD20 hIgG1 KIF bispecific antibody depletes B cells in cynomolgus monkeys. Almost complete and sustained B cell depletion (approximately 98%) was observed in two animals treated with a single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF. Fig.40 ), one animal showed complete depletion, while a second animal showed strong but incomplete depletion (approximately 87%). Partial B cell depletion was observed for one animal in the 2M24 / CD20 hIgG1 inert group ( Fig.39 ), while the second animal showed no depletion on day 7. The 2M24 / CD20 hIgG1 KIF bispecific antibody was well tolerated in cynomolgus monkeys.

[0262] The 2M24 / CD20 hIgG1 KIF bispecific antibody also depleted bone marrow in cynomolgus monkeys ( Fig.41A ) and lymph nodes ( Fig.41B ) B cells. A single dose (5 mg / kg) of 2M24 / CD20hIgG1 KIF induced strong B cell depletion in the bone marrow (approximately 87-88%) and partial B cell depletion in the lymph nodes (60-78%) in both animals. In the rituximab group, B cell depletion was also observed in both tissues. Partial B cell depletion was observed in the 2M24 / CD20 hIgG1 inert group, with the exception of animal CB764A, which had minimal B cell depletion in the lymph nodes.

[0263] The 2M24 / CD20 hIgG1 KIF bispecific antibody also induced a strong depletion of cynomolgus monkey B cells in vitro ( Fig.42 ). 2M24 / CD20 hIgG1KIF induced a strong depletion of B cells compared to rituximab hIgG1 KIF. The maximum depletion achieved by rituximab was about 30% of B cells, while the 2M24 / CD20 hIgG1 KIF bispecific antibody exhibited a maximum depletion of about 50%.

[0264] Example 7: Purification and functional characterization of 2M24 / CD20 bispecific antibody in scFv format

[0265] 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 a scFv fused to a human IgG1 Fc domain with a knob-forming mutation and the CD20 targeting arm is based on rituximab hIgG1 with a hole-forming mutation. Fig.43 As shown. The knob-forming mutation on the Dectin-1 targeting arm is T366W; the hole-forming mutations on the CD20 targeting arm are T366S, L368A, and Y407V. Without wishing to be bound by theory, it is believed that this format provides a general platform for generating anti-Dectin-1 bispecific antibodies with simpler manufacturing requirements (e.g., compared to bispecific antibodies with anti-Dectin-1 arms containing multiple polypeptide chains).

[0266] 2M24 scFv / CD20 hIgG1 was expressed in Hek293 cells by transfection of three plasmids (2M24 scFv hIgG1 plasmid, CD20 heavy chain and CD20 light chain). The supernatant was harvested after four days of expression and purified via protein A. Aggregates were removed by size exclusion chromatography. Fig.44A As shown, the 2M24 scFv / CD20 hIgG1 bispecific antibody was purified as a homogeneous molecule on SEC.

[0267] Next, co-cultures of CD20-expressing Raji cells and Dectin-1-expressing HEK reporter assays were treated with increasing concentrations of the 2M24 scFv / CD20 hIgG1 bispecific antibody. Activation of the reporter gene was assessed by measuring SEAP levels in the culture medium (based on absorbance at 630 nm). As assessed by the NFkB reporter assay, the bispecific molecule promoted targeted immune stimulation ( Fig.44B ).

[0268] To examine B cell depletion, human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. Fig.44C The results showed that the 2M24scFv / CD20hIgG1 bispecific antibody, similar to the 2M24 / CD20 hIgG1 KIF molecule, was able to deplete human B cells ( Fig.44C ).

[0269] Example 8: Characterization of B cell depletion by 2M24×CD20 bispecific binding protein

[0270] Comparison of the anti-Dectin-1 single chain variable fragment (scFv) fused to a conventional anti-CD20 antibody arm with hIgG1 Fc (see Fig.43 The non-fucosylated 2M24 scFv / CD20 bispecific binding protein prepared by combining the anti-CD20 antibody rituximab and the bispecific anti-CD3 / anti-CD20 T cell engager can deplete B cells in PBMCs of healthy donors. The non-fucosylated 2M24 scFv / CD20 bispecific binding protein comprises: a first polypeptide chain, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31; a second polypeptide chain, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32; and a third polypeptide chain, the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33.

[0271] PBMCs were isolated from the buffy coat of healthy donors by Ficoll separation.PBMCs were suspended in ADCC culture medium (RPMI 1640, 10% heat-inactivated FBS, 1x penicillin / streptomycin, 1x non-essential amino acids) in a round-bottomed ultra-low adhesion 96-well plate at a density of 500,000 cells in 50uL.Test articles were prepared with 2X working solution in ADCC culture medium and 3 times of serial dilution were performed.50uL test articles were added to cells.The reactants were gently suspended and incubated at 37°C for 24 hours.Cells were collected, treated with human Fc blocking agents and live / dead dyes, and then stained for the following markers (CD45, CD3, CD16, CD14, CD56 and CD19).B cells were detected according to phenotype CD45+CD3-CD14-CD56-CD19+.B cell levels were expressed as the percentage of CD19+ cells in the treated group relative to the CD19+ cells in the untreated control group.

[0272] like Fig.45 As shown, the non-fucosylated scFv 2M24×CD20 bispecific binding molecule is superior to rituximab and CD3×CD20 T cell engagers in depleting B cells. These data indicate that bispecific antibodies targeting Dectin-1 on myeloid cells and CD20 on B cells have strong efficacy in depleting B cells compared to rituximab or T cell engagers. These findings suggest that engaging myeloid cells via Dectin-1 is an attractive strategy for cancer immunotherapy.

[0273] Next, B cell depletion was tested in prostate cancer tumor biopsies.

