Multispecific binding proteins that bind dectin-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 related methods of use and production. The present disclosure relates to multispecific (e.g., bispecific) binding molecules that bind to human Dectin-1, and related methods of use and production. Disclosed herein are methods of targeted phagocytosis for removing disease-causing agents, including host cells / host cell products, microorganisms, or products thereof, by administering a multispecific (e.g., bispecific) binding molecule comprising a Dectin-1 binding arm and a second arm that specifically binds to the agent, e.g., CD20.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 327,281, filed April 4, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic format of the sequence listing (186542000840SEQLIST.xml, size: 33,783 bytes, created on March 30, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to multispecific (eg, bispecific) binding proteins that bind to human Dectin-1 and human CD20, and related methods of use and production. [Background technology]

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

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

[0006] It would be beneficial to develop targeted removal and degradation of accumulated disease-causing agents without increasing overall phagocytosis. The present disclosure provides a solution to this problem and describes other advantages.

[0007] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] The present disclosure relates to multispecific (e.g., bispecific) binding molecules that bind to human Dectin-1, and related methods of use and production. Disclosed herein is a method of targeted phagocytosis for removing disease-causing agents, including host cells / host cell products, microorganisms, or products thereof, by administering a multispecific (e.g., bispecific) binding molecule comprising a Dectin-1 binding arm and a second arm that specifically binds to the agent, e.g., CD20. The multispecific (e.g., bispecific) binding molecule allows phagocytes to engage the target agent and form a synapse between them, promoting the clustering of Dectin-1 on the phagocyte. This stimulates phagocytosis of the target agent while also stimulating cytokine secretion by the phagocyte via the Dectin-1 / Syk / NfkB pathway. Additionally, antigens of the ingested agent are presented on the surface of dendritic cells / macrophages, enhancing the adaptive immune response to the disease-causing agent. Overall, Dectin-1 agonist multispecific (e.g., bispecific) binding molecules are believed to promote immune stimulation, targeted phagocytosis, and neo-antigen presentation / activation of the adaptive immune system to eliminate disease-causing agents.

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

[0010] In some embodiments, the multispecific binding protein comprises:Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKV DIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31) or QVQ LVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGG SGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSD A first polypeptide chain comprising: KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35);Amino acid sequence: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32) or QVQ LQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL A second polypeptide chain comprising LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36);and a third polypeptide chain comprising the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33). 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 are compositions comprising a mixture of multispecific binding protein species, each of which 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.

[0011] In some embodiments, the multispecific binding protein comprises:Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKV DIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 37) or QVQ LVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGG SGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSD A first polypeptide chain comprising: KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39);Amino acid sequence: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38) or QVQ LQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40);and a third polypeptide chain comprising the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (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. In some embodiments, provided herein are compositions comprising a mixture of multispecific binding protein species, each of which 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.

[0012] In some embodiments, the first, second, and third polypeptide chains are assembled into a multispecific binding protein that comprises a first antigen binding domain that binds human Dectin-1 and a second antigen binding domain that binds human CD20.

[0013] In some embodiments, at least one of the first and second polypeptide chains is non-fucosylated. In some embodiments, both the first and second polypeptide chains 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 2 nM; is capable of binding to human or cyno 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.

[0014] In some embodiments, provided herein is a polynucleotide encoding a multispecific binding protein of any one of the above embodiments. In some embodiments, provided herein is a vector (e.g., an expression vector) comprising a polynucleotide of any one of the above embodiments.

[0015] In some embodiments, provided herein is a host cell (e.g., an isolated host cell or cell line) comprising any one of the polynucleotides or vectors 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 an α1,6-fucosyltransferase (Fut8) or α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1) knockout. In some embodiments, the host cell overexpresses β1,4-N-acetylglucosaminyltransferase III (GnT-III). In some embodiments, the host cell further overexpresses Golgi μ-mannosidase II (ManII).

[0016] In some embodiments, provided herein is a method for producing a multispecific binding protein, 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 production of the antibody or multispecific binding protein, the host cell is treated with kifunensine.

[0017] In some embodiments, provided herein is a pharmaceutical composition comprising a multispecific binding protein of any one of the above embodiments and a pharma- ceutically acceptable carrier. In some embodiments, the composition comprises a mixture of multispecific binding protein species, each of which 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. 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, the composition comprises a mixture of multispecific binding protein species, each species comprising 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.

[0018] In some embodiments, provided herein is a method of treating a disease or disorder comprising administering to an individual in need thereof an effective amount of any one of the multispecific binding proteins or compositions described in the above embodiments. In some embodiments, the disease or disorder is a B cell mediated disease or disorder. In some embodiments, the individual has or has been diagnosed with a B cell mediated disease or disorder. In some embodiments, the B cell mediated disease or disorder 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 disorder is an autoimmune disease or disorder. In some embodiments, the autoimmune disease or disorder is rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and Wegener's granulomatosis. In some embodiments, the individual is a human.

[0019] It is to be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the detailed description that follows. [Brief description of the drawings]

[0020] [Figure 1A] Figure 2 shows binding analysis of anti-human Dectin-1 antibody (clone 2M24) on human and monkey monocytes from peripheral blood mononuclear cells (PBMC) by flow cytometry. To identify monocytes, single live CD14+ cells were gated. Cells were incubated with 2M24 anti-Dectin-1 primary antibody or mIgG1 isotype control antibody followed by incubation with fluorescent anti-mouse secondary antibody. Primary antibodies were used in serial dose titrations. Figure 2 shows binding analysis of anti-human Dectin-1 clone 2M24 on human monocytes. [Figure 1B] Figure 1 shows binding analysis of anti-human Dectin-1 antibody (clone 2M24) on human and monkey monocytes from peripheral blood mononuclear cells (PBMC) by flow cytometry. To identify monocytes, single live CD14+ cells were gated. Cells were incubated with 2M24 anti-Dectin-1 primary antibody or mIgG1 isotype control antibody followed by incubation with fluorescent anti-mouse secondary antibody. Primary antibodies were used in serial dose titration. Figure 1 shows binding analysis of anti-human Dectin-1 clone 2M24 antibody on cynomolgus monkey monocytes. [Figure 1C] Figure 1 shows binding analysis of anti-human Dectin-1 antibody (clone 2M24) on human and monkey monocytes derived from peripheral blood mononuclear cells (PBMC) by flow cytometry. Single viable CD14+ cells were gated to identify monocytes. Cells were incubated with 2M24 anti-Dectin-1 primary antibody or mIgG1 isotype control antibody followed by incubation with fluorescent anti-mouse secondary antibody. Primary antibodies were used in serial dose titrations. Figure 1 shows a comparison of binding to human monocytes, HEK cells overexpressing human Dectin-1, and cynomolgus monocytes between the 2M24 clone and other Dectin-1 antibodies identified from immunization of ATX-Gx Alloy transgenic mice and a commercially available anti-Dectin-1 antibody. The anti-human Dectin-1 clone 2M24 antibody showed high affinity for human Dectin-1 and Cynomolgus Dectin-1 expressed on monocytes, with superior affinity compared to other anti-Dectin-1 antibodies, including the commercially available antibody.

[0021] [Figure 2A]Phagocytosis of pHrodo-labeled polystyrene anti-mouse Fc IgG beads conjugated with anti-Dectin-1 antibody 2M24 or isotype control antibody by HEK-Blue hDectin-1a cells and human monocytes. Polystyrene anti-mouse Fc IgG beads (approximately 3.4 μm) were labeled with a pH-sensitive fluorescent dye (pHrodo Red) and conjugated with 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 (cells:beads). HEK-Blue hDectin-1a cells were labeled with the cell-permeable dye calcein AM. Phagocytosis of the beads was monitored by IncuCyte live cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the overlap of red object counts (pHrodo) over calcein-positive cells. Shown is phagocytosis of beads in HEK-Blue hDectin-1a cells over a period of 2.5 hours (top) and a representative image of pHrodo positive cells 2.5 hours after phagocytosis (bottom). [Figure 2B]Phagocytosis of pHrodo-labeled polystyrene anti-mouse Fc IgG beads conjugated with anti-Dectin-1 antibody 2M24 or isotype control antibody by HEK-Blue hDectin-1a cells and human monocytes. Polystyrene anti-mouse Fc IgG beads (approximately 3.4 μm) were labeled with a pH-sensitive fluorescent dye (pHrodo Red) and conjugated with 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 (cells:beads). HEK-Blue hDectin-1a cells were labeled with the cell-permeable dye calcein AM. Phagocytosis of the beads was monitored by IncuCyte live cell imaging. Phagocytosis was quantified using IncuCyte analysis software and expressed as the overlap of red object counts (pHrodo) over calcein-positive cells. Phagocytosis of beads by human monocytes over a 4-hour period (top) and representative images of pHrodo-positive cells 2.5 hours after phagocytosis (bottom) are shown, in which endocytosed beads are brightly fluorescent in the phagosomes.

[0022] [Diagram 3] Figure 1 shows binding of fully human 2M24 anti-Dectin-1 antibody (hIgG4) or isotype control antibody on HEK-Blue hDectin-1a cells and primary human monocytes. A shows binding analysis of fully human 2M24 anti-Dectin-1 antibody to HEK cells. B shows binding to primary human monocytes. Primary antibodies were used in serial dose titrations followed by fluorescent secondary antibodies against the primary antibodies. Fully human 2M24 anti-Dectin-1 hIgG4 antibody bound with high affinity to Dectin-1 expressing cells.

[0023] [Figure 4]Targeted phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated with fully human 2M24 anti-Dectin-1 antibody (hIgG4) or isotype control antibody by Dectin-1 expressing cells. Polystyrene biotin beads were labeled with pHrodo Red and conjugated to anti-Dectin-1 antibody 2M24 or isotype control via streptavidin. Conjugated beads were mixed with cells in a 1:3 ratio and phagocytosis of beads was monitored by IncuCyte live cell imaging. Phagocytosis of phrodo-biotin beads conjugated to streptavidin 2M24 anti-Dectin-1 hIgG4 antibody is shown for HEK-Blue hDectin-1a cells (top left), human monocytes (top right), and human macrophages (bottom). Fully human 2M24 anti-Dectin-1 antibody (hIgG4) promoted phagocytosis of Dectin-1 expressing cells.

[0024] [Figure 5A] Figure 1 shows the results of a secreted alkaline phosphatase reporter assay of Dectin-1 in HEK-Blue hDectin-1a cells. Figure 2 shows the results of a secreted alkaline phosphatase assay performed with immobilized fully human 2M24 anti-Dectin-1 antibody. Fully human 2M24 (hIgG4) anti-Dectin-1 antibody or isotype control antibody was immobilized overnight at 0.1-10 μg per well on U-bottom polypropylene microtiter plates, then HEK-BLUE hDectin-1a cells were cultured for 22 h and alkaline phosphatase secretion was assessed in the supernatant at OD 630 nm. [Figure 5B]Figure 1 shows the results of a secreted alkaline phosphatase reporter assay of Dectin-1 in HEK-Blue hDectin-1a cells. Figure 2 shows the results of a secreted alkaline phosphatase assay performed with bead-conjugated fully human 2M24 anti-Dectin-1 antibody. Biotin beads of sizes 3, 10, and 16.5 μm were conjugated to streptavidin 2M24 (hIgG4) anti-Dectin-1 antibody. The antibody-conjugated beads were mixed with HEK-Blue hDectin-1a cells for 22 h and the supernatants were assessed for alkaline phosphatase secretion at OD 630 nm. Bars represent the mean ± standard deviation; n=2 replicates. The 2M24 (hIgG4) anti-Dectin-1 antibody induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells both in immobilized and bead-conjugated form.

[0025] [Figure 6A] Figure 1 shows the amount of cytokine secretion by human primary macrophages stimulated with anti-Dectin-1 (15E2) antibody in solution. Primary human macrophages and primary monocytes were stimulated with 15E2 anti-Dectin-1 antibody or isotype antibody 10 μg / ml in solution for 24 hours and the amount of TNFα and IL6 secreted was evaluated by ELISA analysis of the supernatants. Zymosan was used as a positive control for cytokine secretion. Bars represent mean ± standard deviation. n=2 replicates. A shows the results of primary human monocytes stimulated with soluble 15E2 anti-Dectin-1 antibody, B shows the results of stimulated primary human macrophages. Soluble 15E2 anti-Dectin-1 antibody did not induce cytokine secretion in primary human monocytes and macrophages. [Figure 6B]Figure 1 shows the amount of cytokine secretion by human primary macrophages stimulated with anti-Dectin-1 (15E2) antibody in solution. Primary human macrophages and primary monocytes were stimulated with 15E2 anti-Dectin-1 antibody or isotype antibody 10 μg / ml in solution for 24 hours and the amount of TNFα and IL6 secreted was evaluated by ELISA analysis of the supernatants. Zymosan was used as a positive control for cytokine secretion. Bars represent mean ± standard deviation. n=2 replicates. A shows the results of primary human monocytes stimulated with soluble 15E2 anti-Dectin-1 antibody, B shows the results of stimulated primary human macrophages. Soluble 15E2 anti-Dectin-1 antibody did not induce cytokine secretion in primary human monocytes and macrophages.

[0026] [Figure 7A] Cytokine secretion by human primary monocytes and PBMCs stimulated with immobilized 2M24 or 15E2 anti-Dectin-1 antibodies. Anti-Dectin-1 antibodies or isotype control antibodies were immobilized overnight at 10 μg per well on U-bottom polypropylene microtiter plates and human monocytes or human PBMCs were cultured for 24 h. Secretion of TNFα, IL6, and IFNg was assessed by ELISA analysis of the supernatants. A, Cytokine secretion by human monocytes after stimulation with immobilized anti-Dectin-1 antibodies. B, Cytokine secretion by cultured human PBMCs after stimulation. Bars represent mean ± standard deviation; n = 2 replicates. 2M24 anti-Dectin-1 antibody induced cytokine secretion in both primary human monocytes and PBMCs and showed superior immune stimulation to 15E2 Dectin-1 agonist antibody. [Figure 7B]Cytokine secretion by human primary monocytes and PBMCs stimulated with immobilized 2M24 or 15E2 anti-Dectin-1 antibodies. Anti-Dectin-1 antibodies or isotype control antibodies were immobilized overnight at 10 μg per well on U-bottom polypropylene microtiter plates and human monocytes or human PBMCs were cultured for 24 h. Secretion of TNFα, IL6, and IFNg was assessed by ELISA analysis of the supernatants. A, Cytokine secretion by human monocytes after stimulation with immobilized anti-Dectin-1 antibodies. B, Cytokine secretion by cultured human PBMCs after stimulation. Bars represent mean ± standard deviation; n = 2 replicates. 2M24 anti-Dectin-1 antibody induced cytokine secretion in both primary human monocytes and PBMCs and showed superior immune stimulation to 15E2 Dectin-1 agonist antibody.

[0027] [Figure 8] Figure 1 shows the results of a competition assay performed with the 12M4 anti-Dectin-1 antibody clone and the natural ligand of Dectin-1. HEK-Blue hDectin-1a cells were incubated with serial dose titrations of 2M24 (hIgG4) anti-Dectin-1 antibody or 15E2, 259931, GE2 anti-Dectin-1 commercial antibodies starting at 300nM in the presence of 8ug / ml biotin-laminarin for 30 minutes on ice. 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.

[0028] [Figure 9] 1 shows a summary of the functional characterization of the 2M24 and 15E2 anti-Dectin-1 antibodies.

[0029] [Figure 10] Schematic diagram of the generation of bispecific antibodies by click chemistry. (A) Differential labeling of antibodies with MTA or FOL reagents. (B) Covalent cross-linking of antibodies via specific MTA-FOL interactions.

[0030] [Figure 11] We present the potential modes of activity deployed by anti-Dectin-1 agonistic bispecific antibodies to eliminate targeted cancer cells, including immune stimulation, phagocytosis, neoantigen presentation, and activation of T and B lymphocytes of the adaptive immune system.

[0031] [Figure 12A] Characterization of a click chemistry conjugated bispecific containing anti-Dectin-1 (clone 2M24) and anti-hCD70 arms. SDS-PAGE analysis of the covalently linked antibody pair (2M24 / anti-hCD20, 2M24 / anti-hCD70, and isotype control) under non-reducing and reducing conditions. [Figure 12B] Characterization of a click chemistry conjugated bispecific containing anti-Dectin-1 (clone 2M24) and anti-hCD70 arms is shown. Flow cytometry-based characterization of bispecific (2M24 / anti-hCD70 or isotype control) binding to Dectin-1 expressing HEK293 cells (top left) and two renal cancer cell lines - A498 (top right) and 786-0 (bottom left) is shown. Also shown are EC50 concentrations (nM) based on nonlinear regression fitting (bottom right). Anti-Dectin-1 / anti-hCD70 bispecifics bind to Dectin-1 or CD70 expressing cells with affinities of 1.8 nM or 12.34 nM, respectively.

[0032] [Figure 13]Figure 1 shows ligation of Dectin-1 expressing HEK293 and A498 renal cancer cell lines induced by 2M24 / anti-hCD70 bispecific. Flow cytometry 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 or isotype control (left). ligation of HEK293 and A498 cells is indicated by double positive signals (green+red+, square boxes). Also shown is ligation efficiency quantified as a percentage of total target cells (A498) that form doublets with HEK293 cells (right). Bars represent mean ± standard deviation; n=3 replicates. 2M24 / anti-hCD70 bispecific induced ligation of Dectin-1 expressing HEK293 and A498 renal cancer cell lines.

[0033] [Figure 14A] Figure 1 shows the ligation of Dectin-1 expressing cells to B cells induced by anti-Dectin-1 / anti-hCD20 bispecific antibody. Figure 2 shows the ligation of Dectin-1 expressing HEK293 cells to B cells induced by anti-Dectin-1 / anti-hCD20 bispecific antibody. Flow 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 or isotype control (left). ligation of HEK293 to Raji cells is indicated by double positive signals (green + red +; square boxes). Also shown is the ligation efficiency quantified as a percentage of total target cells (Raji) that form doublets with HEK293 cells (right). Bars represent the mean ± standard deviation; n = 2 replicates. [Figure 14B]Figure 1 shows the ligation of Dectin-1 expressing cells to B cells induced by anti-Dectin-1 / anti-hCD20 bispecific antibody. Figure 2 shows the results of a similar experiment performed to evaluate the ligation of human M0 macrophages to Raji cells induced by anti-Dectin-1 / anti-hCD20 bispecific. Bars represent mean ± standard deviation; n = 2 replicates. 2M24 / anti-hCD20 bispecific induced ligation of Dectin-1 expressing cells to CDC20 positive B cells (Raji cells).

[0034] [Figure 15] Figure 1 shows the results of a Dectin-1-induced secreted alkaline phosphatase reporter assay in HEK-Blue hDectin-1a cells using anti-Dectin-1 / anti-CD20 bispecific in the presence of Raji cells. 2M24 (hIgG4) / anti-CD20 bispecific was incubated with Raji cells, followed by two washes 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 h. Secreted alkaline phosphatase was assessed in the supernatant at OD 630 nm. Bars represent the mean ± standard deviation; n=2 replicates. Raji cells coated with anti-Dectin-1 / anti-CD20 bispecific induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells.

[0035] [Figure 16]Figure 1 shows induction of Raji cell phagocytosis by Dectin-1 expressing HEK293 cells by anti-Dectin-1 / anti-hCD20 bispecific antibody. Representative Incucyte images showing phagocytosis of Raji cells (arrows) by HEK cells at 16 h and 0 h are shown (left). Colocalization is indicated by yellow fluorescence. Reduction of calcein red signal in Raji cells after 16 h indicates cell death by phagocytosis. Quantification of HEK (calcein green) and Raji (calcein red) overlap or colocalization in different treatment groups is shown (right). Preincubation of HEK cells with the ADCP inhibitor latrunculin A inhibits 15E2 / anti-hCD20 bispecific antibody mediated phagocytosis (n=2 replicates).

[0036] [Figure 17] Figure 1 shows the ligation of Dectin-1-expressing cells to HER2-expressing cells induced by anti-Dectin-1 / anti-hHER2 bispecific antibody. Flow cytometric analysis of co-cultures of Dectin-1-expressing HEK293 cells (labeled with calcein green) and HER2-expressing SKBR3 cells (labeled with pHrodo red) in the presence of 15E2 / anti-hHER2 bispecific or isotype control is shown (left). ligation of HEK293 to SKBR3 cells is indicated by a double positive signal (green+red+; square box). Also shown is the ligation efficiency quantified as the percentage of total target cells (SKBR3) that form doublets with Dectin-1-expressing cells (right). Bars represent the mean ± standard deviation; n=2 replicates. Anti-Dectin-1 / anti-hHER2 bispecific induces ligation of Dectin-1 to HER2-positive cancer cells.

[0037] [Figure 18]Figure 1 shows the ligation of Dectin-1 expressing HEK293 cells to CD94 expressing BaF3 cells induced by anti-Dectin-1 / anti-hCD94 bispecific. Flow 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 or isotype control is shown (left). Ligation of HEK293 cells to BaF3 cells is indicated by double positive signals (green + red +; square boxes). Also shown is the ligation efficiency quantified as a percentage of total target cells (BaF3) that form doublets with HEK293 cells (right). Bars represent the mean ± standard deviation; n = 2 replicates. Anti-Dectin-1 / anti-hCD94 bispecific induced ligation of Dectin-1 expressing cells to CD94 expressing cells.