[0274] Single cell suspensions were generated from prostate cancer biopsies using a tumor dissociation kit from Miltenyi Biotec. Flow cytometry confirmed the expression of CD20 on CD45+CD3-CD14-CD56-CD19+ cells and Dectin-1 on CD45+CD3-CD19-CD11b+CD163+ tumor-associated macrophages. Cells were suspended in ADCC medium (RPMI 1640, 10% heat-inactivated FBS, 1x penicillin / streptomycin, 1x non-essential amino acids) at a density of 500,000 cells in 100uL in a round-bottom ultra-low adhesion 96-well plate. Test articles were prepared at a 3X concentration of 30ug / mL in ADCC medium and 50uL was added to the cells (final concentration of 10ug / mL). The reaction was gently suspended and incubated at 37°C for 24 hours. Cells were collected, treated with human Fc blocking agent and live / dead dye, and then stained for the following markers (CD45, CD3, CD16, CD14, CD11b, and CD19). B cells were detected according to the phenotype CD45+CD3-CD14-CD56-CD19+. B cell levels were expressed as the percentage of CD19+ cells in the treatment group relative to the CD19+ cells in the control group (RSV hIgG1).

[0275] like Fig.46 As shown, the non-fucosylated scFv 2M24×CD20 bispecific binding molecule is superior to rituximab in depleting B cells. These data indicate that 2M24×CD20 can effectively deplete CD20-expressing B cells present in solid tumor biopsies by engaging with tumor-associated macrophages expressing Dectin-1. In addition, the 2M24×CD20 bispecific antibody containing scFv and non-fucosylated format is superior to rituximab or the traditional IgG2M24×CD20 format (DuetMab). Taken together, these observations confirm that engaging bone marrow cells via Dectin-1 is an attractive cancer immunotherapy strategy and that the use of scFv and non-fucosylated modifications can enhance efficacy.

[0276] Example 9: Comparison of 2M24×CD20 bispecific binding protein and rituximab

[0277] The 2M24×CD20 bispecific binding protein was compared to rituximab with respect to functional properties of interest, including depletion of target cells.

[0278] The effect of 2M24×CD20 bispecific binding protein on CD16 expression on NK cells was examined. Human PBMCs from healthy donors were treated with serial dilutions of 2M24×CD20 bispecific hIgG1 binding protein treated with KIF, rituximab treated with KIF, or isotype control RSV hIgG1 antibody treated with KIF. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. CD16 expression on CD56+ NK cells was quantified and compared with expression levels in isotype control treated groups.

[0279] like Fig.47A As shown, rituximab induced potent and robust shedding of CD16 on NK cells compared to the 2M24×CD20 bispecific hIgG1 binding protein (both treated with KIF). NK cell ADCC activity requires CD16; therefore, loss of CD16 expression reduces the cytotoxic potential of NK cells. These results suggest that the 2M24×CD20 bispecific binding protein has the potential to better preserve NK cytotoxic potential compared to rituximab.

[0280] The effect of 2M24×CD20 bispecific binding protein on CD19 expression on B cells was examined. Human PBMCs from healthy donors were treated with 0.1 nM of 2M24×CD20 bispecific hIgG1 binding protein treated with KIF, rituximab treated with KIF, or isotype control RSV hIgG1 antibody treated with KIF. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. CD19 expression (MFI) on B cells was quantified.

[0281] Preservation of target antigen expression is critical for 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 via trimming / shedding after binding of anti-CD19 antibodies. Here, using antibodies targeting CD20, a bystander effect was observed, where CD19 expression was reduced after treatment with rituximab treated with KIF, but not with 2M24×CD20 bispecific hIgG1 binding protein treated with KIF ( Fig.47B ). Thus, the 2M24×CD20 bispecific antibody was better able to maintain CD19 levels on B cells than rituximab. These results suggest that, therapeutically, the 2M24×CD20 bispecific binding protein may exhibit prolonged activity due to minimal effects on target antigen expression.

[0282] The effect of 2M24×CD20 bispecific binding protein on B cell depletion was examined. Human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. Fig.47C B cells were quantified (indicated by dashed lines in the figure).

[0283] The 2M24 bispecific binding protein against CD20 was generated using variable domain sequences from either rituximab or obinutuzumab. In ADCC / ADCP assays, the 2M24 / CD20 bispecific antibody (anti-CD20 arm derived from rituximab sequence) demonstrated nearly complete depletion of B cells ( Fig.47C ). Thus, the 2M24×CD20 bispecific binding protein with the anti-CD20 arm from rituximab showed better B cell depletion than the format using the anti-CD20 binding arm from obinutuzumab. These data support the development of 2M24 / CD20 bispecific antibodies using the rituximab variable domain.

[0284] Renal cancer biopsies were also used to examine the effects of 2M24×CD20 bispecific binding proteins on B cell depletion. Single cell suspensions were generated from renal cancer biopsies and the cells were treated with 2M24 / CD20 bispecific hIgG1, 2M24 / RSV bispecific hIgG1, rituximab hIgG1, and isotype control RSV hIgG1 antibodies. After 24 hours of treatment, cells were stained with antibodies against lineage-specific markers for flow cytometric analysis. B cells were quantified as the percentage of CD19+ cells in the CD45+ immune cell population.

[0285] The results showed that the 2M24 / CD20 bispecific binding protein induced a substantial depletion of B cells in single-cell suspensions of primary renal tumors ( Fig.47D ). Dectin-1 expression on tumor-associated macrophages in primary renal biopsies was also confirmed. The 2M24 / CD20 bispecific antibody induced better B cell depletion compared with rituximab.

[0286] Example 10: Exploratory study of 2M24×CD20 bispecific binding protein in cynomolgus monkeys

[0287] This example describes the results of an exploratory study of the effects of the 2M24 / CD20 bispecific binding protein in cynomolgus monkeys. Fig.48As shown. Cynomolgus monkeys were divided into three treatment groups, each containing 2 animals (1 male, 1 female). Each group was administered a single dose of 5 mg / kg with a specific test article. The test articles included: 1) 2M24 / CD20hIgG1 KIF; 2) 2M24 / CD20 hIgG1 inert; and 3) rituximab hIgG1 KIF. Animals were monitored daily and samples such as whole blood, bone marrow, lymph nodes, and colorectal tissue were collected as indicated. The study was planned to last for 8 weeks. The time points for sample collection and other measurements are shown in Fig.48 middle.

[0288] Blood B cell levels were examined. Blood was 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 post-dose sample compared to the level before administration of the test article.

[0289] The results showed that 2M24 / CD20 bispecific hIgG1 KIF depleted B cells in vivo. Almost complete and sustained B cell depletion (approximately 98%) was observed in two animals treated with a single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF ( Fig.49A ; upper left). In the rituximab group, one animal showed complete depletion, while the second animal showed strong but incomplete depletion (approximately 87%) ( Fig.49A ; top right). Partial B cell depletion was observed in one animal in the 2M24 / CD20 hIgG1 inert group, while the second animal showed no depletion at day 7 ( Fig.49A ; lower left).

[0290] B cell depletion in the bone marrow and lymph nodes was also examined. 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 post-dose (Day 7) samples compared to the levels before administration of the test article (Day -7).

[0291] The results showed that 2M24 / CD20 bispecific hIgG1 KIF depleted bone marrow and lymph node B cells in vivo. A single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF induced strong B cell depletion in the bone marrow (approximately 87-88%) and partial B cell depletion in the lymph nodes (60-78%) in both animals. Fig.49B ). In the rituximab group, B cell depletion was also observed in both tissues. Partial B cell depletion was observed in the 2M24 / CD20 hIgG1 inert group, with the exception of animal CB764A, which had minimal B cell depletion in the lymph nodes.

[0292] Ex vivo B cell depletion was also examined. PBMCs from healthy cynomolgus monkeys were treated with serial dilutions of 2M24 / CD20 hIgG1KIF, rituximab KIF, and isotype control RSV hIgG1KIF antibodies. After 24 hours of treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometric analysis. B cell depletion was quantified relative to the isotype control group.

[0293] The results showed that 2M24 / CD20 bispecific hIgG1 KIF induced a strong depletion of B cells in cynomolgus monkeys in vitro. Compared with rituximab hIgG1 KIF, 2M24 / CD20 bispecific hIgG1 KIF induced a strong depletion of B cells ( Fig.49C ). The maximum depletion achieved by rituximab was about 30% of B cells, while the 2M24 / CD20 hIgG1 KIF bispecific antibody exhibited a maximum depletion of about 50%.

[0294] Example 11: Replacement study of anti-Dectin-1×CD20 bispecific binding protein in mice

[0295] This example describes the results of an alternative study conducted in mice on the effects of the 2A11 anti-mouse Dectin-1 / mCD20 bispecific binding protein. Fig.50A As shown. Mice were treated with a single dose (10 mg / kg) of bispecific 2A11 / mCd20mIgG1 binding protein, bivalent anti-mCd20 rat IgG2a antibody, or mIgG1 isotype control. Animals were sacrificed on day 8 and tissues including blood, peritoneum, bone marrow, and spleen were harvested for flow cytometry analysis. Single cell suspensions were stained with antibodies against lineage-specific markers. B cells were quantified based on Cd19 expression and reported as a percentage of total Cd45 cells.

[0296] like Fig.50B As shown, the surrogate 2A11 / mCd20 bispecific antibody induced a significant depletion of B cells in the blood and tissues of healthy wild-type mice.

[0297] Example 12: In vitro testing of the efficacy of the 2M24×CD20 bispecific binding protein

[0298] The properties of the 2M24 / CD20 bispecific binding protein were analyzed in vitro. The ability of the 2M24 / CD20 bispecific binding protein to activate the Dectin-1 pathway in the presence of CD20-expressing B-cell lymphoma lines was examined. B-cell lymphoma lines with different CD20 expression levels were treated with serial dilutions of the indicated antibodies in the presence of a reporter HEK cell line expressing Dectin-1. After 24 hours of treatment, supernatants were collected to test Dectin-1-induced NF-kB-driven secreted embryonic alkaline phosphatase (SEAP) activity. D-zymosan, a known ligand for Dectin-1, was used as a positive control.

[0299] The results showed that the 2M24 / CD20 bispecific binding protein induced Dectin-1 pathway activation only in the presence of CD20-expressing B cells. Fig.51A As shown, the 2M24 / CD20 bispecific binding protein showed dose-dependent activation of NF-kB in the presence of B lymphoma lines with CD20 expression levels ranging from 9,000 copies (Sc-1) to 370,000 copies (SU-DHL-6). However, the 2M24 / CD20 bispecific binding protein failed to activate NF-kB reporter cells in a CD20-null B lymphoma line (NALM-6) or in the absence of any target cells. D-zymosan showed activation of the Dectin-1 pathway in reporter HEK cells regardless of the presence or absence of any target cells. Together, these data suggest that Dectin-1 clustering and downstream NF-kB pathway activation require simultaneous Dectin-1 binding and engagement of CD20 (as low as 9,000 copies) on target cells.

[0300] The ability of the 2M24 / CD20 bispecific binding protein to induce phagocytosis was also tested. In the presence of macrophages derived from healthy human donors, B cell lymphoma lines with different CD20 expression levels were treated with serially diluted antibodies. After 24 hours of treatment, B cell depletion was assessed relative to the control group treated with RSV (non-specific).