[0038] [Figure 19] Schematic diagram of Fab 2M24-mSA or full-length 2M24-mSA bound to biotinylated target antibody. A shows a chimeric fusion of monomeric streptavidin (mSA) with Fab 2M24 or full-length 2M24. mSA is genetically fused to either Fab 2M24 or full-length 2M24. B shows the linkage of Fab 2M24-mSA or 2M24-mSA to biotinylated target antibody. The chimeric fusion was incubated with biotinylated target antibody to generate a bispecific comprising a Dectin-1 binding arm and a second arm that binds to a target receptor or protein of interest.

[0039] [Figure 20] Biochemical and functional characterization of Fab 2M24-mSA fusion protein. A, HPLC characterization of recombinant Fab 2M24-mSA. B, SDS-PAGE analysis of purified Fab 2M24-mSA under reducing conditions. C, Flow cytometric characterization of Fab 2M24-mSA binding to HEK293 cells stably overexpressing human Dectin-1 (EC50=1.45 nM). Fab 2M24 fusion to monomeric streptavidin bound to Dectin-1 expressing cells with an affinity of 1.45 nM.

[0040] [Figure 21A] Figure 2 shows phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated with Fab-2M24 anti-Dectin-1 antibody tagged with monomeric streptavidin (Fab-2M24-mSA). Figure 2 shows duplet formation of HEK-Blue hDectin-1a cells with Fab-2M24-mSA conjugated to biotin beads and phagocytosis of the beads assessed by flow cytometry. [Figure 21B] Phagocytosis of pHrodo-labeled polystyrene biotin beads conjugated with Fab-2M24 anti-Dectin-1 antibody tagged with monomeric streptavidin (Fab-2M24-mSA). Phagocytosis of phrodo biotin beads (~3 μm) conjugated to Fab-2M24-mSA as assessed by IncuCyte live imaging (top) and representative images of pHrodo-positive cells (engulfed beads fluorescing bright red in phagosomes) versus no-beads control (bottom) 3 hours after phagocytosis. Fab 2M24-mSA fusions induced bead binding and phagocytosis by Dectin-1 expressing HEK293 cells.

[0041] [Figure 22] A-D show bispecific complexes containing Fab 2M24-mSA and a target biotinylated antibody. HPLC analysis of Fab 2M24-mSA complexed with biotinylated anti-hCD20 (A), biotinylated anti-hCD19 (B), biotinylated anti-hCD70 (C), or biotinylated anti-Aβ 1-42 (D) is shown. Panels include overlays of A280 traces containing Fab 2M24-mSA alone, the target biotinylated antibody alone, and Fab 2M24-mSA complexed with a biotinylated target antibody.

[0042] [Diagram 23]Ligation of Dectin-1-expressing HEK293 cells to CD20-expressing Raji cells induced by Fab 2M24-mSA / biotin anti-hCD20 bispecific antibody. 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 or isotype bispecific control is shown (left). Co-cultures were incubated at 4°C or 37°C. Ligation of HEK293 and Raji cells is indicated by a double positive signal (green + red +; dotted square). Ligation efficiency quantified as a percentage of total target cells (Raji) forming doublets is also shown (right). Bars represent the mean ± standard deviation; n = 4 replicates. The Fab 2M24-mSA / biotin anti-hCD20 bispecific induced binding between Dectin-1 expressing HEK293 cells and Raji cells.

[0043] [Figure 24A] 1 shows a bispecific antibody design of a human bispecific antibody (e.g., a human IgG1 bispecific antibody) targeting Dectin-1 and a disease target or antigen. A schematic diagram of the design is provided. One arm (2M24A.X) with VH domain A and VL domain B targets human Dectin-1, and the other arm (2M24B.X) with VH domain C and VL domain D targets a disease target or antigen. [Figure 24B] 1 shows a bispecific antibody design of a human bispecific antibody (e.g., a human IgG1 bispecific antibody) targeting Dectin-1 and a disease target or antigen. A schematic diagram of an exemplary mechanism of action of an anti-Dectin-1 bispecific antibody with an active Fc domain that targets hDectin-1 on myeloid cells (via the first arm), antigens on target cells / disease agents (via the second arm), and Fc receptors on myeloid cells and NK cells, inducing robust immune stimulation and phagocytosis is provided.

[0044] [Figure 25A]Figure 2 shows that a bispecific antibody with one arm targeting hDectin-1 and the other arm targeting hCD20 (using the variable domains of rituximab) binds to cells expressing human Dectin-1 or human CD20. The top panel shows the binding of a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) or hDectin-1 and RSV (2M24 / RSV) to HEK293 cells stably expressing human Dectin-1, as assessed by flow cytometry. The bottom panel shows the binding of the bispecific antibody 2M24 / RSV hIgG1-FITC conjugated and 2M24 bivalent hIgG1-FITC conjugated to PBMCs, as assessed by flow cytometry. [Figure 25B] Figure 1 shows that a bispecific antibody with one arm targeting hDectin-1 and the other arm targeting hCD20 (using the variable domains of rituximab) binds to cells expressing human Dectin-1 or human CD20. Figure 2 shows the binding of rituximab (human IgG1), 2M24 / CD20 with activating human IgG1 Fc, 2M24 / CD20 with inactive human IgG1 Fc, 2M24 / RSV with activating human IgG1 Fc, or 2M24 / RSV with inactive human IgG1 Fc to the CD20-expressing B cell lymphoma Raji cell line.

[0045] [Figure 26A] Figure 1 shows that bispecific antibodies targeting hDectin-1 and hCD20 (2M24 / CD20) induce ligation of Dectin-1 to CD20-expressing cells. To assess ligation of Dectin-1-expressing HEK293 cells (effector) and CD20-expressing Raji cells (target), cells were differentially labeled with calcein green (effector) or calcein red (target) dyes. Labeled cells were co-cultured and treated with hIgG1 inactive 2M24 / CD20 or 2M24 / RSV (control) bispecific antibodies to induce effector:target ligation. Successful ligation of effector:target cells was indicated by double positive staining (calcein green+, calcein red+, square box). [Figure 26B] Figure 2 shows that bispecific antibodies targeting hDectin-1 and hCD20 (2M24 / CD20) induce ligation of Dectin-1 to CD20-expressing cells. Figure 3 shows dose titration of the bispecific in effector:target cell co-cultures. Ligation efficiency was quantified as the percentage of total target cells bound or ligated to effector cells.

[0046] [Figure 27A] Figure 1 shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an active hIgG1 Fc does not induce monocyte depletion by antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).PBMCs from two healthy donors, donor 76 (A) and donor 77 (B), were treated with increasing concentrations of 2M24 / CD20 bispecific antibody (hIgG1 active or inactive isotype) and rituximab for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD14+ monocytes (as % of isotype control). [Figure 27B] Figure 1 shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an active hIgG1 Fc does not induce monocyte depletion by antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).PBMCs from two healthy donors, donor 76 (A) and donor 77 (B), were treated with increasing concentrations of 2M24 / CD20 bispecific antibody (hIgG1 active or inactive isotype) and rituximab for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD14+ monocytes (as % of isotype control).

[0047] [Figure 28A]Figure 1 shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an activating hIgG1 Fc induces superior B cell depletion compared to rituximab. PBMCs from two healthy donors, donor 83 (A) and donor 84 (B), were treated with increasing concentrations of the indicated antibodies for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as % of B cells in PBMCs treated with isotype control). [Figure 28B] Figure 1 shows that a bispecific antibody targeting hDectin-1 and hCD20 (2M24 / CD20) with an activating hIgG1 Fc induces superior B cell depletion compared to rituximab. PBMCs from two healthy donors, donor 83 (A) and donor 84 (B), were treated with increasing concentrations of the indicated antibodies for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as % of B cells in PBMCs treated with isotype control).

[0048] [Figure 29A] Figure 1 shows that rituximab induces higher B cell shaving (CD19 downregulation) compared to 2M24 / CD20 active IgG1 bispecific antibody. CD19+ expression on B cells from two healthy donors, donor 83 (A) and donor 84 (B), was quantified by flow cytometry after 24 h incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab, or isotype control. The mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugated) was used to assess the effect of 2M24 / CD20 bispecific and rituximab on CD19 expression on B cells. EC50 values ​​were calculated based on nonlinear regression analysis. [Figure 29B]Figure 1 shows that rituximab induces higher B cell shaving (CD19 downregulation) compared to 2M24 / CD20 active IgG1 bispecific antibody. CD19+ expression on B cells from two healthy donors, donor 83 (A) and donor 84 (B), was quantified by flow cytometry after 24 h incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab, or isotype control. The mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugated) was used to assess the effect of 2M24 / CD20 bispecific and rituximab on CD19 expression on B cells. EC50 values ​​were calculated based on nonlinear regression analysis.

[0049] [Diagram 30] Figure 1 shows the difference in cytokine release induced by 2M24 / CD20 activating IgG1 bispecific antibody compared to rituximab. ELISA-based (mesoscale discovery) quantification of cytokines was performed on supernatants isolated from healthy donor PBMCs treated with 2M24 / CD20 activating hIgG1 bispecific, rituximab, or isotype control. PBMCs were stimulated overnight with antibodies and the supernatants were subsequently analyzed by MSD. Cytokines tested were IFNγ, IL-12p70, IL-6, TNFα, IL-1β, IL-4, IL-13, IL-10, and IL-8. Each plot shows the amount of cytokine secretion (pg / mL) as a function of the antibody used for treatment (from left to right: 2M24 / CD20 hIgG1 bispecific, 2M24 / RSV hIgG1 bispecific, rituximab hIgG1, and isotype control hIgG1).

[0050] [Figure 31A]Figure 1 shows that 2M24 / CD20 hIgG1 (active isotype) bispecific antibody induces superior B cell depletion and lower CD19 shaving compared to rituximab in co-cultures of human macrophages and GFP-expressing Raji B cells. 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, 2M24 / RSV control, fucosylated rituximab, or isotype hIgG1 control. Co-cultures were incubated at 37°C for 24 h and then stained with PE anti-CD206 antibody to label macrophages and BV-605 anti-CD19 antibody to label Raji cells. The number of remaining viable / Raji-GFP+ cells was assessed at the end of the experiment. Primary antibodies were used in serial dose titrations. [Figure 31B] Figure 2 shows that 2M24 / CD20 hIgG1 (active isotype) bispecific antibody induces superior B cell depletion and lower CD19 shaving compared to rituximab in co-cultures of human macrophages and GFP-expressing Raji B cells. Assessment of CD19 on Raji-GFP cells after 24 hours. Shaving of B cell receptor is shown as a decrease in CD19 MFI in the presence of anti-Dectin-1 / anti-hCD20 bispecific or rituximab.

[0051] [Figure 32A]Figure 1 shows that 2M24 / CD20 active IgG1 bispecific antibody induces superior tissue B cell depletion compared to rituximab in single cell suspensions of renal cancer specimens. Single cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, 2M24 / RSV hIgG1 control, fucosylated rituximab, and respective isotype controls. Renal cancer tissue specimens 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. Cells were first gated for live cells and further separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells), and then CD19+ (B cells) and CD3+ (T cells) cells were identified within the CD45+ population (A and B). The number of remaining B cells was assessed by anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 32B] Figure 1 shows that 2M24 / CD20 active IgG1 bispecific antibody induces superior tissue B cell depletion compared to rituximab in single cell suspensions of renal cancer specimens. Single cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, 2M24 / RSV hIgG1 control, fucosylated rituximab, and respective isotype controls. Renal cancer tissue specimens 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. Cells were first gated for live cells and further separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells), and then CD19+ (B cells) and CD3+ (T cells) cells were identified within the CD45+ population (A and B). The number of remaining B cells was assessed by anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C). [Figure 32C]Figure 1 shows that 2M24 / CD20 active IgG1 bispecific antibody induces superior tissue B cell depletion compared to rituximab in single cell suspensions of renal cancer specimens. Single cell suspensions from two renal cancer tissue specimens were analyzed by flow cytometry in the presence of 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, 2M24 / RSV hIgG1 control, fucosylated rituximab, and respective isotype controls. Renal cancer tissue specimens 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. Cells were first gated for live cells and further separated into CD45+ cells (immune cells) and CD45- cells (non-immune cells), and then CD19+ (B cells) and CD3+ (T cells) cells were identified within the CD45+ population (A and B). The number of remaining B cells was assessed by anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (C).

[0052] [Diagram 33] A-C show that anti-Dectin-1 antibody (clone 2M24) induces Dectin-1 clustering and TNFα secretion from human macrophages. Cytokine secretion by cultured macrophages and single cell suspensions of renal cancer specimens stimulated with immobilized anti-Dectin-1 antibody (clone 2M24) or 2M24 / CD20 bispecific antibody was examined. Anti-Dectin-1 antibody (clone 2M24), isotype control, or 2M24 / CD20 bispecific antibody were immobilized overnight at 10 μg per well on U-bottom polypropylene microtiter plates, followed by incubation with human monocyte-derived macrophages (A and B) or single cell suspensions from renal cancer specimens (C). Cells were cultured for 24 h, and the amount of TNFα secretion in the supernatants was assessed by ELISA. As a positive control, cells were stimulated with zymosan.

[0053] [Diagram 34]Figure 1 shows that immobilized anti-Dectin-1 antibody (clone 2M24) promotes immune stimulation in single cell suspensions of renal cancer specimens. Single cell suspensions from renal cancer specimens were treated with immobilized anti-Dectin-1 antibody (clone 2M24) or isotype control hIgG4 antibody for 24 hours. Supernatants were analyzed by ELISA for the release of various cytokines including IFNγ, IL-6, TNFα, IL-23, IL-12p70, IL-10, and IL-13. Each plot shows the amount of cytokine (pg / mL) as a function of antibody treatment. Results are shown for treatment with anti-Dectin-1 antibody (clone 2M24) or isotype control hIgG4 antibody using renal cancer donor 3 (left) or donor 4 (right).

[0054] [Diagram 35] Figure 1 shows the effect of 2M24 / CD20 bispecific antibody on CD16 expression on human NK cells compared to rituximab or isotype control (RSV). The results show that CD16 antigen levels on NK cells are better maintained in PBMCs treated with 2M24 / CD20 bispecific compared to rituximab.

[0055] [Diagram 36] Figure 1 shows the effect of 2M24 / CD20 bispecific antibody on CD19 expression on human B cells compared to rituximab or isotype control (2M24 / RSV bispecific). The results show that CD19 antigen levels are better maintained in B cells treated with 2M24 / CD20 bispecific compared to rituximab.

[0056] [Figure 37] 1 shows depletion of human B cells with the 2M24 / CD20 bispecific antibody derived from rituximab or the 2M24 / CD20 bispecific antibody derived from obinutuzumab. The results show that the 2M24 / CD20 bispecific derived from the rituximab arm is superior in depleting B cells compared to the bispecific derived from obinutuzumab.

[0057] [Figure 38] 1 shows the design of an exploratory study of the safety and efficacy of 2M24 / CD20 bispecific antibodies in non-human primates.

[0058] [Figure 39] Figure 1 shows depletion of circulating B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody made from cells treated with kifunensine (KIF). Figure 1 shows depletion of B cells in monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF (top) or 2M24 / CD20 hIgG1 inactive (bottom). [Diagram 40] Figure 1 shows depletion of circulating B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody generated in cells treated with kifunensine (KIF). Figure 1 shows depletion of B cells in monkeys treated with 5 mg / kg rituximab hIgG1 KIF.

[0059] [Figure 41A] Figure 1 shows depletion of tissue resident B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody generated in cells treated with kifunensine (KIF). Figure 1 shows depletion of B cells in bone marrow from monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF. [Figure 41B] Figure 1 shows depletion of tissue resident B cells in cynomolgus monkeys by 2M24 / CD20 hIgG1 bispecific antibody generated in cells treated with kifunensine (KIF). Figure 1 shows depletion of B cells in lymph nodes of monkeys treated with 5 mg / kg 2M24 / CD20 hIgG1 KIF or rituximab hIgG1 KIF.

[0060] [Diagram 42] 1 shows ex vivo depletion of B cells from cynomolgus monkey PBMCs.

[0061] [Diagram 43]Using knobs-into-holes technology, we show the format of a bispecific molecule (2M24 scFv / CD20) that pairs an anti-CD20 conventional half antibody with an anti-Dectin-1 single-chain variable fragment (scFv) Fc fusion arm. H is the 2M24 VH domain and L is the 2M24 VL domain.

[0062] [Figure 44A] Figure 1 shows purification and functional characterization of 2M24 / CD20 bispecific antibodies. Purification of the molecule by size exclusion chromatography (SEC) is shown. [Figure 44B] 1 shows purification and functional characterization of 2M24 / CD20 bispecific antibodies. Purified bispecific antibodies promoted targeted immune stimulation as assessed by NFκB reporter assay. [Figure 44C] Figure 1 shows purification and functional characterization of 2M24 / CD20 bispecific antibody. Figure 2 shows depletion of human B cells by 2M24 scFv / CD20 bispecific antibody.

[0063] [Diagram 45] Figure 1 shows depletion of B cells from healthy donor PBMCs by CD20-targeting antibodies including rituximab, CD3xCD20 bispecific T cell engager, and bispecific binding molecule comprising an anti-CD20 conventional half antibody arm and an anti-Dectin-1 single chain variable fragment (scFv) arm in a non-fucosylated hIgG1 Fc format (2M24 scFv / CD20). PBMCs isolated from healthy donors were incubated with CD20-targeting antibodies for 24 hours, then stained with anti-CD19 (B cell specific marker) antibody and remaining B cells were characterized by flow cytometry. Data are expressed as the percentage of remaining B cells compared to a control untreated group. NF is non-fucosylated.

[0064] [Figure 46]Figure 1 shows depletion of B cells from prostate cancer tumor biopsy specimens with rituximab, a bispecific binding molecule containing an anti-CD20 conventional half antibody arm in a non-fucosylated hIgG1 Fc format and an anti-Dectin-1 single chain variable fragment (scFv) arm (2M24 scFv / CD20), a 2M24xCD20 hIgG1 bispecific (DuetMab format), or an isotype control. Single cell suspensions generated from prostate cancer specimens were treated with CD20-targeting antibodies for 24 hours. Cells were then stained with antibodies against CD45, CD3, CD11b, CD16, CD163, and CD19. B cells were identified as CD45+CD3-CD19+. Data shows the percentage of remaining B cells compared to the B cell counts of an isotype control group (RSV hIgG1).

[0065] [Figure 47A] 1 shows the effect of 2M24xCD20 bispecific binding protein (DuetMab format) treatment on CD16 expression on NK cells from healthy human PBMCs compared to rituximab or control antibody. CD16 levels on NK cells were better maintained by 2M24xCD20 bispecific treatment than by rituximab.

[0066] [Figure 47B] FIG. 1 shows the effect of 2M24×CD20 bispecific binding protein treatment on CD19 expression on B cells from healthy human PBMCs compared to rituximab or control antibody. Results from three donors are shown, and for each donor, the order of data points is 2M24×CD20 bispecific, rituximab, control (left to right). CD19 levels on B cells were better maintained by 2M24×CD20 bispecific treatment than by rituximab.

[0067] [Figure 47C]Figure 1 shows the effect of 2M24xCD20 bispecific binding protein treatment on depletion of B cells from healthy human PBMCs compared to rituximab, obinutuzumab or control antibody. Two formats of 2M24xCD20 bispecific binding protein were tested, one with the variable domains of rituximab and one with the variable domains of obinutuzumab. B cells were quantified relative to an untreated control (dotted line). 2M24xCD20 bispecific binding protein with anti-CD20 arms derived from rituximab showed superior B cell depletion than the equivalent 2M24 bispecific with anti-CD20 arms derived from obinutuzumab.

[0068] [Figure 47D] The effect of 2M24xCD20 bispecific binding protein treatment on B cell depletion using single cell suspensions from renal cancer specimens is shown compared to 2M24 / RSV bispecific, rituximab, isotype control antibody, or no treatment. The 2M24xCD20 bispecific binding protein induced superior B cell depletion compared to rituximab.

[0069] [Figure 48] 1 shows the design of an exploratory study for 2M24xCD20 bispecific binding protein in non-human primates (cynomolgus monkeys). Time points for whole blood collection, tissue collection, whole blood collection for chemistry and coagulation tests, and weight measurement are shown. CBC: complete blood count. PK: pharmacokinetics. BM: bone marrow. LN: lymph node.

[0070] [Figure 49A] 1 shows B cell depletion in non-human primate studies with 2M24×CD20 bispecific binding protein. Depletion of blood CD19+ B cells with 2M24×CD20 hIgG1 bispecific binding protein treated with KIF (top left), 2M24×CD20 bispecific binding protein with inactive hIgG1 Fc (bottom left), or Rituximab hIgG1 treated with KIF (top right). [Figure 49B]1 shows B cell depletion by 2M24×CD20 bispecific binding protein in a non-human primate study. Depletion of B cells (% of CD45+) in bone marrow (top) or lymph nodes (bottom) by 2M24×CD20 hIgG1 bispecific binding protein treated with KIF, 2M24×CD20 bispecific binding protein with inactive hIgG1 Fc, or Rituximab hIgG1 treated with KIF, as indicated. [Figure 49C] 1 shows B cell depletion by 2M24xCD20 bispecific binding protein in a non-human primate study. Ex vivo B cell depletion from cynomolgus monkey PBMCs treated with serial dilutions of KIF-treated 2M24xCD20hIgG1 bispecific binding protein, KIF-treated rituximab hIgG1, or KIF-treated control antibody.

[0071] [Figure 50A] 1 shows the results of a mouse study on the anti-mouse Dectin-1 antibody 2A11 x mCD20 bispecific binding protein for depletion of B cells. The study design is shown. [Figure 50B] Figure 1 shows the results of mouse studies with anti-mouse Dectin-1 antibody 2A11 x mCD20 bispecific binding protein for depletion of B cells. Results of treatment with 2A11 x mCD20 bispecific mIgG1 binding protein, anti-mouse CD20 rat IgG2a antibody, or mIgG1 isotype control on CD19+ B cells (as % of total CD45+ cells) in blood, peritoneum, bone marrow, and spleen are shown. Differences between study groups were analyzed by ordinary one-way ANOVA and Tukey's test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

[0072] [Figure 51A]1 shows the results of ex vivo testing of various properties of 2M24×CD20 bispecific binding proteins. Activation of a Dectin-1 expressing reporter HEK cell line as a function of antibody concentration is shown. Results are shown following treatment with 2M24×CD20 bispecific binding proteins (circles), rituximab (squares), or D-zymosan (triangles), an established ligand of Dectin-1 used as a positive control. [Figure 51B] 1 shows the results of ex vivo testing of various properties of 2M24xCD20 bispecific binding protein. Induction of phagocytosis of B cell lines (% of B cells compared to isotype control treatment) as a function of antibody concentration. Shown are results of treatment with 2M24xCD20 bispecific binding protein (black circles), rituximab (triangles), or a non-specific RSV antibody (grey circles). [Figure 51C] 1 shows the results of ex vivo testing of various properties of 2M24xCD20 bispecific binding protein. B cell depletion (% of B cells compared to untreated control) is shown as a function of antibody concentration. Results are shown following treatment with 2M24xCD20 bispecific binding protein (black squares), rituximab (triangles), anti-CD20 / anti-CD3 bispecific engager (grey squares), or a non-specific RSV antibody (grey circles). [Fig. 51D] Figure 1 shows the results of ex vivo testing of various properties of 2M24xCD20 bispecific binding protein. Ex vivo cytokine stimulation from human PBMCs by 2M24xCD20 bispecific binding protein or comparative anti-CD20 antibody (pg / mL of indicated cytokines) is shown. Results are shown from treatment with 2M24xCD20 bispecific binding protein, rituximab, anti-CD20 / anti-CD3 bispecific engager, non-specific RSV antibody, or untreated control. Cytokines measured included IL-6 (D, top left), TNF-α (D, bottom left), IFN-γ (D, right), IL-12p70 (E, top left), IL-1β (E, bottom left), and IL-2 (E, right). [Figure 51E]Figure 1 shows the results of ex vivo testing of various properties of 2M24xCD20 bispecific binding protein. Ex vivo cytokine stimulation from human PBMCs by 2M24xCD20 bispecific binding protein or comparative anti-CD20 antibody (pg / mL of indicated cytokines) is shown. Results are shown from treatment with 2M24xCD20 bispecific binding protein, rituximab, anti-CD20 / anti-CD3 bispecific engager, non-specific RSV antibody, or untreated control. Cytokines measured included IL-6 (D, top left), TNF-α (D, bottom left), IFN-γ (D, right), IL-12p70 (E, top left), IL-1β (E, bottom left), and IL-2 (E, right).

[0073] [Figure 52A] Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. The effect of surrogate anti-mDectin-1 / anti-mCD20 mIgG2a bispecific antibody (squares) or isotype control (circles) treatment on tumor volume (mm3) over time (days post injection) in in vivo mouse xenograft models using Ramos B cell lymphoma xenografts in SCID mice (top left), Daudi B cell lymphoma xenografts in SCID mice (bottom left), or Raji B cell lymphoma xenografts in SCID-beige mice. Efficacy data are presented as mean + / - SEM. [Figure 52B]Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. Activation of myeloid cells of the indicated cell populations (myeloid cells shown as % of CD45+ cells) in the spleen (left), lymph node (middle), or tumor (right) of mice implanted with MC38 murine colon carcinoma cells expressing human CD20 is shown. For each cell population, cell numbers are provided as % of total CD45+ cells, with data for isotype control on the left and anti-mDectin-1 / anti-mCD20 on the right. GRNs: granulocytes. DCs: dendritic cells. Macs: macrophages. Differences between test groups were analyzed by two-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. [Figure 52C] Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. 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 (right in all panels) or isotype control (left in all panels) is shown. For each cell population, cell numbers are provided as % of the indicated cell population. Differences between test groups were analyzed by two-way ANOVA. *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. [Fig. 52D]Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. Activation of intratumoral CD3+ T cells (as % of tumor CD45+ cells; left), intratumoral CD4+ or CD8+ T cells (as % of tumor T cells; center), and cytokine IL-2 and GmzB production by intratumoral CD8+ T cells in the MC38 xenograft mouse model after treatment with anti-mDectin-1 / anti-mCD20 mIgG1 bispecific (right of all panels) or isotype control (left of all panels) is shown. Differences between test groups were analyzed by two-way ANOVA. *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. [Figure 52E] Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. Depletion of B cells in blood (top left), lymph nodes (top right), spleen (bottom left), and bone marrow (bottom right) after treatment with anti-mDectin-1 / anti-mCD20 mIgG2a bispecific (gray circles) or isotype control (black circles) in naive C57BL / 6 mice is shown. Data are expressed as B cells (% of B cells in isotype control) on the indicated days after injection. [Fig. 52F] Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. Depletion of B cells in lungs (top left), liver (top right), brain (bottom left), and heart (bottom right) after treatment with anti-mDectin-1 / anti-mCD20 mIgG2a bispecific (gray circles) or isotype control (black circles) in naive C57BL / 6 mice is shown. Data are expressed as B cells (% of B cells in isotype control) at the indicated days after injection. [Fig. 52G]Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. Tumor growth inhibition is compared between anti-mDectin-1 / anti-mCD20 bispecifics with active Fc (mIgG2a) and inactive mIgG2a Fc and isotype control in the Daudi xenograft model in SCID mice. Tumor volume (mm3) is plotted over time (days post-implantation). Data are shown for isotype control (circles), anti-mDectin-1 / anti-mCD20 mIgG2a active (squares), and anti-mDectin-1 / anti-mCD20 mIgG2a inactive (triangles). Error bars indicate SEM. [Fig. 52H] Figure 1 shows the results of in vivo testing of surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies in various mouse models. The percentages of resident monocytes (CD11bhiMHCII-Ly6c-; top left), inflammatory monocytes (CD11bhiMHCII-Ly6c+; top right), macrophages (CD11b+F4 / 80+; bottom left), and M1 and M2 macrophages (bottom right) in tumor tissue are shown (all plots show % of CD45+ except bottom right shows % of macrophages) after treatment with mIgG2a isotype control (circles) or anti-mDectin-1 / anti-mCD20 bispecific with mIgG2a inactive Fc (triangles).

[0074] [Figure 53A] 1 shows the results of in vivo testing of 2M24×CD20 bispecific binding protein in a cynomolgus monkey model, showing peripheral blood B cell levels (% compared to baseline) over time after administration of 2M24×CD20 bispecific at 1 mg / kg (top left), 10 mg / kg (bottom left), or 100 mg / kg (right). [Figure 53B] 1 shows the results of in vivo testing of 2M24xCD20 bispecific binding protein in a cynomolgus monkey model, showing B cell levels in lymphoid organs bone marrow (top) and lymph nodes (bottom) over time after administration of 2M24xCD20 bispecific at 1 mg / kg (left), 10 mg / kg (middle), or 100 mg / kg (right). [Figure 53C] 1 shows the results of in vivo testing of 2M24xCD20 bispecific binding protein in a cynomolgus monkey model. B cell levels (as % of tissue CD45+ cells) are shown in the indicated lymphoid (left) and non-lymphoid (right) tissues 15 days after treatment with vehicle control (left for all tissues) or 100 mg / kg 2M24xCD20 bispecific (right for all tissues). [Fig. 53D] 1 shows the results of in vivo testing of 2M24xCD20 bispecific binding protein in a cynomolgus monkey model, showing depletion of the indicated B cell subsets in the spleen 15 days after treatment with vehicle control (D bottom; E left) or 100 mg / kg of 2M24xCD20 bispecific (D top; E right). [Figure 53E] 1 shows the results of in vivo testing of 2M24xCD20 bispecific binding protein in a cynomolgus monkey model, showing depletion of the indicated B cell subsets in the spleen 15 days after treatment with vehicle control (D bottom; E left) or 100 mg / kg of 2M24xCD20 bispecific (D top; E right). [Fig. 53F] FIG. 1 shows the results of in vivo testing of 2M24×CD20 bispecific binding protein in a cynomolgus monkey model. Levels of circulating classical dendritic cells (as % of CD45+ cells) are shown over time (days) after treatment with 2M24×CD20 bispecific (black circles) or rituximab-like anti-CD20 mAb (grey circles). [Figure 53G] 1 shows the results of in vivo testing of 2M24×CD20 bispecific binding protein in a cynomolgus monkey model. Circulating classical dendritic cell levels (as % of CD45+ cells) are shown over time (days) after treatment with 1 mg / kg (black circles) or 10 mg / kg (grey squares) of 2M24×CD20 bispecific. [Fig. 53H]1 shows the results of in vivo testing of 2M24×CD20 bispecific binding protein in a cynomolgus monkey model, showing the levels of proinflammatory cytokines in serum of cynomolgus monkeys over time after three weekly doses of 2M24×CD20 bispecific binding protein at 1 mg / kg (H), 10 mg / kg (H), or 100 mg / kg (I). [Fig. 53I] 1 shows the results of in vivo testing of 2M24×CD20 bispecific binding protein in a cynomolgus monkey model, showing the levels of proinflammatory cytokines in serum of cynomolgus monkeys over time after three weekly doses of 2M24×CD20 bispecific binding protein at 1 mg / kg (H), 10 mg / kg (H), or 100 mg / kg (I). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] Some aspects are described below with reference to illustrative applications. It should be understood that many specific details, relationships, and methods are described to provide a complete understanding of the features described herein. However, a person skilled in the art will readily recognize that the features described herein can be implemented in other ways without one or more specific details. The features described herein are not limited to the order of the illustrated acts or events, since some acts can occur in different orders and / or simultaneously with other acts or events. Furthermore, not all illustrated acts or events are required to implement a method in accordance with the features described herein.

[0076] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, to the extent the terms "including," "includes," "having," "has," "with," or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be inclusive in a similar manner to the term "comprising." As used herein, the term "comprising" is synonymous with "including" or "containing," and is inclusive or open-ended.

[0077] References to "or" herein are intended to encompass "and / or" unless otherwise indicated. As used herein, the term "about" with respect to a numerical value means a numerical value of +10% or -10% of that numerical value. The term "about" with respect to a range refers to the range minus 10% of its minimum value and plus 10% of its maximum value.

[0078] I. Multispecific Binding Proteins In certain aspects, the disclosure provides antigen-binding domains that bind to human Dectin-1, and multispecific (e.g., bispecific) binding molecules comprising same. In certain aspects, the disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments comprising 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 disclosure provides multispecific (e.g., bispecific) antibodies and antibody fragments comprising a first antigen-binding domain that binds to human Dectin-1 and a second antigen-binding domain that binds to CD20.

[0079] In some embodiments, the multispecific binding protein comprises:Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKV DIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31) or QVQ LVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGG SGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSD A first polypeptide chain comprising: KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35);Amino acid sequence: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32) or QVQ LQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL A second polypeptide chain comprising LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36);and a third polypeptide chain comprising the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0080] In some embodiments, the multispecific binding protein comprises:Amino acid sequence: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKV DIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 37) or QVQ LVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGG SGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKRSD A first polypeptide chain comprising: KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39);Amino acid sequence: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38) or QVQ LQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40);and a third polypeptide chain comprising the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0081] As is known in the art, the C-terminal lysine of some antibody heavy chain species may be truncated in some parts of the molecule. In some embodiments, one or both of the antibody Fc domains does not have a C-terminal lysine.

[0082] 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 are compositions comprising a mixture of multispecific binding protein species, each species comprising 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.

[0083] 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 are compositions comprising a mixture of multispecific binding protein species, each species comprising 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.

[0084] In some embodiments, the first, second, and third polypeptide chains are assembled into a multispecific binding protein that comprises a first antigen binding domain that binds human Dectin-1 and a second antigen binding domain that binds human CD20.

[0085] 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 isoforms. In some embodiments, human Dectin-1 isoform A comprises the amino acid sequence MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMAIWRSNSGSNTLENGYFLSRNKENHSQPTQSSLEDSVTPTKAVKTTGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO: 9). In some embodiments, human dectin-1 isoform B comprises the amino acid sequence MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVSEKGSCAASPPWRLIAVILGILCLVILVIAVVLGTMGVLSSPCPPNWIIYEKSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDSSNELGFIVKQVSSQPDNSFWIGLSRPQTEVPWLWEDGSTFSSNLFQIRTTATQENPSPNCVWIHVSVIYDQLCSVPSYSICEKKFSM (SEQ ID NO: 10). In some embodiments, the 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 can bind to human dectin-1 and monkey dectin-1, such as cynomolgus monkey dectin-1.

[0086] In some embodiments, the multispecific binding protein, antigen binding domain, antibody, or antibody fragment binds to 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 number 931. An exemplary, non-limiting human CD20 polypeptide is provided by NCBI Ref. Seq.NP_068769:MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP (SEQ ID NO: 34).

[0087] In some embodiments, antibody and immunoglobulin are used interchangeably and are used herein in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and single domain antibodies (described in more detail herein), so long as they exhibit the desired antigen-binding activity.

[0088] In some embodiments, an antibody (immunoglobulin) refers to a protein having a structure substantially similar to a native antibody structure, or a protein having heavy and light chain variable regions having structures substantially similar to native heavy and light chain variable region structures. A native antibody refers to a naturally occurring immunoglobulin molecule having a diverse structure. For example, the IgG class of native immunoglobulins is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called variable light domain or light chain variable domain, followed by a constant light (CL) domain, also called light chain constant region. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described, for example, in Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies (immunoglobulins) are assigned to different classes. There are five major classes of antibodies: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Based on the amino acid sequence of its constant domain, the light chain of an immunoglobulin can be assigned to one of two types, called kappa (κ) and lambda (λ). An immunoglobulin basically consists of two Fab molecules and an Fc domain linked via an immunoglobulin hinge region.

[0089] In some embodiments, Fc, Fc region, or Fc domain refers to the C-terminal region of an antibody heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Fc can refer to all or part of 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 the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. An IgG Fc region includes the IgG CH2 and IgG CH3 domains, and optionally includes the hinge. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. Human IgG Fc domains are particularly useful in the present disclosure, and can be Fc domains derived from human IgG1, IgG2 or IgG4.

[0090] 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.

[0091] "Fab" (fragment antigen binding) is the portion of an antibody that binds an antigen and contains the variable region and CH1 of the heavy chain linked to a light chain via an interchain disulfide bond.

[0092] In some embodiments, the multispecific binding proteins or antibodies of the present disclosure comprise an Fc region. The antibodies / multispecific binding proteins can be of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD. The immunoglobulin Fc region of the molecule that triggers targeted phagocytosis can play an important role in the process by engaging Fc receptors and inducing further phagocytosis. In some embodiments, the molecule has a modified Fc region (e.g., comprising one or more mutations that reduce effector function as described herein) that has reduced ADCC activity compared to wild-type human IgG1.

[0093] In some embodiments, a multispecific binding protein or antibody of the disclosure comprises an Fc region, and the glycostructures attached to the Fc region are fucose-reduced or fucose-deficient, e.g., at least one or two of the heavy chains of the antibody are nonfucosylated. In some embodiments, a composition is provided herein that comprises a multispecific binding protein or antibody of the disclosure that comprises an Fc region, and the glycostructures attached to the Fc region are fucose-reduced or fucose-deficient, e.g., at least one or two of the heavy chains of the antibody are nonfucosylated. In some embodiments, less than 50% of the N-glycoside-linked glycochains in the composition comprise a fucose residue. In some embodiments, none of the N-glycoside-linked glycochains comprise substantially any fucose residue. In some embodiments, the fucose-reduced or fucose-deficient multispecific binding protein or antibody has improved ADCC function.

[0094] In other embodiments, a multispecific binding protein of the disclosure (e.g., an IgG1 antibody), or a composition comprising a multispecific binding protein of the disclosure (e.g., an IgG1 antibody), comprises wild-type glycosylation of the Fc region. In some embodiments, provided herein is a fucosylated binding protein of the disclosure (e.g., an IgG1 antibody), or a composition comprising a fucosylated binding protein of the disclosure (e.g., an IgG1 antibody).

[0095] Fucosylated or fucosylated linked protein can refer to the presence of fucose residues in the oligosaccharides attached to the peptide backbone of the antibody. Specifically, a fucosylated antibody contains an α(l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or both of the N-linked oligosaccharides attached to the Fc region of the antibody, e.g., at position Asn297 (EU numbering of Fc region residues) of the human IgG1 Fc region. Asn297 can also be located approximately +3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, depending on slight sequence differences in immunoglobulins. A nonfucosylated or fucose-deficient antibody has reduced fucose compared to the amount of fucose of the same antibody produced in a cell line. Antibody fucosylation can be measured, for example, in N-glycosidase F treated antibody compositions assessed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS).

[0096] In some embodiments, the Fc region comprises one or more mutations that reduce or eliminate fucosylation, e.g., a substitution at Asn297 (EU numbering of Fc region residues) of human IgG1 Fc region. Optionally, the Fc region further comprises one or more amino acid substitutions therein that further improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region. Examples of publications related to "defucosylated" or "fucose-deficient" antibodies include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; Okazaki et al. al. J. Mol. Biol. 336:1239-1249(2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614(2004).

[0097] In some embodiments, the defucosylated antibody or nonfucosylated binding protein is produced in a cell line with genetic modifications resulting in a defucosylated or nonfucosylated antibody. Examples of cell lines producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application 2003 / 0157108 A1 (Presta, L), and WO2004 / 056312 A1 (Adams et al.) (especially in Example 11)), as well as knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. al. Biotech. Bioeng. 87:614 (2004)), β1,4-N-acetylglucosaminyltransferase III (GnT-III) and Golgi μ-mannosidase II (ManII), and cells in which mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (MGAT1; see Byrne, G. et al. (2018) PLoS Biol. 16:e2005817) has been knocked out.

[0098] In some embodiments, defucosylated or nonfucosylated binding protein is produced in a cell line treated with an inhibitor of sugar processing enzyme(s), such as kifunensine, an inhibitor of mannosidase I (see, e.g., Elbein, AD et al. (1990) J. Biol. Chem. 265:15599-15605). For example, cells can be centrifuged, resuspended in growth medium containing kifunensine (e.g., at 250 μg / mL), cultured, and used for antibody production.

[0099] In some embodiments, one or both of the first and second antigen binding domains, antibodies, or fragments comprise a tag, e.g., for affinity purification, hi some embodiments, the tag is a polyhistidine tag.

[0100] In some embodiments, a multispecific (e.g., bispecific) binding molecule comprises a single chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds human Dectin-1 and a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region connected to the VH domain. In some embodiments, the scFv arm binds Dectin-1, and a conventional antibody arm having a VH domain and a VL domain on separate polypeptides binds to a target of interest (e.g., as described herein), such as a disease-causing agent. In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. A non-limiting example of this format is shown in FIG. 43.

[0101] In some embodiments, the disease-causing agent is a B cell, a tumor or a cancer cell, e.g., 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 starting from the late pre-B lymphocyte stage. Therefore, therapies that deplete B cells have targeted CD20 for various indications, including cancer (e.g., non-Hodgkin's lymphoma or chronic lymphocytic leukemia) and autoimmune conditions (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and Wegener's granulomatosis).

[0102] In some embodiments, provided herein is a multispecific (e.g., bispecific) binding molecule comprising a single chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region connected to the VH domain, wherein the VH domain and the VL domain of the second antibody arm form an antigen binding domain that binds to a target of interest (e.g., a disease causing agent of the present disclosure). In some embodiments, the first Fc region comprises one or more knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the present disclosure between the VH domain and the VL domain, and a second linker of the present disclosure between the VL domain and the first Fc region.QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGG SGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIE EPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31) or QVQL VQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGG GSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGPGTKVDIEEPKR SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 37).