[0301] The results showed that the 2M24 / CD20 bispecific binding protein induced more potent depletion of B-cell lymphoma cell lines than rituximab in the presence of macrophages. Fig.51B As shown, in the presence of macrophages derived from healthy human donors, treatment with 2M24 / CD20 bispecific binding protein resulted in a dose-dependent increase in the level of depletion of B lymphoma lines. Compared to rituximab, the highest concentration of 2M24 / CD20 bispecific binding protein showed more effective or complete depletion of B lymphoma lines in the presence of CD20 expression ranging from 9,000 to 370,000 receptors.

[0302] The ability of 2M24 / CD20 bispecific binding protein to deplete human primary B cells was also tested. Human PBMCs from n=6 healthy donors were treated with serially diluted 2M24 / CD20 bispecific binding protein and comparative anti-CD20 antibodies (rituximab and anti-CD20 / anti-CD3 bispecific conjugates). 24 hours after treatment, PBMCs were stained with fluorophore-labeled antibodies for flow cytometric analysis. The degree of B cell depletion relative to untreated controls was assessed.

[0303] The results showed that the 2M24 / CD20 bispecific binding protein showed more effective depletion of primary human B cells compared to the anti-CD20 antibody rituximab or the anti-CD20 / anti-CD3 bispecific T cell engager. The 2M24 / CD20 bispecific binding protein showed a dose-dependent increase in the level of primary human B cell depletion, with an average EC50 of 0.16 ( Fig.51C ). In contrast, the other anti-CD20 antibodies tested, rituximab and CD20×CD3 bispecific binders induced B cell depletion with average EC50 values ​​of 0.14 and 0.17 nM, respectively. However, the 2M24 / CD20 bispecific binding protein showed about 91% depletion of primary B cells at the highest concentration, while rituximab and CD20×CD3 binders showed partial depletion of 46% and 47% of total B cells, respectively. Taken together, these data indicate that the 2M24 / CD20 bispecific binding protein exhibits more potent B cell depletion than rituximab and CD20×CD3 binders.

[0304] The ability of 2M24 / CD20 bispecific binding protein to stimulate in vitro cytokine secretion was also tested. Human PBMC from n=8 healthy donors were treated with serially diluted 2M24 / CD20 bispecific binding protein and comparative anti-CD20 antibodies. 24 hours after treatment, supernatants were collected and analyzed by multiple MSD cytokine assays to determine and compare 2M24 / CD20 bispecific binding protein and comparative anti-CD20 antibody induced cytokine secretion. The supernatant of PBMC samples treated with RSV hIgG1 (non-specific antibody) and untreated was used as a negative control.

[0305] The results showed that the 2M24 / CD20 bispecific binding protein showed low to moderate proinflammatory cytokine production. The 2M24 / CD20 bispecific binding protein showed dose-dependent induction of cytokine production after 24 hours of treatment with healthy donor PBMC. At the highest tested concentration of 1.67ug / ml, the 2M24 / CD20 bispecific binding protein showed significant induction of IL-6, TNFa, IFN-g and IL-2 ( Fig.51D and51E Specifically, the 2M24 / CD20 bispecific binding protein showed higher production of cytokines IL-6 and TNF-a compared to rituximab; however, the 2M24 / CD20 bispecific binding protein induced significantly lower production of IL-6, TNF-a, IFN-g, and IL-2 compared to treatment with the CD20×CD3 engager.

[0306] Example 13: Testing the efficacy of the 2M24×CD20 bispecific binding protein in an in vivo mouse model

[0307] The efficacy of alternative anti-mouse Dectin-1 antibodies with a mIgG2a Fc region was tested in a mouse in vivo model.

[0308] First, the anti-tumor activity of anti-mouse Dectin-1 / anti-CD20 (rituximab) mIgG2a antibodies was tested in several B-cell lymphoma xenograft models. The efficacy of alternative anti-mDectin-1 / anti-hCD20 mIgG2a antibodies was evaluated in the Ramos and Daudi xenograft models of SCID mice and the Raji xenograft model of SCID-beige mice. After the establishment of the corresponding tumors in SCID or SCID-beige mice, 4 doses of anti-mDectin-1 / anti-mCD20 mIgG2a were administered at an equivalent dose of 10 mg / kg on days 0, 4, 7, and 10 when the tumor volume reached the 60-100 mm3 range. Tumor progression was monitored by measuring tumor volume twice a week and compared with isotype mIgG2a treatment.

[0309] The results showed that the alternative anti-mDectin-1 / anti-mCD20 mIgG2a antibody showed strong anti-tumor efficacy in several B-cell lymphoma xenograft models. Four doses of 10 mg / kg of alternative anti-mDectin-1 / anti-mCD20 mIgG2a showed 90% tumor growth inhibition in the Ramos xenograft model of SCID mice, and complete tumor regression in the Daudi model of SCID mice (10 / 10) and the Raji model of SCID-beige mice (8 / 8). Fig.52A Complete regression of tumors in SCID mice and especially NK cell-deficient SCID-beige mice demonstrates the efficacy of anti-mDectin-1 / anti-mCD20 in myeloid cell activation and in ameliorating tumor growth in these mouse xenograft models.

[0310] Next, the ability of alternative anti-mDectin-1 / anti-mCD20 bispecific antibodies to induce myeloid cell activation was tested in an immunocompetent mouse model. The murine colon cancer cell line MC38 was modified to express human CD20 and 5×10^6 CD20-MC38 cells were implanted (sc) into C57BL / 6 mice. After tumor establishment, when the tumor volume reached 80-150mm 3 A single dose of anti-mDectin-1 / anti-mCD20 bispecific antibody was administered at an equivalent dose of 10 mg / kg and compared with similar doses of isotype mlgG1 treatment (n=10 per group). Eight days after treatment, spleens, lymph nodes (LNs), and tumors were analyzed by flow cytometry to assess myeloid cell activation.