[0103] In some embodiments, provided herein is a multispecific (e.g., bispecific) binding molecule comprising a single chain variable fragment (scFv) comprising a VH domain and a VL domain of the present disclosure that binds to human Dectin-1, and a first antibody arm comprising a first Fc region, an antibody heavy chain comprising a VH domain associated with an antibody light chain comprising a VL domain, and a second antibody arm comprising a second Fc region connected to the VH domain, wherein the VH domain and 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 knob-forming mutations and the second Fc region comprises one or more cognate hole-forming mutations, or the second Fc region comprises one or more knob-forming mutations and the first Fc region comprises one or more cognate hole-forming mutations. In some embodiments, the scFv comprises a first linker of the disclosure between the VH domain and the VL domain, and a second linker of the disclosure between the VL domain and the first Fc region. In some embodiments, the first antibody arm is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31).In some embodiments, the second antibody arm is QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE a second polypeptide comprising the sequence of PQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (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 is QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGASSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTF It comprises the amino acid sequence of GPGTKVDIEEPKRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 37).In some embodiments, the second antibody arm is QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE a second polypeptide comprising the sequence of PQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 38); and a third polypeptide comprising the amino acid sequence of QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33).

[0104] A multispecific antibody has binding specificities for at least two different epitopes, usually from different antigens. Multispecific or bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0105] To enable targeted removal of disease-causing agents by phagocytosis, the antigen-binding domain of the present disclosure may be selected from IgG, intrabodies, peptibodies, nanobodies, single domain antibodies, SMTPs, and multispecific antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFvs, tandem tri-scFvs, ADAPTIRs).

[0106] Methods for making bispecific antibodies are known in the art. Established methods for making bispecific antibodies include the "knobs-into-holes" or "protuberance-into-cavity" approach. See, for example, U.S. Patent No. 5,731,168. Two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each contain an interface; the interface of one immunoglobulin polypeptide interacts with a corresponding or cognate interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. In some embodiments, the interface can be engineered such that a "knob" or "protuberance" located at the interface of one immunoglobulin polypeptide corresponds to a cognate "hole" or "cavity" located at the interface of the other immunoglobulin polypeptide. In some embodiments, the knobs can be constructed by replacing small amino acid side chains with larger side chains. In some embodiments, the holes can be constructed by replacing large amino acid side chains with smaller side chains. The knobs or holes may be present in the original interface or may be synthetically introduced. Polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding knob- or hole-forming mutations can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, for example, U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; 8,679,785; 8,844,834; U.S. Patent Application Publication No. 2013 / 0089553; Spiess et al., Nature Biotechnology 31:753-758, 2013; and Ridgway and Carter (1996) Protein Eng. 9:617-621. The modified immunoglobulin polypeptides can be produced using prokaryotic host cells, such as E. coli, or eukaryotic host cells, such as mammalian cells (eg, CHO cells) or yeast cells.Immunoglobulin polypeptides with corresponding knobs and holes can be expressed in host cells in co-culture and purified together as heteromultimers, or expressed in monoculture and purified separately and assembled in vitro. Exemplary cognate knob and hole mutations are shown below (numbering according to the EU index). The EU numbering used herein is known in the art. See, for example, the IMGT resources at www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html and www.imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html. As used herein, an "antibody arm" can refer to a pair of antibody heavy and light chains, where the variable domains of the heavy and light chains form an antigen binding site that binds to a target antigen. [Table 2]

[0107] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences.

[0108] In some embodiments, the multispecific (e.g., bispecific) antibody further comprises one or more mutations in only one of the antibody arms to improve heavy / light chain pairing. For example, amino acid substitutions can be used to replace the native disulfide bond at the CH1-CL interface of one antibody arm with an engineered disulfide bond. See, for example, Mazor, Y. et al. (2015) MAbs 7:377-389 and EP3452089A2. In some embodiments, the multispecific or bispecific antibody comprises two antibody light chains and two antibody heavy chains, where only one of the antibody heavy chains comprises the amino acid substitutions F126C and C220V according to EU numbering, and only the corresponding or cognate light chain comprises the amino acid substitutions S121C and C214V.

[0109] Multispecific (e.g., bispecific) antibodies also include cross-linked or "heteroconjugate" antibodies. Techniques for producing bispecific antibodies from antibody fragments are also described in this document. For example, bispecific antibodies can be prepared using chemical linkage. In some embodiments, a bispecific antibody comprises a first IgG antibody comprising a first antigen-binding domain covalently linked to a second IgG antibody comprising a second antigen-binding domain.

[0110] In some embodiments, the multispecific (e.g., bispecific) antibody further comprises one or more mutations in only one of the antibody arms to reduce binding affinity to Protein A. See, e.g., Ollier, R. et al. (2019) MAbs 11:1464-1478 and AU2018204314. In some embodiments, the multispecific or bispecific antibody comprises two antibody light chains and two antibody heavy chains, where only one of the antibody heavy chains comprises the amino acid substitutions H435R and Y436F according to EU numbering.

[0111] In some embodiments, the monospecific or multispecific (e.g., bispecific) antibody further comprises one or more mutations to reduce effector function, e.g., to reduce or eliminate binding of the Fc region to an Fc receptor. In some embodiments, the antibody comprises two antibody Fc regions, where the antibody Fc regions comprise amino acid substitutions at one or more of positions 234, 235, and 237 according to EU numbering. In some embodiments, the antibody comprises two antibody Fc regions, where the antibody Fc regions comprise L234A, L235E, and G237A substitutions according to EU numbering.

[0112] In some embodiments, a monospecific or multispecific (e.g., bispecific) antibody comprises two antibody heavy chains and two antibody light chains, wherein the VH domain of a first antibody heavy chain forms an antigen-binding domain with the VL domain of a first antibody light chain, and the VH domain of a second antibody heavy chain forms an antigen-binding domain with the VL domain of a second antibody light chain, wherein the first antibody heavy chain comprises F126C, C220V, and T366W substitutions according to EU numbering, the first antibody light chain comprises S121C and C214V substitutions, and the second antibody heavy chain comprises T366S, L368A, Y407V, H435R, and Y436F substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain further comprise L234A, L235E, and G237A substitutions according to EU numbering. In some embodiments, the first antibody heavy chain and the second antibody heavy chain comprise a human IgG1 Fc domain.

[0113] In some embodiments, provided herein is a polynucleotide encoding an antibody or multispecific binding protein of any one of the above embodiments. In some embodiments, provided herein is a vector (e.g., an expression vector) comprising a polynucleotide of any one of the above embodiments. In some embodiments, provided herein is a host cell (e.g., an isolated host cell or cell line) comprising a polynucleotide or vector of any one of the above embodiments. In some embodiments, provided herein is a pharmaceutical composition comprising a multispecific binding protein of any one of the above embodiments and a pharma- ceutically acceptable carrier. Any of these can be used in the production and / or processing methods disclosed herein.

[0114] In some embodiments, provided herein is a method of producing a multispecific binding protein, comprising culturing a host cell of any one of the above embodiments under conditions suitable for production of the multispecific binding protein. In some embodiments, the method further comprises recovering the multispecific binding protein. Multispecific binding proteins can be produced using standard recombinant techniques, as described herein and / or exemplified below.

[0115] Antibodies and antibody fragments can be produced using recombinant methods. For example, nucleic acids encoding the antibody / fragment can be isolated and inserted into a replicable vector for further cloning or for expression. DNA encoding the antibody / fragment can be easily isolated and sequenced using conventional procedures (e.g., via oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody / fragment). Many vectors are known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Suitable host cells for cloning or expressing the DNA in the vectors herein are prokaryote, yeast, or higher eukaryote cells. When using recombinant techniques, the antibody / fragment can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody / fragment is produced intracellularly, particulate debris (either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. If the antibody / fragment is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter.

[0116] In some embodiments, the multispecific binding proteins of the disclosure are part of a pharmaceutical composition, for example, comprising an antibody and one or more pharma- ceutically acceptable carriers. The pharmaceutical compositions and formulations described herein can be prepared in the form of a lyophilized formulation or an aqueous solution by mixing the active ingredient (e.g., a fusion protein) having the desired purity with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers, e.g., phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants, e.g., polyethylene glycol (PEG).

[0117] Certain aspects of the disclosure relate to kits or articles of manufacture that include 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 disorder, such as cancer.

[0118] Suitable containers include, for example, bottles, vials, syringes, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating a condition and may have a sterile access port (e.g., the container can be an intravenous solution bag, or a vial with a stopper pierceable by a hypodermic injection 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 particular condition. The label or package insert further comprises instructions for administering the multispecific binding protein composition to a subject. Articles of manufacture and kits containing the combination therapeutic agents described herein are also contemplated.

[0119] II.How to use In certain aspects, the disclosure provides a method of treating a disease or disorder comprising administering an effective amount of an antibody, antibody fragment, multispecific (e.g., bispecific) binding molecule, or composition of the disclosure to an individual in need thereof. In some embodiments, the individual is a human. In some embodiments, the individual is diagnosed with or has been diagnosed with cancer (e.g., non-Hodgkin's lymphoma or chronic lymphocytic leukemia) or a B-cell-associated autoimmune condition (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and Wegener's granulomatosis).

[0120] In some embodiments, the method includes using a multispecific (e.g., bispecific) binding molecule of the present disclosure having a first antigen-binding domain that binds human Dectin-1 and a second antigen-binding domain that binds a disease-causing agent, e.g., CD20 or hCD20. In some embodiments, the disease-causing agent is a B cell, a tumor or a cancer cell, e.g., a malignant B cell. In some embodiments, CD20 is expressed on the surface of a B cell, such as a malignant B cell. Binding of a molecule that mediates targeted removal of a disease-causing agent by phagocytosis can be with or without avidity, i.e., with or without induction of dimerization of a phagocytic receptor, such as Dectin-1, or a target antigen present on the disease-causing agent.

[0121] In addition to the beneficial removal of disease-causing agents by phagocytosis, the molecule may induce the production of inflammatory mediators to alter the microenvironment of diseases such as tumors, cancers, and lymphomas. Without wishing to be bound by theory, it is believed that molecules that perform targeted phagocytosis may show clear benefits to patients with, for example, cancer, inflammatory, or immune diseases (e.g., autoimmune diseases, inflammatory bowel disease, multiple sclerosis), degenerative diseases (e.g., joints and cartilage), rheumatoid arthritis, Felty's syndrome, aggressive NK leukemia, IBM, IBD, and the like. In addition, targeted phagocytic antibody therapy may be more active in depleting cells in tissues than ADCC, which relies on NK cells. This treatment targets bone marrow cells and may have selective activity for removing specific disease-causing agents rather than treatments that improve phagocytosis in general. For example, a target of interest for cancer treatment is CD20.

[0122] The following description is presented to enable those skilled in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein can be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described and shown herein, but should be accorded the scope consistent with the claims. EXAMPLES

[0123] Example 1: Functional characterization of 2M24 anti-Dectin-1 antibody This example describes the production of monoclonal antibodies specific for human Dectin-1. This example describes the characterization of novel anti-human Dectin-1 antibodies.

[0124] Materials and Methods Production of anti-Dectin-1 antibodies Four-week-old ATX-Gx transgenic mice were subcutaneously inoculated with recombinant human dectin-1 isoform B for 5 weeks and boosted with antigen once a week. Antibody titers in mouse serum were assessed before and after the booster vaccination via ELISA and flow cytometry. Mice with the highest serum antibody titers were selected to provide B cells for hybridoma generation.

[0125] Prior to cell fusion, mice were boosted once with recombinant human Dectin-1 isoform B. Mice were sacrificed and spleens were harvested. Spleen cells were mixed with SP2 / 0-Ag14 myeloma cells and incubated at 37°C, and fusion was induced in the presence of polyethylene glycol (PEG) or by electroporation. Cells were then harvested and plated at limiting dilution in 96-well plates at one cell per well. Cells were then treated with hypoxanthine, aminopterin, and thymidine (HAT) medium and cultured for more than two weeks for selection.

[0126] To identify candidates specific for Dectin-1, hybridoma supernatants were screened by flow cytometry on cells overexpressing Dectin-1 and on human primary monocytes. Cross-reactivity of cynomolgus Dectin-1 was assessed using flow cytometry with antibodies that bind to cynomolgus primary monocytes.

[0127] Healthy donor samples Fresh healthy donor buffy coats were obtained from Stanford Blood Center. Peripheral blood mononuclear cells were isolated by ficoll paque (GE Healthcare, Chicago, IL) separation and cryopreserved in Bambanker cell freezing medium (Bulldog Bio, Portsmouth, NH). Briefly, buffy coats were diluted with phosphate-buffered saline (1:1 ratio), followed by layering the diluted buffy coat on ficoll and centrifuging at 760 g. The PBMC layer was isolated and washed with PBS before downstream analysis. Peripheral blood leukocytes were isolated by red blood cell lysis. Cryopreserved cynomolgus monkey PBMCs were obtained from Human Cells Biosciences.

[0128] Primary cells and cell culture Human monocytes were isolated from healthy donor PBMCs using a pan-monocyte isolation kit (Miltenyi Biotec, Inc., Auburn, CA) according to the manufacturer's instructions. For macrophage and dendritic cell differentiation, monocytes were cultured in RPMI containing 10 human serum (Millipore Sigma) in the presence of 50 ng / ml MCSF (Peprotech, Rocky Hill, NJ) for 6 days to fully differentiate into macrophages, or in the presence of 50 ng / ml GMCSF and 50 ng / ml IL-4 (Peprotech, Rocky Hill, NJ) for 6 days to fully differentiate into dendritic cells. Cytokine-containing medium was changed every 3 days.

[0129] HEK Blue hDectin-1-a and HEK Blue hDectin-1-b cells (Invivogen, San Diego, CA) were maintained in DMEM / 10% FBS supplemented with mormocin and puromycin according to the manufacturer's instructions. Freestyle 293F cells were transiently transfected according to the manufacturer's instructions (Thermo Fisher, Waltham, MA). Briefly, viable cell density and viability were measured. Cells were cultured in Freestyle 293 Expression Medium at a final density of 11 × 10 6The Freestyle Max reagent was diluted to 1000 viable cells / mL. The Freestyle Max reagent was diluted in OptiPro SFM medium, mixed, and incubated at room temperature for 5 minutes. The diluted Freestyle Max reagent was added to the plasmid DNA diluted in OptiPro SFM medium and mixed. The Freestyle Max reagent / plasmid DNA complex was incubated at room temperature for 10-20 minutes. The complex was slowly transferred to the cells, with gentle rotation of the culture flask during addition, and the cells were incubated on an orbital shaker in a 37°C incubator with 80% relative humidity and 8% CO2.

[0130] Binding of Dectin-1 Antibody to Dectin-1-Expressing Cells Dectin-1 expressing cells (HEK Blue hDectin-1-a, HEK Blue hDectin-1-b, HEK293F hDectin-1 a FL, human monocytes, or cynomolgus monkey monocytes) were plated at 1 × 10 per well in non-tissue culture treated 96-well V-bottom plates. 5 ~2×10 5 Human monocytes were plated with 100 mM glycerol (GlcNAc) and 10 mM ethanol. Human monocytes were further incubated with human FcgR blocking antibody (Biolegend, San Diego, CA) for 10 min at room temperature to reduce antibody binding to Fc receptors. Cells were then stained with eFluor 506 viability dye (ThermoFisher, Waltham, MA) at 1:1000 dilution for 30 min on ice, followed by a washing step with FACS buffer (PBS containing 2% fetal bovine serum). Primary Dectin-1 antibodies or isotypes were used at titrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41, and 0.14 nM and incubated for 30 min on ice, followed by a further washing step with FACS buffer.

[0131] For detection of mouse primary antibodies, cells were incubated with fluorescently labeled AF647 anti-mouse Fc-specific secondary antibody (Jackson Immuno). For detection of human IgG4 primary antibodies, cells were incubated with Alexa Fluor 647 anti-human Fc-specific secondary antibody (Jackson Immuno) (detection in HEK cells) or FITC anti-human IgG4 antibody (Sigma) (detection in primary monocytes) for 30 min on ice. Data were acquired using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA) and analyzed using Graphpad Prism 8.4.

[0132] Dectin-1 antibody inhibition of laminarin HEK Blue hDectin-1a cells were cultured at 1 × 10 per well in non-tissue culture treated 96-well V-bottom plates. 5 Cells were plated with 100 μg / ml of biotin-laminarin. Primary anti-Dectin-1 antibodies were used at titrations of 300, 100, 33.3, 11.1, 3.7, 1.23, 0.41, 0.14, 0.05, 0.015, and 0.005 nM and incubated in the presence of 8 μg / ml of biotin-laminarin for 30 min on ice. After a washing step with FACS buffer, binding of biotin-laminarin on HEK cells was detected using streptavidin-AF647 for 30 min on ice. For analysis, 4000 cell events were acquired on a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA) and analyzed using Graphpad Prism 8.4.

[0133] Labeling of polystyrene beads with pHrodo and conjugation to antibodies Polystyrene beads of different sizes coated with goat anti-mouse IgG (or biotin) (Spherotech, Lake Forest, IL) were washed twice with PBS / Tween® 20 0.05%. pHrodo Red, succinimidyl ester (pHrodo Red, SE) (ThermoFisher, Waltham, MA) was added to the beads at 10 μM and incubated for 60 minutes at room temperature with shaking. The beads were then washed with PBS / BSA 0.1% to remove excess pHrodo Red.

[0134] After pHrodo labeling, antibodies were conjugated to the beads according to the manufacturer's recommendations. Briefly, based on the beads' binding capacity to the antibodies, a 5-fold excess of antibodies was added to the beads and incubated with shaking at room temperature for 60 min. The beads were then washed with PBS / BSA 0.1% to remove unbound antibodies. To assess the quality of the beads, pHrodo red activation was assessed in low pH buffer by flow cytometry. Antibody binding to the beads was assessed using fluorescently labeled AF647 anti-mouse Fc-specific antibody or FITC anti-human IgG4 antibody secondary antibodies.

[0135] Antibody-dependent targeted phagocytosis of Phrodo-labeled beads For phagocytosis experiments, 50,000 HEK cells or primary cells (macrophages or dendritic cells) overexpressing Dectin-1 were seeded in 96-well plates in RPMI containing ultra-low IgG FBS at 10. 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 spun down briefly.

[0136] In some experiments, cells were labeled by adding the cell tracker calcein AM (Thermo Fisher, Waltham, MA). Phagocytosis was monitored by taking images at desired time points with an IncuCyte S3 live imaging system (Germany) and analyzed using IncuCyte S3 software. Phagocytosis was quantified as the overlap of bright red fluorescence (engulfed beads) with calcein AM positive cells or the integrated red intensity of bright red fluorescence.

[0137] SEAP reporter assay in HEK cells overexpressing dectin-1 using anti-dectin-1 antibody Anti-Dectin-1 monoclonal antibody 2M24 (VH and VL domains contain SEQ ID NOs: 7 and 8, respectively) or 15E2 and control isotypes were immobilized by coating on the surface of wells of untreated 96-well U-bottom polypropylene microtiter plates. For coating, 10, 2, 1, 0.5, and 0.1 μg of anti-Dectin-1 antibody diluted in 50 μl of sterile PBS were added to each well. The plates were left overnight in a class II laminar flow cabinet with the lid removed to allow the solution to evaporate. The coated plates were washed twice with 200 μl of sterile PBS to remove salt crystals and unbound antibody. HEK Blue hDectin-1-a cells were then cultured on the plates in RPM1 with 10% ultra-low IgG FBS (VWR) for 22 hours, and alkaline phosphatase levels were assessed in the supernatants at OD 630 nm using QUANTI Blue Solution (Invivogen, San Diego, CA) according to the manufacturer's instructions.

[0138] To measure the amount of HEK cell SEAP secretion induced by anti-Dectin-1 antibody-conjugated beads, streptavidin-2M24 (hIgG4) was conjugated to biotin polystyrene beads of 3, 10, and 16 μm size (Spherotech, Lake Forest, IL) by incubating the beads with the antibody for 30 min at room temperature and washing twice with PBS to remove unbound antibody. Anti-Dectin-1 antibody-conjugated beads were added at 1 × 10 5 After mixing with HEK Blue hDectin-1-a cells at a cell:bead ratio of 1:3 in RPM1 containing 10% ultra-low IgG FBS for 22 h, alkaline phosphatase secretion was assessed in the supernatant at OD 630 nm as described above.

[0139] Cytokine secretion Anti-Dectin-1 monoclonal antibodies 2M24 or 15E2 clones and control isotypes were immobilized by coating 10 μg on the surface of wells of untreated 96-well U-bottom polypropylene microtiter plates as described above. Freshly isolated monocytes or peripheral blood mononuclear cells were then cultured on the plates with immobilized antibodies at 200,000 cells / well in RPM1 with 10% ultra-low IgG FBS for 24 h. In other wells, cells were treated with 10 μg / ml Dectin-1 antibodies in solution instead of immobilized antibodies. TNFα, IL-6, and IFNg in the supernatants were assessed using the U-PLEX assay platform (Meso Scale Discovery), and their levels were expressed as fold change in Dectin-1 antibody-induced cytokine secretion relative to isotype control. As a positive control, cells were stimulated with 25 μg / ml zymosan.

[0140] result To generate Dectin-1 antibodies, 4-week-old ATX-Gx Alloy transgenic mice were subcutaneously inoculated with recombinant Dectin-1 isoform B protein and boosted weekly with antigen, and the antibodies generated by this inoculation have human variable domains and mouse constant domains.

[0141] Of the 56 anti-Dectin-1 antibody clone candidates generated in this study, the 2M24 clone was the only one that showed binding to both Dectin-1 isoforms A and B on HEK cells as well as monocytes. As shown in Figure 1A, the 2M24 anti-Dectin-1 antibody clone showed high affinity for Dectin-1-expressing human monocytes. In contrast, other clones either bound only to Dectin-1 isoform A (e.g., 2M08, 2M12, 2M38) or showed no binding at all (2M49). Furthermore, the affinity of 2M24 for Dectin-1 was superior to that shown by other clones and commercially available Dectin-1 antibodies (15E2, 259931, GE2). Figure 1C shows a comparison of binding to human monocytes and to HEK cells overexpressing Dectin-1 between the 2M24 clone and other Dectin-1 clones identified from immunization of Alloy transgenic mice and commercially available Dectin-1 clones.

[0142] The 2M24 antibody was also evaluated for cross-reactivity to cynomolgus dectin-1. Binding was assessed by flow cytometric analysis of PBMC-derived cynomolgus monocytes. As shown in Figure 1B, the anti-human dectin-1 clone 2M24 antibody showed cross-reactivity and high affinity to cynomolgus dectin-1 expressed on monocytes. The 2M24 anti-dectin-1 antibody had affinity superior to the commercial antibodies tested, showing an EC50 of 0.3 nM. The agonist 15E2 and 255931 commercial antibodies showed EC50s of 14 nM and 16 nM, respectively, on cynomolgus monocytes. Figure 1C shows a comparison of binding to cynomolgus monocytes between the 2M24 clone and the commercial clones 15E2 and 259931.

[0143] To evaluate the functionality of the 2M24 Dectin-1 antibody in promoting phagocytosis, polystyrene beads were coated with the 2M24 antibody and mixed with HEK-Blue hDectin-1a cells or primary human monocytes. The 2M24 antibody efficiently induced phagocytosis of the beads. As shown in Figures 2A-2B, the 2M24 anti-Dectin-1 antibody linked to polystyrene beads promoted phagocytosis in both HEK-Blue hDectin-1a cells and primary human monocytes.

[0144] The fully human 2M24 antibody of IgG4 isotype was generated from the mIgG1 2M24 clone. This antibody has human constant and variable regions. The functionality of hIgG4 2M24 was then assessed for binding to two Dectin-1 expressing cell types, HEK-Blue hDectin-1a cells and human monocytes. As shown in Figures 3A-3B, the fully human 2M24 showed high affinity binding to Dectin-1 in transfected HEK cells (EC50=1.6 nM) and human monocytes (EC50=0.7 nM).

[0145] Next, the hIgG4 2M24 antibody was tested for its ability to promote phagocytosis of beads in dectin-1 expressing cells. As shown in Figure 4, the hIgG4 2M24 antibody exhibited efficient phagocytosis in HEK cells, human monocytes, and human macrophages that overexpress dectin-1. Thus, the fully human IgG4 2M24 antibody can promote phagocytosis of dectin-1 expressing cells.

[0146] The fully human 2M24(hIgG4) anti-Dectin-1 antibody was further tested for its ability to promote signaling through Dectin-1. Activation of Dectin-1 signaling by the antibody can be assessed in a secreted alkaline phosphatase assay using HEK-Blue hDectin-1a cells. HEK-Blue hDectin-1a cells have been engineered to express Dectin-1 isoform A and genes involved in the Dectin-1 / NF-κB / SEAP signaling pathway, and therefore express secreted alkaline phosphatase (SEAP) in response to stimulation with Dectin-1 ligand. As shown in Figures 5A-5B, the 2M24(hIgG4) anti-Dectin-1 antibody induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells in both immobilized and bead-conjugated form. These observations support the idea that 2M24(hIgG4) antibody engages cell surface dectin-1 to promote SEAP secretion, and demonstrate receptor clustering and agonistic activity by this antibody. Furthermore, efficient clustering signaling of dectin-1 can be promoted by beads conjugated to 2M24(hIgG4). Signaling was better induced on larger beads, reflecting better receptor clustering. This supports the idea that dectin-1 clustering promoted by bispecific antibodies, including an anti-dectin-1 antibody targeting phagocytes and an antibody targeting another cell, such as a cancer cell, may promote clustering and signaling by dectin-1 on phagocytes.

[0147] The natural ligand of dectin-1 clusters the receptors and signals downstream of dectin-1 / Syk / NFkB, inducing inflammatory gene expression. To assess whether engagement of dectin-1 antibodies in solution can induce cytokine secretion, monocytes or macrophages were treated with 10 μg / ml of a commercial anti-dectin-1 antibody. As shown in Figure 6A-6B, 15E2 commercial anti-dectin-1 antibody did not induce cytokine secretion in primary human macrophages and monocytes, indicating insufficient clustering of dectin-1 receptors. This data supports that free dectin-1 antibodies in solution do not induce immune stimulation due to insufficient dectin-1 clustering.

[0148] To evaluate whether cytokine secretion could be induced by the 2M24 (hIgG4) anti-Dectin-1 antibody, the antibody was immobilized on beads and cultured with monocytes or PBMCs. As shown in Figures 7A-7B, the 2M24 anti-Dectin-1 antibody induced cytokine secretion in primary human monocytes and PBMCs. The 2M24 antibody not only promoted cytokine secretion but also showed superior immune stimulation compared to that promoted by the 15E2 anti-Dectin-1 agonist antibody. Of 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. Because T cells do not express Dectin-1, they are not directly activated by the anti-Dectin-1 antibody, but are activated by cytokines secreted by monocytes in PBMCs stimulated by Dectin-1 antibody. Thus, the differential effect of Dectin-1 antibody on IFNg was more pronounced in PBMCs than in pure monocytes.

[0149] Finally, we tested the activation of dectin-1 by its natural ligand in the presence of anti-dectin-1 antibodies. HEK-Blue hDectin-1a cells were incubated with 2M24 (hIgG4) dectin-1 antibody or 15E2, 259931, GE2 anti-Dectin-1 commercial antibodies in the presence of 8ug / ml biotinylated laminarin at 1 / 3 serial dose titrations starting at 300nM. As shown in Figure 8, binding of 2M24 (hIgG4) antibody to dectin-1 did not inhibit binding of laminarin, the natural ligand of dectin-1. Thus, engagement of dectin-1 with 2M24 anti-Dectin-1 antibody does not inhibit pathogen clearance and is unlikely to increase susceptibility to potential fungal infections.

[0150] In conclusion, the 2M24 anti-Dectin-1 antibody can induce phagocytosis by Dectin-1 expressing cells and can induce activation of Dectin-1 signaling without competing with the natural ligand of Dectin-1. The properties of the 2M24 and 15E2 antibodies are summarized in Figure 9.

[0151] Example 2: Bispecific anti-Dectin-1 antibodies This example describes the generation and characterization of a bispecific antibody that contains a Dectin-1-binding arm and a second arm that binds to a specific tumor antigen.

[0152] Materials and Methods Creation of bispecifics Antibodies were differentially labeled with MTA or FOL reagents according to the manufacturer's (AAT Bioquest) guidelines. The labeled antibodies were mixed and incubated to allow for covalent assembly via MTA and FOL interactions. The following antibodies were used for biotin:streptavidin-derived bispecific antibodies: Anti-Dectin-1 15E2 antibody heavy chain:mSA fusion QWQLQQSGAELARPGASWKMSCKASGYTFTTYTMHWWKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTASMQLSSLTSEDSAWYYCARERAVLVPYAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH (SEQ ID NO: 18) Anti-Dectin-1 15E2 antibody light chain QIVLTQSPAVMSASPGEKWTITCTASSSLSYMHWFQQKPGTSPKLWLYSTSILASGVPTRFSGSGSGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) Avi-tagged anti-CD20 Fab heavy chain (CH1 domain based on hIgG4 sequence) QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE (SEQ ID NO: 20) Anti-CD20 Fab light chain QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 21) Avi-tagged anti-HER2 Fab heavy chain (CH1 domain based on hIgG4 sequence) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVAAAGASHHHHHHGSGLNDIFEAQKIEWHE (SEQ ID NO: 22) Anti-HER2 Fab light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 23)

[0153] Cell ligation assay Dectin-1 expressing cells were labeled with calcein green and target cells were labeled with calcein red. Cells were incubated in the presence of bispecific or isotype control antibodies and analyzed by flow cytometry. Cell ligation was indicated by a double positive signal (green+red+). Ligation efficiency was quantified as the percentage of total target cells that formed doublets with Dectin-1 expressing cells.

[0154] Five million effector (Dectin-1 expressing cells) or target cells (cells expressing a target of interest, e.g., CD20-positive Raji cells or HER2-positive SKBR3 cells) were differentially labeled with either calcein green (0.5 nM) or calcein red / pHrodo-red (0.5 nM). Cells were washed extensively with PBS and placed on ice. Effector and target cells were then co-cultured at a 3:1 ratio (effector:target) in the presence of 2M24 bispecific antibody or isotype control and incubated at 37 °C for 30 min. After incubation, samples were gently resuspended and analyzed by flow cytometry. PMT voltages were adjusted accordingly and cells were gated based on FITC and / or PE fluorescence corresponding to calcein green or red fluorescence. Ligation efficiency was reported as the number of PE-positive cells (target cells) in the doublet population divided by the total number of PE-positive target cells in the reaction.

[0155] SEAP reporter assay in HEK cells overexpressing dectin-1 using anti-dectin-1 antibody To measure the amount of SEAP secretion from HEK cells induced by Raji cells (expressing CD20), Raji cells were coated with 2M24 / anti-hCD20 or hIgG4 / anti-CD20 bispecifics for 30 min on ice, followed by two washes with PBS to remove unbound bispecifics. Bispecific-coated Raji cells were cultured at 1 × 10 in RPM1 containing 10% ultra-low IgG FBS. 5 The cells were mixed with HEK Blue hDectin-1-a cells at a ratio of 1:2 (HEK cells:Raji cells). After 22 hours, the amount of alkaline phosphatase secreted in the supernatant was evaluated at OD 630 nm as described in Example 2.

[0156] result Dectin-1 agonist bispecific antibodies can exploit various modes of activity (e.g., immune activation, phagocytosis, neo-antigen presentation, and adaptive immune activation) for targeted depletion of cancer cells (Figure 11). As a proof of concept for engaging dectin-1 antibodies (15E2 or 2M24) with targeting antibodies, a click chemistry approach was used to generate bispecifics containing an anti-dectin-1 targeting arm and a second arm targeting a protein of interest. This approach allowed the generation of bispecifics for a variety of assays. A schematic of this approach is shown in Figures 10A-10B. Because binding of dectin-1 by dectin-1-specific antibodies can induce phagocytosis of targets (see Examples 1 and 2), bispecific antibodies were evaluated for their ability to promote phagocytosis of specific target cells. First, bispecifics were evaluated for their ability to eliminate CD70-expressing cancer cells by phagocytosis. CD70 is a type II transmembrane glycoprotein that belongs to the tumor necrosis factor (TNF) superfamily. CD70 is expressed at low levels in normal tissues, but is highly overexpressed in a variety of diseases, including acute myeloid leukemia (AML), renal cell carcinoma, rheumatoid arthritis, and lupus.

[0157] Click chemistry was used to generate bispecific molecules containing a dectin-1 targeting arm (anti-dectin-1; clone 2M24) and a CD70 targeting arm (anti-hCD70; clone 113-16). The purity of the bispecific (2M24 / anti-hCD70) antibody was assessed by SDS-PAGE analysis (Figure 12A) and the binding was assessed by flow cytometry analysis (Figure 12B). As shown in Figure 12B, binding studies on cells revealed that 2M24 / anti-hCD70 bound to a dectin-1 expressing HEK293 cell line with an EC50 of 1.8 nM and to a CD70 positive kidney cancer cell line with an EC50 of 12.34 nM (A498 cells) or 11.62 nM (786-0 cells). The bispecific was then evaluated for its ability to induce cell ligation. As shown in FIG. 13, the 2M24 / anti-hCD70 bispecific induced ligation between Dectin-1-expressing HEK293 cells and CD70-expressing renal carcinoma cells, resulting in cell doublets of HEK293 cells (labeled with calcein green) and A498 cells (labeled with calcein red).

[0158] Next, targeting of CD20-expressing cells by the bispecific was evaluated. CD20 is a transmembrane protein present on almost all B cells from the stage of their commitment to B cell development until it is downregulated during differentiation into antibody-secreting plasma cells, and is considered a pan-B cell antigen marker. As shown in Figures 14A-14B, the 2M24 / anti-hCD20 bispecific induced ligation of Dectin-1 expressing cells (both Dectin-1 expressing HEK293 cells and human M0 macrophages) with CD20 expressing B cells (Raji cell line). This intercellular ligation mediated by the bispecific may induce synapse formation between effector and target cells, alter cytokine signaling, activate phagocytosis, and ultimately perform target antigen presentation.

[0159] To test the induction of signaling resulting from stimulation with bispecific antibodies that bind Dectin-1, a secretory alkaline phosphatase assay was performed. As shown in FIG. 15, Raji cells coated with anti-Dectin-1 / anti-CD20 bispecifics induced alkaline phosphatase secretion in HEK-Blue hDectin-1a cells. Thus, using bispecific antibodies to connect target cells to cells that express Dectin-1 (such as phagocytes) can promote signaling by Dectin-1 expressing cells. In the case of phagocytes, signaling can result in cytokine production and immune stimulation.

[0160] It has been previously demonstrated that Dectin-1 expression in HEK293 cells is necessary and sufficient to induce phagocytosis of beads of various sizes coated with anti-Dectin-1 targeting antibodies (see Examples 1 and 2). To demonstrate phagocytosis of live target cells, a bispecific was generated that contains a Dectin-1 targeting arm and a CD20 targeting arm. In co-culture assays of HEK293 cells and CD20-expressing Raji cells, phagocytosis of cells treated with the anti-Dectin-1 / anti-hCD20 bispecific was observed in contrast to an isotype control bispecific (Figure 16). Furthermore, pre-incubation of cells with latrunculin A, an inhibitor of phagocytosis that inhibits actin polymerization, inhibited phagocytosis of cells treated with the anti-Dectin-1 / anti-hCD20 bispecific. These findings indicate that expression of Dectin-1 is sufficient to induce phagocytosis and that co-targeting Dectin-1 and a target of interest with a Dectin-1 agonist bispecific is sufficient to induce phagocytosis of target cells.

[0161] We performed a proof-of-concept experiment to co-target Dectin-1 expressing cells and HER2 positive breast cancer cells using an anti-Dectin-1 / anti-HER2 bispecific antibody. Approximately 20%-25% of invasive breast cancers overexpress the human epidermal growth factor receptor HER2 tyrosine kinase receptor. As shown in Figure 17, the anti-Dectin-1 (15E2) / anti-HER2 bispecific induced the association of Dectin-1 with HER2 expressing cells. This interaction is thought to promote synapse formation between effector and target cells, as Dectin-1 clustering induces cytokine secretion by effector cells and induces phagocytosis of target cells, resulting in neo-antigen presentation and activation of adaptive immune cells (B and T cells).

[0162] Finally, the anti-Dectin-1 (2M24) / anti-hCD94 bispecific was also evaluated. Large granular lymphocytic (LGL) leukemia is a rare chronic lymphoproliferative disorder of the T-cell and natural killer (NK) cell lineage. CD94 / NKG2 is a family of C-type lectin receptors expressed primarily on the surface of NK cells and a subset of CD8+ T lymphocytes. As shown in Figure 18, the anti-Dectin-1 (2M24) / anti-hCD94 bispecific induced the linkage of Dectin-1-expressing cells to CD94-expressing cells. Thus, bispecific antibodies that bind Dectin-1 can mediate the linkage of Dectin-1-expressing cells to various target cells.

[0163] Example 3: Generation of bispecific anti-Dectin-1 antibodies using streptavidin-biotin This example describes the biochemical and functional characterization of bispecific antibodies that bind Dectin-1, generated using streptavidin-biotin conjugation.

[0164] Materials and Methods Creation of bispecifics mSA was genetically fused to either Fab 2M24 or full-length 2M24. The chimeric fusion was incubated with a biotinylated targeting antibody to generate a bispecific containing a Dectin-1-binding arm and a second arm that binds to a target receptor or protein of interest. Full length 2M24 sequence fused to mSA: (SEQ ID NO:15) Fab 2M24 sequence fused to mSA: QVQLVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPSSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVGGGSGGGSGGGSEFASAEAGITGTWYNQHGSTFTVTAGADGNLTGQYENRAQGTGCQNSPYTLTGRYNGTKLEWRVEWNNSTENCHSRTEWRGQYQGGAEARINTQWNLTYEGGSGPATEQGQDTFTKVKPSAASGSAAAGASHHHHHH (SEQ ID NO: 17)

[0165] Antibody-dependent targeted phagocytosis of Phrodo-labeled beads Antibody-dependent targeted phagocytosis of Phrodo-labeled beads was performed as described in Example 2. To monitor phagocytosis by flow cytometry, HEK cells overexpressing Dectin-1 were incubated with biotin beads conjugated to Fab 2M24-mSA for 30 min on ice or at 37° C. for 30 min, followed by two washes with PBS. Phagocytosis was assessed by detecting activated Phrodo red within the HEK cell / bead duplet population by flow cytometry in the PE channel using a CytoFlex flow cytometer (Beckman Coulter, Atlanta, GA).

[0166] result To enable efficient generation of bispecific antibodies, a new strategy was developed that exploits the high affinity interaction of streptavidin and biotin. A monomeric streptavidin (mSA) construct was fused to the Fc domain of 2M24 or to the CH1 domain of Fab 2M24. The recombinant fusion protein was incubated with various biotinylated antibodies of interest to allow assembly of bispecifics. A schematic of this strategy is shown in Figures 19A-19B.

[0167] This fusion technology allows for high-throughput production and screening of bispecific antibodies. To test this approach, Fab 2M24-mSA fusion protein was generated and purified. As shown in Figures 20A-20C, Fab 2M24-mSA fusion showed high affinity binding to Dectin-1 expressing cells (EC50=1.45 nM). This Fab 2M24-mSA fusion protein can be combined with various biotinylated antibodies against targets of interest. Furthermore, Fab 2M24-mSA fusion also induced bead binding and phagocytosis by Dectin-1 expressing HEK 293 cells (Figures 21A-21B), indicating that the Fab version of the 2M24 antibody can efficiently promote phagocytosis in cells expressing Dectin-1.

[0168] Anti-Dectin-1-streptavidin fusions were used to generate bispecifics against various targets (e.g., CD20, CD19, CD70, amyloid B(1-42)). As shown in Figures 22A-22D, these bispecifics showed high homogeneity based on HPLC analysis. These data indicate the robust feasibility of this technology in generating bispecific antibodies.

[0169] Next, the anti-Dectin-1 bispecific generated using Fab 2M24-mSA fusion protein was evaluated for its ability to induce cell ligation. As shown in Figure 23, the Fab 2M24-mSA / biotin anti-hCD20 bispecific induced ligation between Dectin-1 expressing HEK293 cells and CD20 expressing B cells (Raji cell line). This interaction can induce cytokine secretion by effector cells, trigger phagocytosis of target cells, and promote Dectin-1 clustering leading to neo-antigen presentation and activation of adaptive immune cells (B and T cells).

[0170] Example 4: Bispecific design to generate human bispecific antibodies targeting Dectin-1 and a disease target or antigen To allow for the assembly and efficient production of highly purified and active bispecifics, design principles were employed based on previously reported strategies such as "knobs-into-holes" (Ridgway, 1996; patent US8679785B2), DuetMab (Mazor, 2015; patent EP3452089A2), single-step Protein A and G avidity purification methods (Ollier, 2019; AU2018204314B2), as well as mutations to eliminate FcR binding (patent WO2016 / 081746A2). Assembly of the complete bispecific involves expression of the four individual subunits cloned into an expression vector such as pFUSE. A diagram of an exemplary anti-Dectin-1 bispecific antibody is shown in Figure 24A.

[0171] Bispecific antibodies using this design were constructed for proof-of-concept studies, as shown in Table 1. These bispecific antibodies have one arm that targets hDectin-1 and a second arm that targets hCD20. The bispecific antibodies listed in Table 1 were generated by expressing all four chains and purifying them to 95% purity and homogeneity. All bispecifics were confirmed to bind to their respective targets. [Table 1]

[0172] The variable domains of the opposing antibody arms of anti-Dectin-1 in Table 1 were as follows: CD20 VH: QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 24) CD20 VL: QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 25)

[0173] These hDectin-1 bispecific antibodies engage three targets: Dectin-1 on myeloid cells, CD20 on target cells or disease-causing agents, and Fc receptors on myeloid cells and NK cells, inducing robust immune stimulation and phagocytosis (Figure 24B). In particular, bispecific antibodies with a non-fucosylated, active hIgG1 Fc domain can recruit myeloid cells (e.g., monocytes, macrophages, and dendritic cells) and natural killer (NK) cells to eliminate disease-causing target cells, such as tumor cells, that express a specific antigen. In the context of cancer, without wishing to be bound by theory, dual engagement of Dectin-1 and Fcγ receptors on myeloid and NK cells is believed to elicit a potent immune response that ultimately eliminates cancer cells through the following actions: (1) bispecific antibody-induced crosslinking of Dectin-1 with Fcγ receptors leads to ITAM-dependent activation of downstream inflammatory pathways and release of immunomodulatory cytokines and cytotoxic proteins (proteases, perforin) that can modulate the tumor microenvironment and directly kill target cells; (2) bispecific antibody-induced clustering of Dectin-1 and Fcγ receptors leads to phagocytosis and elimination of targeted cancer cells by monocytes, macrophages, and dendritic cells; and (3) presentation of phagocytosed antigens by macrophages and DCs, a process that triggers a T cell immune response aimed at eliminating cancer cells.