[0311] The results showed that the anti-mDectin-1 / anti-mCD20 bispecific antibody induced the activation of dendritic cells in lymphoid organs (spleen and lymph nodes) and the polarization of macrophages in tumors. Fig.52B As shown, a single administration of 10 mg / kg anti-mDectin-1 / anti-mCD20 bispecific mIgG1 resulted in significantly higher dendritic cell expansion in LNs compared to the isotype-treated control group. In tumor tissue, anti-mDectin-1 / anti-mCD20 bispecific antibody-induced myeloid cell activation was observed through a significantly higher influx of monocytes in the tumor compared to the isotype-treated control, with improved polarization toward pro-inflammatory M1 macrophages.

[0312] Anti-mDectin-1 / anti-mCD20 bispecific antibody-induced T cell activation was also demonstrated in the MC38 xenograft model. After CD20-MC38 tumors were established in C57BL / 6 mice, T cell activation was induced in mice with a TV of 80-150 mm 3 A single dose of anti-mDectin-1 / anti-mCD20 bispecific antibody was administered at 10 mg / kg and compared with isotype mIgG1 and anti-CD20 mIgG1 treatment. Eight days after treatment, spleens and tumors were analyzed by flow cytometry to assess T cell activation.

[0313] The results showed that the anti-mDectin-1 / anti-mCD20 bispecific antibody caused activation of CD4+ and CD8+ T cells in the spleen, as well as intratumoral activation of cytotoxic CD8+ T cells in tumors. Fig.52C As shown, a single administration of 10 mg / kg anti-mDectin-1 / anti-mCD20 bispecific mIgG1 showed significantly higher T cell expansion in the spleen, as shown by staining for the proliferation marker Ki67. The significantly higher expansion of both T cell subsets was associated with increased IL-2 production, CD4 + Higher formation of effector T cells and CD8+ The anti-mDectin-1 / anti-mCD20 bispecific antibody induced significantly higher levels of T cells, especially CD8 + T cell influx ( Fig.52D In addition, CD8 + T cells showed robust granzyme B expression in mice induced by anti-mDectin-1 / anti-mCD20 bispecific antibodies, indicating cytotoxicity. In summary, anti-mDectin-1 / anti-mCD20 bispecific antibodies demonstrated robust T cell activation in lymphoid organs, as well as cytotoxic CD8 + Intratumoral infiltration of T lymphocytes.

[0314] Test the ability of anti-mDectin-1 / anti-mCD20 bispecific antibody to deplete B cells in untreated C57BL / 6 mouse lymphoid organs.HCD20 Tg mice (hCD20 expressed under mouse CD20 promoter) were treated with a single dose of anti-mDectin-1 / anti-mCD20 bispecific mIgG2a and isotype mIgG2a control.Mice were killed on the 7th, 14th and 28th days after treatment.The lymphoid organs and non-lymphoid organs indicated in the harvest were determined to determine the B cell depletion relative to the isotype treatment control.The B cell population is marked by the dual expression of CD19 and B220 and is identified by flow cytometry.

[0315] The results showed that the anti-mDectin-1 / anti-mCD20 bispecific antibody showed strong depletion of B cells in the periphery and lymphoid organs (spleen, lymph nodes and bone marrow). A single administration of 10 mg / kg anti-mDectin-1 / anti-mCD20 bispecific mIgG2a showed significant depletion of B cells in the periphery and lymphoid organs. On day 7 after treatment, approximately 90% B cell depletion was observed in the periphery and secondary lymphoid organs (spleen and lymph nodes). Fig.52E In the bone marrow, mature B cells (showing high CD20 expression) showed about 85% depletion at day 7 after treatment. Immature B cells with low CD20 expression showed greater resistance to cell depletion induced by anti-mDectin-1 / anti-mCD20 bispecific antibody (not shown). After a single dose of treatment, the recovery of B cells was slowly monitored over time.

[0316] The ability of anti-mDectin-1 / anti-mCD20 bispecific antibodies to deplete B cells in non-lymphoid organs of untreated C57BL / 6 mice was also tested. hCD20 Tg mice (hCD20 expressed under the mouse CD20 promoter) were treated as described above. The indicated non-lymphoid organs were harvested to determine B cell depletion relative to the isotype treated control. Non-lymphoid tissues were processed into single cell populations and enriched with immune cells. B cell populations were marked by the dual expression of CD19 and B220 and identified by flow cytometry.

[0317] The results showed that the anti-mDectin-1 / anti-mCD20 bispecific antibody showed strong depletion of B cells in the non-lymphoid tissues tested. A single administration of 10 mg / kg of anti-mDectin-1 / anti-mCD20 bispecific mIgG2a showed strong depletion of B cells in non-lymphoid organs ( Fig.52F ). At day 7 after treatment, almost complete depletion of B cells was observed in non-lymphoid organs (lungs, liver, brain and heart). B cell recovery was monitored and even at day 28 after a single dose, B cells recovered only to 50% of the isotype treated control.

[0318] The ability of anti-mDectin-1 / anti-mCD20 bispecific antibodies with an inert Fc region to inhibit tumor growth was tested. The efficacy of mouse surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies with mIgG2a activity and an inert Fc region was evaluated in the Daudi xenograft model in SCID mice. After tumors were established in SCID, when tumor volumes reached 60-100 mm 3 Four doses of anti-mDectin-1 / anti-mCD20 bispecific antibody were administered at an equivalent dose of 10 mg / kg on days 0, 4, 7, and 10 when the range was within 500 μg / kg. Tumor progression was monitored by measuring tumor volume twice weekly and compared to isotype mIgG2a-treated controls. Tumors from the anti-mDectin-1 / anti-mCD20 bispecific Fc-inert and isotype control groups were further evaluated for myeloid cell activation on day 14 after the initial dose; the anti-mDectin-1 / anti-mCD20 bispecific active group showed complete tumor regression and was therefore not included in the evaluation.