[0174] 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 in all assays as an isotype control for the target binding arm. The bispecific variants tested here contained mutations (L234A, L235E, and G237A according to EU numbering) in the hIgG1 Fc domain (hIgG1 inactive) that eliminate Fc binding to Fc receptors. Binding of 2M24 / CD20 or 2M24 / RSV bispecifics to HEK293 cells stably expressing human Dectin-1 was assessed by flow cytometry (Figure 25A). The 2M24 / CD20 hIgG1 inactive bispecific antibodies were able to bind with similar affinity to cells expressing human Dectin-1 (cell-based binding EC50 values ​​of 1.4 and 1.7 nM, respectively). Thus, the 2M24 / CD20 bispecific antibody exhibited high affinity binding to Dectin-1-expressing HEK293 cells.

[0175] The binding of rituximab, 2M24 / CD20, or 2M24 / RSV hIgG1 active or inactive bispecific antibodies was also evaluated using the CD20-expressing B cell lymphoma Raji cell line (Figure 25B). The 2M24 / CD20 bispecific (active or inactive hIgG1 isotype) antibodies were able to bind to CD20-expressing Raji cells, but with at least 10-fold lower affinity compared to rituximab. Without wishing to be bound by theory, it is believed that the difference in CD20 binding affinity of the 2M24 / CD20 bispecific and rituximab is caused by the loss of avidity (monovalent vs. bivalent binding) in the bispecific antibodies.

[0176] Next, the ability of the 2M24 / CD20 bispecific antibody to induce ligation of cells expressing hDectin-1 with cells expressing hCD20 was assayed. Dectin-1-expressing HEK293 cells (effector) and CD20-expressing Raji cells (target) were differentially labeled with calcein green (effector) or calcein red (target) dyes. The labeled cells were co-cultured and treated with hIgG1 inactive 2M24 / CD20 or 2M24 / RSV (control) bispecific antibodies to induce effector:target ligation. Successful effector:target cell ligation was indicated by double positive staining (calcein green+, calcein red+, square box; Figure 26A). Engagement efficiency (quantified as the percentage of total target cells bound or ligated to effector cells) was assayed using dose titration of the bispecific antibody in the effector:target cell co-cultures (Figure 26B).

[0177] These results show that the 2M24 / CD20 bispecific antibody can ligate Dectin-1-expressing "effector" cells and CD20-expressing "target" cells with a potent EC50 of 0.17 nM. Despite the low affinity binding of the 2M24 / CD20 bispecific to CD20 on Raji cells (Figure 25B), 2M24 / CD20 ligated very efficiently. These findings suggest that the binding affinity of 2M24 / CD20 is enhanced (avidity) by high expression of Dectin-1 or CD20 on both effector and target cells, thereby facilitating efficient ligation of the two cells. Based on these findings, it is believed that the 2M24 / CD20 bispecific antibody can effectively engage Dectin-1-expressing monocytes, macrophages, or dendritic cells with target disease cells, such as B-cell lymphoma, that express high levels of CD20. Effector:target engagement is the first step in the MOA of the 2M24 bispecific antibody.

[0178] Human IgG1 activating isotype binds to Fcγ receptors on NK cells or monocytes. Therefore, we evaluated whether the hIgG1 activating isotype of 2M24 / CD20 can trigger monocyte killing by NK cells (via antibody-dependent cellular cytotoxicity, ADCC) or other monocytes (fratricide or antibody-dependent cellular phagocytosis, ADCP). In this scenario, the activating hIgG1 domain of 2M24 / CD20 engages Fcγ receptors on NK cells or monocytes and the Dectin-1 receptor on monocytes, inducing Fcγ-mediated activation and target depletion. PBMCs from two healthy donors, donor 76 (Figure 27A) and donor 77 (Figure 27B), were treated with increasing concentrations of 2M24 / CD20 bispecific (hIgG1 active or inactive isotype) and rituximab for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD14+ monocytes (as % of isotype control). No reduction in monocyte numbers was observed in either donor, indicating that 2M24 / CD20 active IgG1 did not induce monocyte depletion. Without wishing to be bound by theory, it is believed that 2M24 / CD20 hIgG1 (active isotype) does not affect monocyte levels and therefore the risk of infection is minimal.

[0179] Based on the proposed MOA of the 2M24 / CD20 bispecific antibody (as shown in FIG. 24B), B cell depletion by the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody or rituximab was evaluated to compare B cell depletion. PBMCs from two healthy donors, donor 83 (FIG. 28A) and donor 84 (FIG. 28B), were treated with increasing concentrations of the indicated antibodies for 24 hours and then analyzed by flow cytometry to quantify the levels of remaining viable CD19+ B cells (reported as % of B cells in isotype control treated PBMCs). Thus, in two healthy donors, high concentrations of the 2M24 / CD20 bispecific antibody induced superior B cell depletion (about 80% reduction) compared to rituximab (about 40% reduction), despite the bivalent binding of rituximab and the approximately 10-fold difference in binding affinity (as shown in FIG. 25B). The unique mechanism of action of 2M24 / CD20-activated IgG1 (Figure 24B), which involves binding to Dectin-1 on myeloid cells, Fcγ receptors on NK cells and monocytes, and CD20 on target B cells, results in an overall superior B cell depletion compared to Rituximab. These data support the concept that Dectin-1-induced immune stimulation via the 2M24 / CD20 bispecific promotes target cell depletion.

[0180] The ability of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody or rituximab (hIgG1) to downregulate CD19 expression on B cells in a process known as shaving or trogocytosis was evaluated. CD19+ expression on B cells from two healthy donors, donor 83 (Figure 29A) and donor 84 (Figure 29B), was quantified by flow cytometry after 24 hours of incubation with increasing concentrations of 2M24 / CD20 hIgG1 (active isotype) bispecific antibody, rituximab, or isotype control. The mean fluorescence intensity (MFI) of CD19 staining with anti-CD19 (BV605 conjugated) was used to evaluate the effect of 2M24 / CD20 bispecific and rituximab on CD19 expression on B cells. In PBMCs from donor 83, the EC50 for CD19 expression was 0.014 nM for rituximab and 0.080 nM for 2M24 / CD20 hIgG1 bispecific (Figure 29A). In PBMCs from donor 84, the EC50 for CD19 expression was 0.013 nM for rituximab and 0.090 nM for 2M24 / CD20 hIgG1 bispecific (Figure 29B). Both the 2M24 / CD20 active IgG1 bispecific and rituximab caused downregulation of CD19 expression on B cells. Interestingly, rituximab showed at least 5-fold more potent shaving compared to the 2M24 / CD20 bispecific. Downregulation of the target CD20 on B cells has already been reported as a mechanism by which malignant B cells evade rituximab-mediated depletion (Beum, PV et al. (2006) J. Immunol. 176:2600-2609). Thus, these findings suggest that the 2M24 / CD20 active IgG1 bispecific may have an advantage in B cell depletion due to its reduced shaving ability compared to rituximab.

[0181] Immune stimulation caused by the 2M24 / CD20 active IgG1 bispecific antibody resulted in the secretion of a unique repertoire of cytokines compared to rituximab (Figure 30). ELISA-based (mesoscale discovery) quantification of cytokines was performed in supernatants isolated from healthy donor PBMCs treated with the 2M24 / CD20 active hIgG1 bispecific, rituximab, or isotype control. PBMCs were stimulated overnight with the antibodies, and the supernatants were subsequently analyzed by MSD. Cytokines tested were IFNγ, IL-12p70, IL-6, TNFα, IL-1β, IL-4, IL-13, IL-10, and IL-8. Results showed that the 2M24 / CD20 active IgG1 caused higher and more distinct cytokine activation in PBMCs compared to rituximab. Furthermore, engagement of Dectin-1 and Fc receptors by the 2M24 / RSV bispecific alone did not induce cytokine release, precluding the possibility of systemic cytokine activation. These findings highlight the unique MOA that distinguishes the 2M24 / CD20 activating IgG1 bispecific antibody from rituximab. Without wishing to be bound by theory, these findings further indicate that 2M24 / CD20 may be able to trigger the release of Th1 and Th2 type responses and promote immune stimulation of the tumor microenvironment.

[0182] It was also found that the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody induced superior B cell depletion and lower CD19 shaving compared to rituximab in co-cultures of human macrophages and GFP-expressing Raji B cells. Co-cultures of human macrophages and Raji-GFP cells (3:1 ratio) were analyzed by flow cytometry in the presence of the 2M24 / CD20 hIgG1 (active isotype) bispecific, 2M24 / RSV control, fucosylated rituximab, or isotype hIgG1 control (Figure 31A). The co-cultures were incubated at 37°C for 24 h and then stained with PE anti-CD206 antibody to label macrophages and BV-605 anti-CD19 antibody to label Raji cells. The number of remaining viable / Raji-GFP+ cells was assessed at the end of the experiment. Primary antibodies were used in serial dose titrations. CD19 was assessed after 24 hours on Raji-GFP cells (Figure 31B), and B cell receptors were shown as a reduction in CD19 MFI in the presence of anti-Dectin-1 / anti-hCD20 bispecific or rituximab. The EC50 for CD19 expression was 0.020 nM for rituximab and 0.95 nM for 2M24 / CD20 hIgG1 bispecific. These results indicate enhanced B cell depletion (mediated by Fcγ receptors) by 2M24 / CD20 bispecific antibody compared to rituximab. Rituximab reduced B cell receptor CD19 surface levels more potently than anti-Dectin-1 / anti-hCD20 bispecific antibody. Similarly, B cell receptor shaving of CD20 by rituximab has been observed, limiting the effectiveness of rituximab in depleting B cells by CD20 reduction. Without wishing to be bound by theory, these data suggest that the superiority of the 2M24 / CD20 hIgG1 (active isotype) bispecific for B cell depletion is due to the reduced shaving of B cell receptors compared to rituximab, highlighting a different mechanism of cell depletion by the 2M24 / CD20 hIgG1 (active isotype) bispecific.

[0183] B cell depletion was also analyzed in single cell suspensions from kidney cancer tissue specimens. Single cell suspensions from two kidney cancer tissue specimens were analyzed by flow cytometry in the presence of 2M24 / CD20 hIgG1 (active or inactive) bispecific antibody, 2M24 / RSV hIgG1 control, fucosylated rituximab, and the respective isotype controls. Kidney cancer tissue specimens 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 (Figures 32A and 32B). The number of remaining viable B cells was assessed by anti-CD19 antibody and expressed as a percentage of the CD45+ immune cell population (Figure 32C). The 2M24 / CD20 active IgG1 bispecific antibody induced superior tissue B cell depletion compared to rituximab in single cell suspensions of kidney cancer specimens. The 2M24 / CD20 hIgG1 (active isotype) bispecific antibody depleted B cells by 44% and 46% (respectively) in two kidney cancer donor specimens, whereas rituximab induced B cell depletion of 33% and 18%, respectively (Figure 32C). This data supports the ability of the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody to deplete 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 Dectin-1 is predominantly expressed on tumor-associated macrophages (TAMs) in the above specimens, and therefore the 2M24 / CD20 hIgG1 (active isotype) bispecific antibody may engage TAMs to facilitate depletion of target cells.

[0184] Cytokine secretion by cultured macrophages and single cell suspensions of renal cancer specimens stimulated with immobilized anti-Dectin-1 antibody (clone 2M24) or 2M24 / CD20 bispecific antibody was examined. Anti-Dectin-1 antibody (clone 2M24), isotype control, or 2M24 / CD20 bispecific antibody were immobilized overnight at 10 μg per well on U-bottom polypropylene microtiter plates, followed by incubation with human monocyte-derived macrophages (Figures 33A and 33B) or single cell suspensions from renal cancer specimens (Figure 33C). Cells were cultured for 24 hours, and the amount of TNFα secreted in the supernatants was assessed by ELISA. As a positive control, cells were stimulated with zymosan. Anti-Dectin-1 antibody (clone 2M24) was found to induce Dectin-1 clustering and TNFα secretion from human macrophages. These data provide evidence that the parental anti-Dectin-1 antibody (clone 2M24) can promote immune stimulation in primary macrophage cultures and single cell homogenates of cancer specimens. Because Dectin-1 is expressed in myeloid cells, tumor-associated macrophages in cancer specimens are expected to produce cytokines in response to anti-Dectin-1 antibody stimulation. This would promote the transition of tumor-associated macrophages from anti-inflammatory to pro-inflammatory, resulting in a potent anti-tumor effect. Furthermore, monovalent binding of the 2M24 / CD20 bispecific antibody to Dectin-1 was sufficient to promote Dectin-1 clustering and immune stimulation to macrophages.

[0185] Immune stimulation by immobilized anti-Dectin-1 antibody in single cell suspensions from renal cancer specimens was also analyzed (FIG. 34). Single cell suspensions from renal cancer specimens were treated with immobilized anti-Dectin-1 antibody (clone 2M24) or isotype control hIgG4 antibody for 24 hours. Supernatants were analyzed by ELISA for the release of various cytokines, including IFNγ, IL-6, TNFα, IL-23, IL-12p70, IL-10, and IL-13. These results indicate that activation of Dectin-1 in myeloid cells (in this example Dectin-1 is expressed primarily by tumor-associated macrophages, TAMs) triggered the release of a specific repertoire of cytokines downstream of the Dectin-1 signaling pathway directly or indirectly through activation of other immune cells. Without wishing to be bound by theory, it is believed that engagement of Dectin-1 by the 2M24 bispecific antibody promotes immune stimulation and may modulate the tumor microenvironment to support the elimination of target-expressing cancer cells.

[0186] Example 5: Characterization of a bispecific antibody targeting Dectin-1 and CD20 This example describes further characterization of bispecific antibodies targeting human Dectin-1 and human CD20. The anti-Dectin-1 arm contained the variable domains of 2M24 and the anti-CD20 arm contained the variable domains of Rituximab (see SEQ ID NOs: 24 and 25 for the VH and VL domains, respectively).

[0187] Materials and Methods CD16 expression on NK cells Human PBMCs from healthy donors were treated with serial dilutions of 2M24 / CD20 hIgG1 KIF, rituximab KIF, and isotype control RSV hIgG1 KIF antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. CD16 expression on CD56+ NK cells was quantified and compared to expression levels in the isotype control treatment group.

[0188] CD19 expression on B cells Human PBMCs from healthy donors were treated with 0.1 nM 2M24 / CD20 hIgG1 KIF, rituximab KIF, and isotype control RSV hIgG1 KIF antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. CD19 expression (MFI) on B cells was quantified.

[0189] B cell depletion in PBMCs Human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified relative to the untreated control group (shown by the dotted line in Figure 37).

[0190] B cell depletion in kidney cancer specimens Single cell suspensions were generated from kidney cancer specimens and cells were treated with 2M24 / CD20 hIgG1, 2M24 / RSV hIgG1, rituximab hIgG1, and isotype control RSV hIgG1 antibodies. 24 hours after treatment, cells were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified as a percentage of CD19+ cells within the CD45+ immune cell population.

[0191] result First, the effect of the 2M24 / CD20 bispecific on CD16 expression was examined in human NK cells. Since CD16 is required for ADCC activity by NK cells, loss of CD16 expression may reduce the cytotoxicity of NK cells. Rituximab induced strong and robust shedding of CD16 on NK cells compared to 2M24 / CD20 hIgG1 KIF (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 the 2M24 / CD20 bispecific may better preserve the cytotoxicity of NK cells.

[0192] Next, the effect of the 2M24 / CD20 bispecific on CD19 expression was examined in human B cells. Maintaining expression of the target antigen is important for the therapeutic activity of monoclonal antibodies. B cell antigens such as CD20, CD19, and BCMA are validated immuno-oncology targets. CD19 is known to be downregulated by shaving / shedding after binding of anti-CD19 antibodies. A bystander effect was observed using a CD20-targeting antibody, where CD19 expression was reduced upon treatment with rituximab, but not with the 2M24 / CD20 hIgG1 KIF bispecific (Figure 36). CD19 levels on B cells were better maintained by the 2M24 / CD20 bispecific compared to rituximab. Without wishing to be bound by theory, it is believed that therapeutically, the 2M24 / CD20 bispecific may exhibit long-term activity due to its minimal effect on target antigen expression.

[0193] To compare rituximab with the anti-CD20 antibody obinutuzumab, the variable domain sequences of rituximab or obinutuzumab were used to generate the 2M24 bispecific antibody against CD20. The obinutuzumab variable domain sequences were as follows: VH: QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS (SEQ ID NO: 46); VL: DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK (SEQ ID NO: 47).

[0194] In ADCC / ADCP assays, 2M24 / CD20 (derived from the rituximab sequence) demonstrated near complete depletion of B cells and was superior to 2M24 / CD20 (derived from obinutuzumab) or the parental bivalent antibody and isotype control (Figure 37). These data support the use of the rituximab sequence to generate 2M24 / CD20 bispecifics.

[0195] Example 6: Characterization of a bispecific antibody targeting Dectin-1 and CD20 in an exploratory study in non-human primates This example describes the results of an exploratory study in cynomolgus monkeys regarding the safety and efficacy of a bispecific antibody targeting human Dectin-1 and human CD20, as described in Example 9.

[0196] Materials and Methods Three groups of cynomolgus monkeys (one male and one female per group) were administered a single dose of test article (5 mg / kg): A) 2M24 / CD20 hIgG1 KIF, B) 2M24 / CD20 hIgG1 inactive, and C) Rituximab hIgG1 KIF. Blood was collected at the indicated time points. Test article abbreviations are as follows (2M24 / CD20 KIF, 2M24 / CD20 inactive, RTX KIF).

[0197] B cell levels were assessed by flow cytometry. Depletion was quantified by the number of CD19+ B cells remaining in the sample after administration compared to the levels before 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 sample after administration (day 7) compared to the levels before test article was administered (day -7).

[0198] 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. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cell depletion was quantified compared to the isotype control group.

[0199] result This exploratory study was designed to test the safety and efficacy of 2M24 / CD20 bispecific antibodies in non-human primates. The study design is shown in Figure 38. Cynomolgus monkeys were divided into three treatment groups consisting of two animals per group (one male, one female). Each group was administered a specific test article at a single dose of 5 mg / kg. The test articles were: 1) 2M24 / CD20 hIgG1 KIF, 2) 2M24 / CD20 hIgG1 inactive, and 3) Rituximab hIgG1 KIF. Animals were monitored daily and samples including whole blood, bone marrow, lymph node, and colorectal tissue were collected as indicated. The study was scheduled for 8 weeks.

[0200] As shown in Figure 39 (top), the 2M24 / CD20 hIgG1 KIF bispecific antibody depleted B cells in vivo in cynomolgus monkeys. Near-complete and sustained B cell depletion (about 98%) was observed in both animals treated with a single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF. In the Rituximab group (Figure 40), one animal showed complete depletion, while the second showed robust but incomplete depletion (about 87%). Partial B cell depletion was observed in one animal in the 2M24 / CD20 hIgG1 inactive group (Figure 39 bottom), while the second animal showed no depletion at day 7. The 2M24 / CD20 hIgG1 KIF bispecific antibody was well tolerated in cynomolgus monkeys.

[0201] The 2M24 / CD20 hIgG1 KIF bispecific also depleted B cells in bone marrow (Figure 41A) and lymph nodes (Figure 41B) in cynomolgus monkeys in vivo. A single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF induced robust B cell depletion in bone marrow (approximately 87-88%) and partial depletion in lymph nodes (60-78%) in both animals. In the rituximab group, B cell depletion was also observed in both tissues. In the 2M24 / CD20 hIgG1 inactive group, partial B cell depletion was observed, except for animal CB764A, which showed minimal B cell depletion in lymph nodes.

[0202] The 2M24 / CD20 hIgG1 KIF bispecific also induced robust depletion of cynomolgus B cells ex vivo (FIG. 42). 2M24 / CD20 hIgG1 KIF induced robust depletion of B cells compared to rituximab hIgG1 KIF. The maximal depletion achieved by rituximab was about 30% of B cells, whereas the 2M24 / CD20 hIgG1 KIF bispecific showed a maximal depletion of about 50%.

[0203] Example 7: Purification and functional characterization of 2M24 / CD20 bispecific antibodies in scFv format This example describes the generation, purification, and characterization of a 2M24 / CD20 bispecific antibody in which the Dectin-1 targeting arm (based on the 2M24 variable domain) is 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. A diagram of this molecule is shown in FIG. 43. The knob-forming mutation on the Dectin-1 targeting arm was T366W, and the hole-forming mutations on the CD20 targeting arm were T366S, L368A, and Y407V. Without wishing to be bound by theory, it is believed that this format provides a universal platform for generating anti-Dectin-1 bispecific antibodies with simpler manufacturing requirements (e.g., compared to bispecific antibodies with anti-Dectin-1 arms with multiple polypeptide chains).