[0319] The results showed that the anti-mDectin-1 / anti-mCD20 bispecific antibody with an inert Fc still exhibited a strong tumor growth inhibition effect in SCID mice and induced an increased influx of immune cells in tumor tissue, highlighting the Dectin-1-dependent activity. Compared with the isotype mIgG2a control in the Daudi model, treatment with anti-mDectin-1 / anti-mCD20 bispecific mIgG2a (active) and Fc-inert induced 95% and 40% tumor growth regression and inhibition, respectively ( Figure 52G ). The data showed that targeting Dectin-1 alone was sufficient to achieve tumor growth inhibition; however, simultaneous engagement of Dectin-1 and the Fc receptor led to significant tumor elimination.

[0320] The ability of anti-mDectin-1 / anti-mCD20 bispecific antibodies with an inert Fc to reprogram myeloid cells was also tested. The efficacy of mice replacing the anti-mDectin-1 / anti-mCD20 bispecific mIgG2a activity and Fc inertness was evaluated in the Daudi xenograft model in SCID mice. After tumors were established in SCID, when tumor volumes reached 60-100 mm 3 Four doses of anti-mDectin-1 / anti-mCD20 bispecific antibody were administered at an equivalent dose of 10 mg / kg on days 0, 4, 7, and 10 when the range was within 500 μg / kg. Tumor progression was monitored by measuring tumor volume twice weekly and compared to isotype mIgG2a-treated controls. Tumors from the anti-mDectin-1 / anti-mCD20 bispecific Fc-inert and isotype control groups were further evaluated for myeloid cell activation on day 14 after the initial dose; the anti-mDectin-1 / anti-mCD20 bispecific active group showed complete tumor regression and was therefore not included in the evaluation.

[0321] Results showed that Dectin-1 stimulation induced by anti-mDectin-1 / anti-mCD20 bispecific Fc inert drives myeloid reprogramming in tumors, leading to higher monocyte recruitment and pro-inflammatory macrophage differentiation. Four doses of 10 mg / kg of anti-mDectin-1 / anti-mCD20 bispecific mIgG2a Fc inert administration demonstrated significant improvement in resident and inflammatory monocyte recruitment to tumor tissues compared to isotype-treated controls ( Fig.52H Although the frequency of intratumoral macrophages was lower in the anti-mDectin-1 / anti-mCD20 bispecific Fc-inert treatment group compared with the control, the macrophages showed better polarization toward the inflammatory M1 phenotype rather than the tumorigenic M2 phenotype, suggesting that they have antitumor efficacy.

[0322] Example 14: Testing the efficacy of the 2M24×CD20 bispecific binding protein in an in vivo cynomolgus monkey model

[0323] The efficacy of the 2M24 / CD20 bispecific binding protein was tested in an in vivo cynomolgus monkey model.

[0324] Analysis of B cell depletion caused by different doses of 2M24 / CD20 bispecific binding proteins. Four groups of untreated cynomolgus monkeys (n=2; 1 male and 1 female per group) were treated with three doses of vehicle, and 1mg / kg, 10mg / kg and 100mg / kg of 2M24 / CD20 bispecific binding proteins per week. According to the dosing regimen, samples were collected from the peripheral blood of the 1mg / kg and 10mg / kg groups at several time points to assess the presence of B cells by flow cytometry. Animals in the vehicle group and the 100mg / kg group were sacrificed on the 15th day, one day after the third dose. B cells are marked by the dual expression of HLA-DR and CD19.

[0325] The results showed that the 2M24 / CD20 bispecific binding protein induced strong B cell depletion in cynomolgus monkeys. All dose levels of the 2M24 / CD20 bispecific antibody showed strong B cell depletion in the peripheral blood of cynomolgus monkeys ( Fig.53A ). The 2M24 / CD20 bispecific antibody at the 1 mg / kg dose level showed complete B cell depletion after the first dose; peripheral B cells began to recover, with B cell levels reaching 15% of baseline values ​​at the end of the third dose and 75% of baseline by day 56. At the 10 mg / kg dose level, the 2M24 / CD20 bispecific antibody induced almost complete depletion after the first dose, which lasted until the third dose on day 15. After the third dose, B cell levels began to recover slowly, reaching approximately 15% of baseline by day 56. Finally, the 100 mg / kg dose caused complete B cell depletion, and this phenomenon persisted in all three doses.

[0326] B cell levels in lymphoid organs (bone marrow and lymph nodes) were also analyzed. Following the above dosing regimen, samples were collected from lymph nodes and bone marrow in the 1 mg / kg and 10 mg / kg groups at several time points to assess the presence of B cells by flow cytometry and to assess depletion compared to baseline levels of B cells.

[0327] The results showed that all dose levels of 2M24 / CD20 bispecific antibodies induced strong B cell depletion in the bone marrow and lymph nodes of cynomolgus monkeys. At the end of three doses per week, both 1mg / kg and 10mg / kg doses of 2M24 / CD20 bispecific antibodies showed about 50% depletion of B cells in the bone marrow and lymph nodes ( Fig.53B ). A 100 mg / kg dose of the 2M24 / CD20 bispecific antibody showed approximately 50% depletion of B cells in the bone marrow, but nearly 90% depletion of B cells in the lymph nodes.

[0328] B cell levels in lymphoid and non-lymphoid tissues were further analyzed. Two groups of untreated cynomolgus monkeys (n=2; 1 male and 1 female per group) were treated with three doses of vehicle and 100 mg / kg of 2M24 / CD20 bispecific antibody per week. According to the dosage regimen, on the 15th day, i.e., after the third dose, the animals were sacrificed and several lymphoid and non-lymphoid organs were harvested to assess the depletion of tissue B cells by flow cytometry. B cells were marked by the dual expression of HLA-DR and CD19.

[0329] The results showed that the 2M24 / CD20 bispecific binding protein induced strong B cell depletion in the periphery and lymphoid organs. Fig.53C As shown, 100mg / kg of 2M24 / CD20 bispecific antibodies showed almost complete depletion of B cells in peripheral blood and secondary lymphoid organs. Compared with the vehicle-treated control, the 2M24 / CD20 bispecific antibody showed that all B cells in the spleen, tertiary lymph nodes, and mesenteric lymph nodes were depleted by about 90%, 85%, and 74%, respectively; B cell depletion in the bone marrow was limited and confined to the mature B cell compartment (not shown). In non-lymphoid organs, compared with the vehicle-treated group, 100mg / kg of 2M24 / CD20 bispecific antibodies showed about 94%, 77%, 90%, 88%, and 95% B cell depletion in the brain, kidney, liver, lung, and heart, respectively. In summary, these data show that 100mg / kg of 2M24 / CD20 bispecific binding proteins have a strong effect and are able to reach the deep microenvironment of cynomolgus monkey lymphoid organs and non-lymphoid organs.

[0330] We also examined the depletion of B cell subsets in the spleen. Two groups of untreated cynomolgus monkeys (n=2; 1 male and 1 female per group) were treated with three doses of vehicle per week, as well as 100 mg / kg of 2M24 / CD20 bispecific antibody. Following the dosing regimen, animals were sacrificed on day 15, i.e., after the third dose, and spleens were harvested to assess the extent of depletion of different B cell subsets by flow cytometry. B cells are marked by dual expression of HLA-DR and CD19.

[0331] The results showed that 100mg / kg of 2M24 / CD20 bispecific antibody induced strong deep tissue depletion of naive B cells and activated B cell subsets in the spleen of cynomolgus monkeys. 100mg / kg of 2M24 / CD20 bispecific antibody showed almost complete depletion of naive B cells in the spleen. Compared with the vehicle-treated group, 2M24 / CD20 bispecific antibody showed 74% and 99% depletion of transitional and mature naive B cells, respectively; the transitional subpopulation was distinguished from the mature subpopulation by the expression of the CD10 marker ( Fig.53D and 53E). 2M24 / CD20 bispecific antibody completely depleted splenic naive B cells, which was reflected in naive follicles (CD21 + IgD hi ) B cell compartment, also in the marginal zone (CD21 hi IgM + ) B cell compartment. Compared with the vehicle-treated group, the 2M24 / CD20 bispecific antibody induced approximately 55% and 93% depletion in the activated B cell compartment and memory B cells; B cell depletion in the activated B cell compartment included germinal center B cells or pathogenic DN2 B cell populations (with CD21 - CD27 - CD11c + as a sign).

[0332] Peripheral myeloid cell activation was also examined and compared to that induced by rituximab-like anti-cynomolgus CD20 antibodies. Two groups of untreated cynomolgus monkeys (n=2; 1 male and 1 female per group) were treated with a single dose of 5 mg / kg of 2M24 / CD20 bispecific antibody (Fab-Fab format) and rituximab-like anti-CD20 antibody. Samples were collected from peripheral blood at several time points according to the dosing schedule to assess the dynamics of myeloid cells in the peripheral blood. In another experiment, two groups of untreated cynomolgus monkeys (n=2; 1 male and 1 female per group) were treated with three doses of 1 mg / kg and 10 mg / kg 2M24 / CD20 bispecific antibody (scFv-Fab format) per week. Samples were collected from peripheral blood of the 1 mg / kg and 10 mg / kg groups at several time points according to the dosing schedule to assess the dynamics of myeloid cells in the peripheral blood.

[0333] The results showed that the 2M24 / CD20 bispecific antibody exhibited robust expansion of circulating bone marrow cells. A single dose of the 2M24 / CD20 bispecific (Fab-Fab) antibody showed an increase in the number of bone marrow cells (HLA-DR + CD19 - ) expansion (not shown). Specifically, a subset of dendritic cells, classical dendritic cells 1 (cDC1, characterized by HLA-DR hi CD11c + CD1c - ) showed significant expansion in the circulation at both doses, with cDC1 cells reaching a peak at day 10 after the start of treatment; no expansion of myeloid cells and cDC1 populations was observed in the rituximab-like CD20-mAb-treated group ( Fig.53FSimilarly, three doses of 2M24 / CD20 bispecific antibody (scFv-Fab) at 1 mg / kg and 10 mg / kg showed a near-significant expansion of bone marrow cells, including the cDC1 cell population; cDC1 cells peaked on day 40 after the start of treatment ( Figure 53G Together, these data support the stimulation of myeloid cells and active expansion of the dendritic cell population cDC1 in the peripheral blood of cynomolgus monkeys mediated by the 2M24 / CD20 bispecific antibody (Fab-Fab and scFv-Fab formats).

[0334] The levels of pro-inflammatory cytokines in serum were also analyzed. Two groups of untreated cynomolgus monkeys (n=2; 1 male and 1 female in each group) were treated with three doses of 1 mg / kg and 10 mg / kg 2M24 / CD20 bispecific antibodies per week. According to the dosing schedule, serum samples were collected from peripheral blood at several time points to evaluate the pro-inflammatory cytokine response by MSD cytokine array.

[0335] The results showed that both doses of 2M24 / CD20 bispecific antibody showed moderate pro-inflammatory cytokine responses in cynomolgus monkeys. Fig.53H As shown, at 1 mg / kg and 10 mg / kg doses of 2M24 / CD20 bispecific antibodies, cytokine responses were moderate, and all 6 cytokines showed relatively stable levels from baseline to day 14. Specifically, a transient increase in MIP-1a was seen after treatment with 1 mg / kg and 10 mg / kg 2M24 / CD20 bispecific antibodies. However, no sustained increase in any proinflammatory cytokines was seen. No detectable levels of IL-1b and TNF-a were seen at baseline or after treatment with 2M24 / CD20 bispecific antibodies (not shown).