[0204] 2M24 scFv / CD20 hIgG1 was expressed in Hek293 cells by transfecting three plasmids (2M24 scFv hIgG1 plasmid, CD20 heavy chain, and CD20 light chain). Supernatants were harvested 4 days after expression and purified via Protein A. Aggregates were removed by size exclusion chromatography. As shown in Figure 44A, the 2M24 scFv / CD20 hIgG1 bispecific antibody was purified as a homogenous molecule on SEC.

[0205] Co-cultures of CD20-expressing Raji cells and Dectin-1-expressing HEK reporter assays were then treated with increasing concentrations of the 2M24 scFv / CD20 hIgG1 bispecific. Reporter activation was assessed by measuring SEAP levels (based on absorbance at 630 nm) in the medium. The bispecific molecule promoted targeted immune stimulation as assessed by this NFkB reporter assay (Figure 44B).

[0206] To test for B cell depletion, human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified relative to an untreated control group (shown by the dotted line in FIG. 44C). The results demonstrated that the 2M24 scFv / CD20 hIgG1 bispecific antibody was able to deplete human B cells, similar to the 2M24 / CD20 hIgG1 KIF molecule (FIG. 44C).

[0207] Example 8: Characterization of B cell depletion by 2M24xCD20 bispecific binding proteins A non-fucosylated 2M24 scFv / CD20 bispecific binding protein with an anti-Dectin-1 single chain variable fragment (scFv) fused to a conventional anti-CD20 antibody arm with hIgG1 Fc (see FIG. 43) was compared to the anti-CD20 antibody rituximab and the bispecific anti-CD3 / anti-CD20 T cell engager for their ability to deplete B cells in healthy donor PBMCs. The non-fucosylated 2M24 scFv / CD20 bispecific binding protein comprised a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:31, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:32, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:33.

[0208] PBMCs were isolated from buffy coats of healthy donors by Ficoll separation. PBMCs were resuspended in ADCC medium (RPMI 1640, 10% heat inactivated FBS, 1x pen / strep, 1x non-essential amino acids) at a density of 500,000 cells in 50uL in round bottom ultra low attachment 96 well plates. Test articles were prepared in 3-fold serial dilutions in 2x working solutions in ADCC medium. 50uL of test article was added to the cells. The reaction was gently resuspended and incubated at 37°C for 24 hours. Cells were harvested, treated with human Fc block and live / dead dye, and then stained with the following markers (CD45, CD3, CD16, CD14, CD56, and CD19). B cells were detected based on the phenotype of CD45+CD3-CD14-CD56-CD19+. B cell levels were expressed as the percentage of CD19+ cells in the treated group compared with CD19+ cells in the untreated control group.

[0209] As shown in Figure 45, the non-fucosylated scFv 2M24xCD20 bispecific binding molecule outperformed rituximab and CD3xCD20 T cell engager in depleting B cells. These data indicate that a bispecific antibody targeting Dectin-1 on myeloid cells and CD20 on B cells is highly potent in depleting B cells compared to rituximab or T cell engager. These findings suggest that engaging myeloid cells via Dectin-1 is an attractive cancer immunotherapy strategy.

[0210] Next, we assayed B cell depletion in prostate cancer tumor specimens.

[0211] Single cell suspensions were generated from prostate cancer specimens using Miltenyi Biotec's tumor dissociation kit. Flow cytometry confirmed CD20 expression on CD45+CD3-CD14-CD56-CD19+ cells and Dectin-1 expression on CD45+CD3-CD19-CD11b+CD163+ tumor associated macrophages. Cells were resuspended in ADCC media (RPMI 1640, 10% heat inactivated FBS, 1x pen / strep, 1x non-essential amino acids) at a density of 500,000 cells in 100uL in round bottom ultra low attachment 96 well plates. Test articles were prepared at 3x concentration of 30ug / mL in ADCC media and 50uL was added to the cells (final concentration 10ug / mL). Reactions were gently resuspended and incubated at 37°C for 24 hours. Cells were harvested, treated with human Fc block and live / dead stain, and then stained for the following markers (CD45, CD3, CD16, CD14, CD11b, and CD19). B cells were detected based on the phenotype CD45+CD3-CD14-CD56-CD19+. B cell levels were expressed as the percentage of CD19+ cells in the treatment group compared to CD19+ cells in the control group (RSV hIgG1).

[0212] As shown in Figure 46, the non-fucosylated scFv 2M24xCD20 bispecific binding molecule performed better than rituximab in depleting B cells. These data indicate that 2M24xCD20 was effective in depleting CD20-expressing B cells present in solid tumor specimens through engagement of tumor-associated macrophages expressing Dectin-1. Furthermore, the 2M24xCD20 bispecific composed of scFv and non-fucosylated format outperformed either rituximab or the conventional IgG 2M24xCD20 format (DuetMab). Collectively, these observations confirm that engaging myeloid cells via Dectin-1 is an attractive cancer immunotherapy strategy and that efficacy can be enhanced by utilizing scFv and non-fucosylated modifications.

[0213] Example 9: Comparison of 2M24xCD20 bispecific binding protein with rituximab The 2M24xCD20 bispecific binding protein was compared to rituximab for functional properties of interest, including target cell depletion.

[0214] The effect of 2M24xCD20 bispecific binding protein on CD16 expression on NK cells was examined. Human PBMCs from healthy donors were treated with serial dilutions of 2M24xCD20 bispecific hIgG1 binding protein treated with KIF, rituximab treated with KIF, or isotype control RSV hIgG1 antibody treated with KIF. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. CD16 expression on CD56+ NK cells was quantified and compared to expression levels in the isotype control-treated group.

[0215] As shown in Figure 47A, rituximab induced strong and robust shedding of CD16 on NK cells compared to 2M24xCD20 bispecific hIgG1 binding protein (both treated with KIF). Since CD16 is required for ADCC activity by NK cells, loss of CD16 expression may reduce the cytotoxicity of NK cells. These results suggest that 2M24xCD20 bispecific binding protein may better preserve the cytotoxicity of NK cells compared to rituximab.

[0216] The effect of 2M24xCD20 bispecific binding protein on CD19 expression on B cells was examined. Human PBMCs from healthy donors were treated with 2M24xCD20 bispecific hIgG1 binding protein treated with KIF, rituximab treated with KIF, or isotype control RSV hIgG1 antibody treated with KIF at 0.1 nM. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. CD19 expression (MFI) on B cells was quantified.

[0217] Maintaining expression of target antigens is important 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 by shaving / shedding after binding of anti-CD19 antibodies. Herein, a bystander effect was observed using CD20-targeting antibodies, with CD19 expression reduced by treatment with KIF-treated rituximab, but not with KIF-treated 2M24×CD20 bispecific hIgG1 binding protein (Figure 47B). Thus, CD19 levels on B cells were better maintained by 2M24×CD20 bispecific binding protein compared to rituximab. These results suggest that therapeutically, 2M24×CD20 bispecific binding protein may exhibit long-term activity due to minimal impact on target antigen expression.

[0218] The effect of 2M24xCD20 bispecific binding protein on B cell depletion was examined. Human PBMCs from healthy donors were treated with serial dilutions of the indicated antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified relative to an untreated control group (shown by the dotted line in Figure 47C).

[0219] The 2M24 bispecific binding protein against CD20 was generated using variable domain sequences of rituximab or obinutuzumab. In ADCC / ADCP assays, the 2M24 / CD20 bispecific (anti-CD20 arms derived from rituximab sequences) showed nearly complete depletion of B cells compared to 2M24 / CD20 (anti-CD20 arms derived from obinutuzumab) or the parental bivalent antibody and isotype control (Figure 47C). Thus, the 2M24xCD20 bispecific binding protein with anti-CD20 arms derived from rituximab showed superior B cell depletion compared to the format using anti-CD20 binding arms derived from obinutuzumab. These data support the use of rituximab variable domains to generate 2M24 / CD20 bispecifics.

[0220] The effect of 2M24xCD20 bispecific binding protein on B cell depletion was further examined using kidney cancer specimens. Single cell suspensions were generated from kidney cancer specimens and cells were treated with 2M24 / CD20 bispecific hIgG1, 2M24 / RSV bispecific hIgG1, rituximab hIgG1, and isotype control RSV hIgG1 antibodies. 24 hours after treatment, cells were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cells were quantified as a percentage of CD19+ cells within the CD45+ immune cell population.

[0221] The results showed that the 2M24 / CD20 bispecific binding protein induced robust depletion of B cells in single cell suspensions from primary renal tumors (Figure 47D). Dectin-1 expression on tumor-associated macrophages in primary renal specimens was also confirmed. The 2M24 / CD20 bispecific induced superior B cell depletion compared to rituximab.

[0222] Example 10: Exploratory testing of 2M24xCD20 bispecific binding proteins in cynomolgus monkeys This example describes the results of an exploratory study of the effect of 2M24 / CD20 bispecific binding protein in cynomolgus monkeys. The study design is shown in Figure 48. Cynomolgus monkeys were divided into three treatment groups, consisting of two animals per group (one male, one female). Each group was administered a specific test article at a single dose of 5 mg / kg. The test articles were: 1) 2M24 / CD20 hIgG1 KIF, 2) 2M24 / CD20 hIgG1 inactive, and 3) Rituximab hIgG1 KIF. Animals were monitored daily and samples such as whole blood, bone marrow, lymph node, and colorectal tissue were collected as indicated. The study was scheduled for 8 weeks. Time points for sample collection and other measurements are shown in Figure 48.

[0223] B cell levels were examined in the blood. Blood was drawn at the indicated time points and B cell levels were assessed by flow cytometry. Depletion was quantified by the number of CD19+ B cells remaining in the sample after administration compared to levels before the test article was administered.

[0224] The results showed that 2M24 / CD20 bispecific hIgG1 KIF depleted B cells in vivo. Near-complete and sustained B cell depletion (about 98%) was observed in both animals treated with a single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF (Figure 49A; top left). In the rituximab group, one animal showed complete depletion, while the second showed robust but incomplete depletion (about 87%) (Figure 49A; top right). Partial B cell depletion was observed in one animal in the 2M24 / CD20 hIgG1 inactive group, while the second animal showed no depletion at day 7 (Figure 49A; bottom left).

[0225] B cell depletion in 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 samples after administration (day 7) compared to levels before test article was administered (day -7).

[0226] The results showed that 2M24 / CD20 bispecific hIgG1 KIF depleted B cells in bone marrow and lymph nodes in vivo. A single dose (5 mg / kg) of 2M24 / CD20 hIgG1 KIF induced robust B cell depletion in bone marrow (approximately 87-88%) and partial depletion in lymph nodes (60-78%) in both animals (Figure 49B). In the rituximab group, B cell depletion was also observed in both tissues. In the 2M24 / CD20 hIgG1 inactive group, partial B cell depletion was observed, except for animal CB764A, which showed minimal B cell depletion in lymph nodes.

[0227] Ex vivo B cell depletion was also examined. PBMCs from healthy cynomolgus monkeys were treated with serial dilutions of 2M24 / CD20 hIgG1 KIF, rituximab KIF, and isotype control RSV hIgG1 KIF antibodies. 24 hours after treatment, PBMCs were stained with antibodies against lineage-specific markers for flow cytometry analysis. B cell depletion was quantified compared to the isotype control group.

[0228] The results showed that 2M24 / CD20 bispecific hIgG1 KIF induced robust depletion of cynomolgus B cells ex vivo. 2M24 / CD20 bispecific hIgG1 KIF induced robust depletion of B cells compared to rituximab hIgG1 KIF (FIG. 49C). The maximal depletion achieved by rituximab was about 30% of B cells, whereas 2M24 / CD20 hIgG1 KIF bispecific showed a maximal depletion of about 50%.

[0229] Example 11: Surrogate testing of anti-Dectin-1 x CD20 bispecific binding protein in mice This example describes the results of a surrogate study on the effect of 2A11 anti-mouse Dectin-1 / mCD20 bispecific binding protein in mice. The study design is shown in Figure 50A. Mice were treated with a single dose (10 mg / kg) of bispecific 2A11 / mCD20 mIgG1 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 % of total Cd45 cells.

[0230] As shown in FIG. 50B, the surrogate 2A11 / mCd20 bispecific antibody induced significant depletion of B cells in the blood and tissues of healthy wild-type mice.

[0231] Example 12: Testing the efficacy of 2M24xCD20 bispecific binding proteins ex vivo The properties of the 2M24 / CD20 bispecific binding protein were analyzed ex vivo. 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 dectin-1-expressing reporter HEK cell line. 24 hours after treatment, supernatants were harvested to test for dectin-1-induced NF-kB-driven secreted embryonic alkaline phosphatase (SEAP) activity. D-zymosan, an established ligand of dectin-1, was used as a positive control.

[0232] The results showed that 2M24 / CD20 bispecific binding protein induced activation of the dectin-1 pathway only in the presence of CD20-expressing B cells. As shown in Figure 51A, 2M24 / CD20 bispecific binding protein showed dose-dependent activation of NF-κB in the presence of B lymphoma lines expressing CD20 levels ranging from 9,000 (Sc-1) to 370,000 copies (SU-DHL-6). However, 2M24 / CD20 bispecific binding protein failed to activate NF-κB reporter cells in a CD20-null B lymphoma line (NALM-6) or in the absence of target cells. D-zymosan showed activation of the reporter HEK cell dectin-1 pathway regardless of the presence of target cells. Taken together, these data indicate that simultaneous binding of dectin-1 and engagement of CD20 (around 9,000 copies) on target cells is required for dectin-1 clustering and activation of the downstream NF-kB pathway.

[0233] The ability of the 2M24 / CD20 bispecific binding protein to induce phagocytosis was also tested. B cell lymphoma lines with different CD20 expression levels were treated with serial dilutions of the indicated antibodies in the presence of macrophage cells from healthy human donors. 24 hours after treatment, B cell depletion was assessed by comparing with an RSV (non-specific) treated control group.

[0234] The results showed that 2M24 / CD20 bispecific binding protein induced depletion of B cell lymphoma cell lines more efficiently than rituximab in the presence of macrophage cells. As shown in Figure 51B, 2M24 / CD20 bispecific binding protein treatment resulted in a dose-dependent increase in the depletion level of B lymphoma lines in the presence of macrophage cells from healthy human donors. Compared to rituximab, the highest concentration of 2M24 / CD20 bispecific binding protein showed more efficient or complete depletion of B lymphoma lines with CD20 expression ranging from 9,000 to 370,000 receptors.

[0235] The ability of 2M24 / CD20 bispecific binding protein to deplete primary human B cells was also tested. Human PBMCs from n=6 healthy donors were treated with serial dilutions of 2M24 / CD20 bispecific binding protein and comparative anti-CD20 antibodies (rituximab and anti-CD20 / anti-CD3 bispecific engager). 24 hours after treatment, PBMCs were stained with fluorescently labeled antibodies for flow cytometric analysis. The extent of B cell depletion was assessed relative to untreated controls.

[0236] The results showed that the 2M24 / CD20 bispecific binding protein exhibited more efficient 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 exhibited a dose-dependent increase in the level of depletion of primary human B cells with an average EC50 of 0.16 (Figure 51C). In comparison, the other anti-CD20 antibodies tested, rituximab and the CD20xCD3 bispecific engager, induced B cell depletion with average EC50 values ​​of 0.14 nM and 0.17 nM. However, the 2M24 / CD20 bispecific binding protein exhibited approximately 91% depletion of primary B cells at the highest concentration, whereas rituximab and the CD20xCD3 engager exhibited partial depletion of 46% and 47% of total B cells, respectively. Taken together, these data demonstrate that the 2M24 / CD20 bispecific binding protein exhibited more robust B cell depletion compared to rituximab and the CD20xCD3 engager.

[0237] The ability of 2M24 / CD20 bispecific binding proteins to stimulate cytokine secretion ex vivo was also tested. Human PBMCs from n=8 healthy donors were treated with serial dilutions of 2M24 / CD20 bispecific binding proteins and comparative anti-CD20 antibodies. 24 hours after treatment, supernatants were collected and analyzed by multiplex MSD cytokine assay to measure and compare cytokine secretion induced by 2M24 / CD20 bispecific binding proteins and comparative anti-CD20 antibodies. Supernatants from RSV hIgG1 (non-specific antibody) treated and untreated PBMC samples were used as negative controls.

[0238] The results showed that 2M24 / CD20 bispecific binding protein exhibited low to moderate production of proinflammatory cytokines. 2M24 / CD20 bispecific binding protein showed dose-dependent induction of cytokine production after 24 hours of treatment with healthy donor PBMCs. At the highest concentration tested, 1.67ug / ml, 2M24 / CD20 bispecific binding protein showed significant induction of IL-6, TNFa, IFN-g, and IL-2 compared to untreated control (Figures 51D and 51E). Specifically, 2M24 / CD20 bispecific binding protein showed higher production of cytokines IL-6 and TNF-a compared to rituximab, while the production levels of IL-6, TNF-a, IFN-g, and IL-2 induced by 2M24 / CD20 bispecific binding protein were significantly lower compared to treatment with CD20xCD3 engager.

[0239] Example 13: Testing the efficacy of 2M24xCD20 bispecific binding proteins in an in vivo mouse model The efficacy of a surrogate anti-mouse Dectin-1 antibody with an mIgG2a Fc region was tested in an in vivo mouse model.

[0240] 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 surrogate anti-mDectin-1 / anti-hCD20 mIgG2a antibodies was evaluated in Ramos and Daudi xenograft models in SCID mice, as well as in the Raji xenograft model in SCID-beige mice. After corresponding tumors were established in SCID or SCID-beige mice, anti-mDectin-1 / anti-mCD20 mIgG2a was administered four times at an equivalent dose of 10 mg / kg on days 0, 4, 7, and 10, when tumor volumes reached the range of 60-100 mm3. Tumor progression was monitored by measuring tumor volumes twice weekly and compared to isotype mIgG2a treatment.

[0241] Results showed that the surrogate anti-mDectin-1 / anti-mCD20 mIgG2a antibody demonstrated robust anti-tumor efficacy across several B cell lymphoma xenograft models. Four 10 mg / kg doses of surrogate anti-mDectin-1 / anti-mCD20 mIgG2a demonstrated 90% tumor growth inhibition in the Ramos xenograft model in SCID mice and complete tumor regression in the Daudi model (10 / 10) and the Raji model (8 / 8) in SCID-beige mice (Figure 52A). Complete regression of tumors in SCID mice, particularly in NK cell-deficient SCID-beige mice, demonstrated the efficacy of anti-mDectin-1 / anti-mCD20 in activating myeloid cells in abolishing tumor growth in these mouse xenograft models.

[0242] Next, the ability of the surrogate anti-mDectin-1 / anti-mCD20 bispecific antibody 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 implanted (subcutaneously) into C57BL / 6 mice with 5x10^6 CD20-MC38 cells. After tumor establishment, tumors with a volume of 80-150 mm were cultured in a 10-well plate with 10% IgG4-dependent markers. 3 Once this range was reached, anti-mDectin-1 / anti-mCD20 bispecific was administered once at an equivalent dose of 10 mg / kg and compared to a similar dose of isotype mIgG1 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.

[0243] The results showed that anti-mDectin-1 / anti-mCD20 bispecific antibody induced activation of dendritic cells in lymphoid organs (spleen and lymph nodes) and polarization of macrophages in tumors. As shown in Figure 52B, a single dose of anti-mDectin-1 / anti-mCD20 bispecific mIgG1 at 10 mg / kg showed significantly higher proliferation of dendritic cells in LNs compared to the isotype-treated control group. In tumor tissues, the activation of myeloid cells induced by anti-mDectin-1 / anti-mCD20 bispecific was observed by a significantly increased influx of monocytes in the tumors, along with improved polarization towards proinflammatory M1 macrophages compared to the isotype-treated control group.

[0244] T cell activation induced by anti-mDectin-1 / anti-mCD20 bispecific antibodies was also demonstrated in the MC38 xenograft model. After CD20-MC38 tumors were established in C57BL / 6 mice, tumors of 80–150 mm 3 At tumor volumes of 100-200 mg / kg, anti-mDectin-1 / anti-mCD20 bispecific antibody was administered as a single dose 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.

[0245] The results showed that anti-mDectin-1 / anti-mCD20 bispecific antibody led to activation of splenic CD4+ and CD8+ T cells, as well as intratumoral activation of cytotoxic CD8+ T cells in the tumor. As shown in Figure 52C, a single dose of anti-mDectin-1 / anti-mCD20 bispecific mIgG1 at 10 mg / kg demonstrated significantly higher T cell proliferation in the spleen, as shown by proliferation marker Ki67 staining. The significantly higher proliferation of both T cell subsets was accompanied by improved IL-2 production in both T cell subsets, CD4 + Increased formation of effector T cells and CD8 + This coincides with an increase in granzyme B production by T cells, indicating their cytotoxic potential. In tumor tissues, the anti-mDectin-1 / anti-mCD20 bispecific inhibited T cells, especially CD8 +In addition, anti-mDectin-1 / anti-mCD20 bispecific-induced mice showed significantly increased intratumoral CD8 + T cells showed robust granzyme B expression, indicating their cytotoxicity. In summary, the anti-mDectin-1 / anti-mCD20 bispecific induced robust T cell activation and cytotoxic CD8 + Intratumoral infiltration of T lymphocytes was demonstrated.