[0336] Two groups of untreated cynomolgus monkeys (n=2; 1 male and 1 female per group) were also treated with three weekly doses of 1 mg / kg and 100 mg / kg of the 2M24 / CD20 bispecific antibody, and cytokines in the serum were analyzed as described above. Fig.53I As shown, at the 100 mg / kg dose, the cytokine responses were all moderate, with all six cytokines showing relatively stable levels from baseline to day 14. IP-10 levels appeared to be most affected, with levels consistently above baseline at all time points, and peaking at approximately 560 pg / ml on day 7. MIP-1α levels also showed a sustained increase relative to baseline at all time points, reaching a high level of approximately 57 pg / ml on day 1. No detectable levels of IL-1b and TNF-a were seen at baseline or after 2M24 / CD20 bispecific antibody treatment (not shown).

[0337] Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. A multispecific binding protein having a first antigen binding domain that binds to human Dectin-1 and a second antigen binding domain that binds to human CD20, comprising: The first polypeptide chain,The first polypeptide chain comprises the amino acid sequence QVQLVQSGAEV KKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:31) or QVQLVQSGAEV KKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:35); A second polypeptide chain, said second polypeptide chain comprising the amino acid sequence QVQLQQPGAEL VKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:32) or QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:36); and The third polypeptide chain comprises the amino acid sequence QIVLSQSPAILS ASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

2. The multispecific binding protein of claim 1, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:31, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:32, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

3. The multispecific binding protein of claim 1, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:35, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:36, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

4. A multispecific binding protein having a first antigen binding domain that binds to human Dectin-1 and a second antigen binding domain that binds to human CD20, comprising: The first polypeptide chain,The first polypeptide chain comprises the amino acid sequence QVQLVQSGAEV KKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:37) or QVQLVQSGAEVK KPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:39); A second polypeptide chain, said second polypeptide chain comprising the amino acid sequence QVQLQQPGAEL VKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:38) or QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNM HWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:40); and The third polypeptide chain comprises the amino acid sequence QIVLSQSPAILS ASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

5. The multispecific binding protein of claim 4, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:37, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:38, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

6. The multispecific binding protein of claim 4, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:39, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:40, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

7. The multispecific binding protein of any one of claims 1-6, wherein at least one of the first polypeptide chain and the second polypeptide chain is non-fucosylated.

8. The multispecific binding protein of claim 7, wherein both the first polypeptide chain and the second polypeptide chain are non-fucosylated.

9. The multispecific binding protein of any one of claims 1-8, wherein the first antigen binding domain: (a) binds to human Dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells or granulocytes; (b) binds to human Dectin-1 expressed on the cell surface with an EC50 of less than 2 nM; (c) capable of binding to human or cynomolgus monkey Dectin-1; and / or (d) Does not compete with the natural ligand of human Dectin-1.

10. The multispecific binding protein of any one of claims 1-9, wherein the second antigen binding domain binds to human CD20 expressed on the surface of B cells.

11. A polynucleotide encoding the multispecific binding protein of any one of claims 1-10. A vector comprising the polynucleotide according to claim 11 .

13. An isolated host cell comprising the polynucleotide of claim 11 or the vector of claim 12.

14. The isolated host cell of claim 13, wherein the host cell is a yeast, insect, plant or prokaryotic cell.

15. The isolated host cell of claim 13, wherein the host cell is a mammalian cell.

16. The isolated host cell of claim 15, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

17. The isolated host cell of claim 15 or claim 16, wherein the host cell comprises an α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosamine transferase (MGAT1) knockout.

18. The isolated host cell of claim 15 or claim 16, wherein the host cell overexpresses β1,4-N-acetylglucosamine transferase III (GnT-III).

19. The isolated host cell of claim 18, wherein the host cell further overexpresses Golgi μ-mannosidase II (ManII).

20. A method of producing an antibody or a multispecific binding protein, the method comprising culturing the host cell of any one of claims 13-19 under conditions suitable for production of the multispecific binding protein.

21. The method of claim 20, further comprising recovering the antibody or multispecific binding protein.

22. The method of claim 20 or claim 21, wherein the host cell is treated with kifunine prior to producing the antibody or multispecific binding protein.

23. A multispecific binding protein produced by the method of any one of claims 20-22.

24. A pharmaceutical composition comprising the multispecific binding protein of any one of claims 1-10 and 23 and a pharmaceutically acceptable carrier.

25. A composition as described in claim 24, wherein the composition comprises a mixture of multispecific binding protein species, wherein each species includes a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:35, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:36, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:

33.

26. The composition of claim 24, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:31, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:32, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

27. The composition of claim 24, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:35, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:36, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

28. A composition as described in claim 24, wherein the composition comprises a mixture of multispecific binding protein species, wherein each species includes a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:37 or SEQ ID NO:39, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:40, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:

33.

29. The composition of claim 24, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:37, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:38, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

30. The composition of claim 24, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:39, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:40, and the third polypeptide chain comprises the amino acid sequence of SEQ ID NO:

33.

31. A method of treating a disease or disorder, the method comprising administering to an individual in need thereof an effective amount of the multispecific binding protein of any one of claims 1-10 and 23 or the composition of any one of claims 24-30.

32. The method of claim 31 , wherein the individual suffers from a B cell mediated disease or disorder.

33. The method of claim 32, wherein the B cell mediated disease or condition is cancer.

34. The method of claim 33, wherein the cancer is non-Hodgkin's lymphoma or chronic lymphocytic leukemia.

35. The method of claim 32, wherein the B cell-mediated disease or disorder is an autoimmune disease or disorder.

36. The method of claim 35, wherein the autoimmune disease or disorder is rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and Wegener's granulomatosis.

37. The method of any one of claims 31-36, wherein the individual is a human.

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