[0246] The ability of anti-mDectin-1 / anti-mCD20 bispecific antibodies to deplete B cells in lymphoid organs of naive C57BL / 6 mice was tested. hCD20 Tg mice (hCD20 expressed under the mouse CD20 promoter) were treated with a single dose of anti-mDectin-1 / anti-mCD20 bispecific mIgG2a and isotype mIgG2a control. Mice were sacrificed 7, 14, and 28 days after treatment. Designated lymphoid and non-lymphoid organs were harvested to measure B cell depletion compared to isotype-treated controls. B cell populations were marked by dual expression of CD19 and B220 and identified by flow cytometry.

[0247] The results showed that anti-mDectin-1 / anti-mCD20 bispecific showed robust depletion of B cells in peripheral and lymphoid organs (spleen, lymph nodes, and bone marrow). A single dose of anti-mDectin-1 / anti-mCD20 bispecific mIgG2a at 10 mg / kg showed significant depletion of B cells in peripheral and lymphoid organs. Approximately 90% B cell depletion was observed in peripheral and secondary lymphoid organs (spleen and lymph nodes) at day 7 after treatment (Figure 52E). In the bone marrow, mature B cells (showing high CD20 expression) showed approximately 85% depletion by day 7 after treatment. Immature B cells with low CD20 expression showed higher resistance to cell depletion induced by anti-mDectin-1 / anti-mCD20 bispecific (not shown). B cell recovery was monitored over time after single dose treatment.

[0248] The ability of anti-mDectin-1 / anti-mCD20 bispecific antibodies to deplete B cells in non-lymphoid organs of naive C57BL / 6 mice was also tested. hCD20 Tg mice (hCD20 expressed under the mouse CD20 promoter) were treated as described above. Designated non-lymphoid organs were harvested and B cell depletion was measured compared to isotype-treated controls. Non-lymphoid tissues were processed into single cell populations and enriched for immune cells. B cell populations were marked by dual expression of CD19 and B220 and identified by flow cytometry.

[0249] The results showed that anti-mDectin-1 / anti-mCD20 bispecific antibody showed robust depletion of B cells in non-lymphoid tissues tested. A single dose of anti-mDectin-1 / anti-mCD20 bispecific mIgG2a at 10 mg / kg showed robust depletion of B cells in non-lymphoid organs (Figure 52F). Seven days after treatment, nearly complete depletion of B cells was observed in non-lymphoid organs (lung, liver, brain, and heart). B cell recovery was monitored and 28 days after single dose treatment, B cells had only recovered to 50% of the isotype-treated control group.

[0250] The ability of anti-mDectin-1 / anti-mCD20 bispecific antibodies with inactive Fc to inhibit tumor growth was tested. The efficacy of murine surrogate anti-mDectin-1 / anti-mCD20 bispecific antibodies with mIgG2a active and inactive Fc regions was evaluated in the Daudi xenograft model in SCID mice. After tumor establishment in SCID mice, tumors with tumor volumes between 60 and 100 mm were treated with IgG2a ... 3 Once tumor volume had reached the range of 100 mg / kg, the anti-mDectin-1 / anti-mCD20 bispecific was administered four times at an equivalent dose of 10 mg / kg on days 0, 4, 7, and 10. 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 inactive treated and isotype control groups were further evaluated for myeloid cell activation 14 days after the first dose. The anti-mDectin-1 / anti-mCD20 bispecific active group showed complete tumor regression and was therefore not included in the evaluation.

[0251] Results showed that anti-mDectin-1 / anti-mCD20 bispecific with inactive Fc still showed robust tumor growth inhibition in SCID mice and induced improved immune cell influx in tumor tissue, highlighting Dectin-1-dependent activity. Treatment with anti-mDectin-1 / anti-mCD20 bispecific mIgG2a (active) and Fc inactive induced 95% and 40% tumor growth regression and inhibition, respectively, compared to isotype mIgG2a control in the Daudi model (Figure 52G). The data indicate that targeting Dectin-1 alone is sufficient for tumor growth inhibition, but engaging both Dectin-1 and Fc receptors contributes to significant tumor elimination.

[0252] The ability of anti-mDectin-1 / anti-mCD20 bispecifics with inactive Fc to reprogram myeloid cells was also tested. The efficacy of mouse surrogate anti-mDectin-1 / anti-mCD20 bispecific mIgG2a active and Fc inactive was evaluated in the Daudi xenograft model in SCID mice. After tumor establishment in SCID mice, tumors with a volume between 60 and 100 mm were reprogrammed. 3 Once tumor volume had reached the range of 100 mg / kg, the anti-mDectin-1 / anti-mCD20 bispecific was administered four times at an equivalent dose of 10 mg / kg on days 0, 4, 7, and 10. 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 inactive treated and isotype control groups were further evaluated for myeloid cell activation 14 days after the first dose. The anti-mDectin-1 / anti-mCD20 bispecific active group showed complete tumor regression and was therefore not included in the evaluation.

[0253] The results showed that anti-mDectin-1 / anti-mCD20 bispecific Fc inactive induced Dectin-1 stimulation caused significant reprogramming of myeloid cells in tumors, increasing monocyte recruitment and proinflammatory macrophage differentiation. Four doses of 10 mg / kg anti-mDectin-1 / anti-mCD20 bispecific mIgG2a Fc inactive significantly improved recruitment of resident and inflammatory monocytes to tumor tissues compared to the isotype-treated control group (Figure 52H). Although the anti-mDectin-1 / anti-mCD20 bispecific Fc inactive treated group had a lower frequency of intratumoral macrophages compared to the control group, macrophages showed better polarization toward the inflammatory M1 phenotype rather than the tumorigenic M2 phenotype, indicating its antitumor efficacy.

[0254] Example 14: Testing the efficacy of 2M24xCD20 bispecific binding protein in an in vivo cynomolgus monkey model The efficacy of the 2M24 / CD20 bispecific binding protein was tested in an in vivo cynomolgus monkey model.

[0255] B cell depletion was analyzed in response to various doses of 2M24 / CD20 bispecific binding protein. Four groups of naive cynomolgus monkeys (n=2, 1 male and 1 female per group) were treated with vehicle and 1, 10, and 100 mg / kg of 2M24 / CD20 bispecific binding protein, 3 times per week. Peripheral blood samples were taken at several time points from the 1 mg / kg and 10 mg / kg groups according to the dosing regimen to assess the presence of B cells by flow cytometry. Animals in the vehicle and 100 mg / kg groups were sacrificed on day 15, 1 day after the third dose. B cells were marked by dual expression of HLA-DR and CD19.

[0256] Results showed that the 2M24 / CD20 bispecific binding protein induced robust B cell depletion in vivo in cynomolgus monkeys. All dose levels of the 2M24 / CD20 bispecific demonstrated robust B cell depletion in the peripheral blood of cynomolgus monkeys in vivo (Figure 53A). The 2M24 / CD20 bispecific at the 1 mg / kg dose level demonstrated complete depletion of B cells after the first dose. Peripheral B cells began to recover, with B cell levels reaching 15% of baseline values ​​by the end of the third dose and 75% of baseline by day 56. At the 10 mg / kg dose level, the 2M24 / CD20 bispecific induced near complete depletion after the first dose, which persisted until the third dose on day 15. After the third dose, B cell levels began to slowly recover to approximately 15% of baseline by day 56. Finally, the 100 mg / kg dose induced complete B cell depletion that persisted across all three doses.

[0257] B cell levels in lymphoid organs (bone marrow and lymph nodes) were also analyzed. Following the dosing regimen described above, lymph nodes and bone marrow samples were taken at several time points from the 1 mg / kg and 10 mg / kg groups to assess the presence of B cells by flow cytometry and to assess depletion relative to baseline levels of B cells.

[0258] Results showed that all dose levels of the 2M24 / CD20 bispecific induced robust B cell depletion in both bone marrow and lymph nodes of cynomolgus monkeys in vivo. At the end of three weekly doses, the 2M24 / CD20 bispecific showed approximately 50% depletion in bone marrow and lymph nodes at both the 1 mg / kg and 10 mg / kg doses (Figure 53B). At the 100 mg / kg dose, the 2M24 / CD20 bispecific showed approximately 50% B cell depletion in bone marrow but nearly 90% B cell depletion in lymph nodes.

[0259] B cell levels were further analyzed in lymphoid and non-lymphoid tissues. Two groups of naive cynomolgus monkeys (n=2, one male and one female in each group) were treated with vehicle and 100 mg / kg of 2M24 / CD20 bispecific three times weekly. On day 15, one day after the third dose according to the dosing regimen, animals were sacrificed and several lymphoid and non-lymphoid organs were harvested to assess tissue B cell depletion by flow cytometry. B cells were marked by dual expression of HLA-DR and CD19.

[0260] The results showed that the 2M24 / CD20 bispecific binding protein induced robust B cell depletion in peripheral and lymphoid organs. As shown in FIG. 53C, at 100 mg / kg, the 2M24 / CD20 bispecific showed nearly complete depletion of B cells in peripheral blood and secondary lymphoid organs. Compared to vehicle-treated controls, the 2M24 / CD20 bispecific showed approximately 90%, 85%, and 74% depletion of all B cells in the spleen, tertiary lymph nodes, and mesenteric lymph nodes. B cell depletion in the bone marrow was limited and restricted to the mature B cell compartment (not shown). In non-lymphoid organs, the 2M24 / CD20 bispecific at 100 mg / kg showed approximately 94%, 77%, 90%, 88%, and 95% B cell depletion in the brain, kidney, liver, lung, and heart, respectively, compared to vehicle-treated cohorts. Taken together, these data demonstrated robust efficacy of the 2M24 / CD20 bispecific binding protein at 100 mg / kg and penetration into deep niches of lymphoid and non-lymphoid organs in cynomolgus monkeys.

[0261] The depletion of B cell subsets in the spleen was also examined. Two groups of naive cynomolgus monkeys (n=2, one male and one female in each group) were treated three times weekly with vehicle and 100 mg / kg of the 2M24 / CD20 bispecific. On day 15, one day after the third dose according to the dosing regimen, the animals were sacrificed and the spleens were harvested to assess the degree of depletion of the different B cell subsets by flow cytometry. B cells were marked by dual expression of HLA-DR and CD19.

[0262] Results showed that 2M24 / CD20 bispecific at 100 mg / kg induced robust deep tissue depletion of naive and activated B cell subsets in the spleen of cynomolgus monkeys. 2M24 / CD20 bispecific at 100 mg / kg showed nearly complete depletion of naive B cells in the spleen. Compared to vehicle-treated cohorts, 2M24 / CD20 bispecific showed 74% and 99% depletion of transitional and mature naive B cells, respectively. The transitional subset is distinguished from the mature subset by expression of the CD10 marker (Figures 53D and 53E). Complete depletion of splenic naive B cells by 2M24 / CD20 bispecific was consistent with the expression of naive follicular (CD21 + IgD hi ) and marginal zone (CD21 hi IgM + ) B cell compartment. Compared to vehicle-treated groups, the 2M24 / CD20 bispecific 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 the pathogenic DN2 B cell population (CD21 - CD27 - CD11c + (marked with ).

[0263] Activation of peripheral bone marrow cells was also examined and compared to that induced by a rituximab-like anti-cynomolgus CD20 antibody. Two groups of naive cynomolgus monkeys (n=2, one male and one female in each group) were treated with 5 mg / kg of 2M24 / CD20 bispecific antibody (Fab-Fab format) and a single dose of rituximab-like anti-CD20 antibody. Following the dosing regimen, samples were taken from peripheral blood at several time points to evaluate the dynamics of bone marrow cells in the peripheral blood. In another experiment, two groups of naive cynomolgus monkeys (n=2, one male and one female in each group) were treated with 1 mg / kg and 10 mg / kg of 2M24 / CD20 bispecific (scFv-Fab format) three times a week. Following the dosing regimen, samples were taken from peripheral blood at several time points from the 1 mg / kg and 10 mg / kg groups to evaluate the dynamics of bone marrow cells in the peripheral blood.

[0264] The results showed that the 2M24 / CD20 bispecific antibody demonstrated robust myeloid cell proliferation in the circulating blood. A single dose of the 2M24 / CD20 bispecific (Fab-Fab) antibody significantly increased myeloid cell proliferation (HLA-DR1) in the peripheral blood of cynomolgus monkeys. + CD19 - Specifically, we showed proliferation of classical dendritic cells 1 (cDC1, HLA-DR hi CD11c + CD1c - 2M24 / CD20 bispecific (characterized by scFv-Fab) showed significant proliferation in the circulation at both doses, with cDC1 cells peaking at day 10 after the start of treatment. No proliferation of bone marrow cells and cDC1 populations was observed in the cohort treated with rituximab-like CD20-mAb (Figure 53F). Similarly, three doses of 2M24 / CD20 bispecific (scFv-Fab) at 1 mg / kg and 10 mg / kg showed almost significant proliferation of bone marrow cells, including the cDC1 cell population, with cDC1 cells peaking at day 40 after the start of treatment (Figure 53G). Taken together, these data confirm that 2M24 / CD20 bispecific (both Fab-Fab and scFv-Fab formats) mediated stimulation of bone marrow cells and robust proliferation of the dendritic cell population cDC1 in the peripheral blood of cynomolgus monkeys.

[0265] Proinflammatory cytokine levels in serum were also analyzed. Two groups of naive cynomolgus monkeys (n=2, one male and one female in each group) were treated with 1 mg / kg and 10 mg / kg of 2M24 / CD20 bispecific three times weekly. Following the dosing regimen, serum samples were taken from peripheral blood at several time points to assess proinflammatory cytokine responses by MSD cytokine array.

[0266] The results showed that the 2M24 / CD20 bispecific at both doses induced a moderate proinflammatory cytokine response in cynomolgus monkeys. As shown in FIG. 53H, both the 1 mg / kg and 10 mg / kg doses of the 2M24 / CD20 bispecific induced a moderate cytokine response, with all six cytokines exhibiting relatively stable levels from baseline to day 14. Specifically, a transient increase in MIP-1a was observed after 1 mg / kg and 10 mg / kg 2M24 / CD20 bispecific treatment. However, no sustained increase in any of the proinflammatory cytokines was observed. There were no detectable levels of IL-1b and TNF-a at baseline or after 2M24 / CD20 bispecific treatment (not shown).

[0267] Two groups of naive cynomolgus monkeys (n=2, one male and one female in each group) were also treated with 2M24 / CD20 bispecific at 1 mg / kg and 100 mg / kg three times weekly, and serum cytokines were analyzed as described above. As shown in FIG. 53I, at the 100 mg / kg dose, the cytokine response was moderate, with all six cytokines showing relatively stable levels from baseline to day 14. IP-10 levels appeared to be most affected, being consistently elevated above baseline at all time points, reaching a peak of approximately 560 pg / ml on day 7. MIP-1α levels also showed a consistent increase from baseline at all time points, reaching a high of approximately 57 pg / ml on day 1. There were no detectable levels of IL-1b and TNF-a at baseline or after 2M24 / CD20 bispecific treatment (not shown).

[0268] The present disclosure has been described in some detail by way of illustrative illustrations and examples for clarity of understanding, but these illustrations and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent 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, Amino acid sequence QVQLVQSGAEVKKPGASVKVSSCKSSGYTFTDYYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGGGSGGGGGSGGGGGSDIQMTQSPSSVSSASVGDRVTITCRASQGISSWLAWYQQQKPGKAAPKLLIFGASSLQSGVPSRFSSGSGSGTDFTLTVSSSLQPEDFATYYCQQAYSFPFTFGPGTKV DIEEEPKRSDKTHTCPPPPAPELLGGGPSVFLFPPKPKDTLMISRTPEPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 31) or QVQ LVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQ GRITMTRDTSISTAYLELSRLRSDDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGG SGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGA SSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGGPGTKVDIEEPKRSD A first polypeptide chain containing KTHTCPPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35), Amino acid sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPPGRGLEWIGAIYPGNGDTSYNQKFKGKAATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTTPPVLDSDGSFFLVSKLTTVDKSRWQQGNVFSSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 32) or QVQ LQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYN QKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL A second polypeptide chain containing LGGPSVFLFPPKPKDTLMISRTPEPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLVSSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36), A third polypeptide chain containing the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSSVSYIHWFQQKPGSSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33), The multispecific binding protein, including the above.

2. The multispecificity binding protein according to 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 multispecificity binding protein according to 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, Amino acid sequence QVQLVQSGAEVKKPGASVKVSSCKSSGYTFTDYYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQGRITMTRDTSISTAYLELSRLRSDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGGGSGGGGGSGGGGGSDIQMTQSPSSVSSASVGDRVTITCRASQGISSWLAWYQQQKPGKAAPKLLIFGASSLQSGVPSRFSSGSGSGTDFTLTVSSSLQPEDFATYYCQQAYSFPFTFGPGTKV DIEEEPKRSDKTHTCPPPPAPELLGGGPSVFLFPPKPKDTLMISRTPEPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTTPPVLDSDGSFFLVSSKLTVDKSRWQQGNVFSSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 37) or QVQ LVQSGAEVKKPGASVKVSCKSSGYTFTDYYIHWVRQAPGQGLEWMGWINPNSGDTNYAQKFQ GRITMTRDTSISTAYLELSRLRSDDDTAVFYCARNSGSYSFGYWGQGTLVTVSSGGGGGSGGGG SGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIFGA SSLQSGVPSRFSGSGSGTDFTLTVSSLQPEDFATYYCQQAYSFPFTFGGPGTKVDIEEPKRSD A first polypeptide chain containing KTHTCPPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLVSSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 39), Amino acid sequence QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPPGRGLEWIGAIYPGNGDTSYNQKFKGKAATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence ID 38) or QVQ LQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYN QKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL A second polypeptide chain containing LGGPSVFLFPPKPKDTLMISRTPEPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 40), A third polypeptide chain containing the amino acid sequence QIVLSQSPAILSASPGEKVTMTCRASSSSVSYIHWFQQKPGSSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 33), The multispecific binding protein, including the above.

5. The multispecific binding protein according to 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 according to 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 multispecificity binding protein according to any one of claims 1 to 6, wherein at least one of the first polypeptide chain and the second polypeptide chain is not fucosylated.

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

9. The first antigen-binding domain described above is (a) Binds to star dectin-1 expressed on the surface of macrophages, monocytes, dendritic cells, or granulocytes; (b) It binds to human dectin-1 expressed on the surface of cells with an EC50 of less than 2 nM; (c) Capable of binding to human or cynomolgus dectin-1; and / or (d) Not competing with the natural ligand of star dectin-1, The multispecific binding protein according to claim 1 or claim 4.

10. The multispecific binding protein according to claim 1 or claim 4, 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 according to claim 1 or claim 4.

12. A vector comprising a polynucleotide as described in claim 11.

13. An isolated host cell comprising the polynucleotide described in claim 11 or a vector comprising the polynucleotide described in claim 11.

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

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

16. The isolated host cell according to claim 15, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

17. The isolated host cell according to claim 15, wherein the host cell comprises α1,6-fucosyltransferase (Fut8) or α-1,3-mannosylglycoprotein 2-β-N-acetylglucosaminetransferase (MGAT1) knockout.

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

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

20. A method for producing a multispecific binding protein, comprising culturing the host cell described in claim 13 under conditions suitable for the production of the multispecific binding protein.

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

22. The method according to claim 20, wherein the host cells are treated with kifunensin before the production of the multispecific binding protein.

23. A multispecific binding protein produced by the method of claim 20.

24. A pharmaceutical composition comprising a multispecific binding protein according to claim 1 or claim 4 and a pharmaceutically acceptable carrier.

25. The composition according to claim 24, wherein the composition comprises a mixture of multispecific binding protein species, each comprising 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 according to 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 according to 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. The composition according to claim 24, wherein the composition comprises a mixture of multispecific binding protein species, each comprising 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 according to 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 according to 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 composition comprising the multispecific binding protein according to claim 1 or claim 4 for treating a disease or disorder in an individual requiring treatment of the disease or disorder, or a pharmaceutical composition comprising the multispecific binding protein according to claim 1 or claim 4 and a pharmaceutically acceptable carrier.

32. The composition or pharmaceutical composition according to claim 31, wherein the individual has a B cell-mediated disease or disorder.

33. The composition or pharmaceutical composition according to claim 32, wherein the B cell-mediated disease or disorder is cancer.

34. The composition or pharmaceutical composition according to claim 33, wherein the cancer is non-Hodgkin lymphoma or chronic lymphocytic leukemia.

35. The composition or pharmaceutical composition according to claim 32, wherein the B cell-mediated disease or disorder is an autoimmune disease or disorder.

36. The composition or pharmaceutical composition according to claim 35, wherein the autoimmune disease or disorder is rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, or Wegener's granulomatosis.

37. The composition or pharmaceutical composition according to claim 31, wherein the individual is a human.

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