Antibodies targeting c-Kit and / or siglec and their use

Abstract

Undesirable mast cell activity is associated with various diseases or disorders such as allergies, inflammatory diseases, autoimmune diseases, and cancer. There is still a need for new and improved therapeutic agents (e.g., therapeutic agents for mast cell-related diseases or disorders) that can target c-Kit and / or siglec-6. Disclosed herein are molecules such as antibodies and antibody fragments that can bind to c-Kit and / or siglec-6.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 350,832, filed on June 9, 2022, and U.S. Provisional Patent Application No. 63 / 481,257, filed on January 24, 2023, the entire contents of each of which are hereby incorporated by reference for all purposes.

Background Art

[0002] Mast cells (MCs) are innate immune cells that play important roles in inflammatory processes. When activated, MCs secrete various immune mediators such as cytokines, leukotrienes, and numerous proteases into the environment. The differentiation, proliferation, and survival of mast cells are strongly regulated by local tissue environmental factors. Stem cell factor (SCF) and IL-3 are the most well-characterized factors. Undesirable mast cell activity is associated with various diseases or disorders such as allergies, inflammatory diseases, autoimmune diseases, and cancer.

[0003] c-Kit is a type III receptor tyrosine kinase and a well-known cell surface receptor that binds to its physiological ligand, SCF. c-Kit is involved in various intracellular signaling pathways and has multiple functions during embryogenesis and adulthood. c-Kit is highly expressed in MCs, and MCs depend on c-Kit-dependent signaling for survival, function, and proliferation upon complete differentiation. c-Kit antibodies such as CDX-0158 have been developed.

[0004] siglec-6 is a member of the CD33-related subfamily of sialic acid-binding immunoglobulin-like lectins (siglecs). siglec-6 is found on human mast cells, some B cells, and the cytotrophoblast and syncytiotrophoblast of the placenta. siglec-6 has been suggested to have inhibitory activity against both IgE-mediated and non-IgE-mediated mast cell responses.

[0005] There is still a need for new and improved therapeutic agents (e.g., therapeutic agents for mast cell-related diseases or disorders) that can target c-Kit and / or siglec-6.

SUMMARY OF THE INVENTION

[0006] This application provides an antigen-binding site that can bind to c-Kit and / or siglec-6.

[0007] In one aspect, an antibody or an antigen-binding fragment thereof is provided, which comprises a first antigen-binding domain capable of binding to siglec-6 and a second antigen-binding domain capable of binding to c-Kit.

[0008] In some embodiments, the first antigen-binding domain can bind to siglec-6 with a higher affinity than the second antigen-binding domain can bind to c-Kit. In some embodiments, the first antigen-binding domain has a K D value characterized by an affinity that is less than about 100 uM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 100 pM, less than about 10 pM, less than about 1 pM, less than about 100 fM, or less than about 10 fM and can bind to siglec-6. In some embodiments, the first antigen-binding domain has a K D value characterized by an affinity that is from about 1 nM to about 900 uM, from about 5 nM to about 500 uM, from about 10 nM to about 100 uM, from about 20 nM to about 1010 uM, or from about 25 to about 500 nM and can bind to c-Kit.

[0009] In some embodiments, the antibody or antigen-binding fragment can bind to cells expressing siglec-6.

[0010] In some embodiments, the antibody or antigen-binding fragment can bind to mast cells. In some embodiments, the antibody or antigen-binding fragment preferentially binds to mast cells over other immune cells. In some embodiments, the immune cell is a T cell, dendritic cell, macrophage, neutrophil, or basophil.

[0011] In some embodiments, the antibody or its antigen-binding fragment does not induce mast cell degranulation.

[0012] In some embodiments, the antibody or its antigen-binding fragment has an internalization rate equal to or lower than that of a reference antibody that can bind to siglec-6. In some embodiments, the reference antibody that can bind to siglec-6 comprises a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 2, and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 10.

[0013] In some embodiments, the antibody or its antigen-binding fragment has a toxicity equal to or lower than that of a reference antibody that can bind to c-Kit. In some embodiments, the reference antibody that can bind to c-Kit comprises a VH region comprising the amino acid sequence of SEQ ID NO: 40 and a VL region comprising the amino acid sequence of SEQ ID NO: 45.

[0014] In some embodiments, the first antigen-binding domain comprises: (a) a heavy chain complementarity-determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 3, or a sequence that differs from it by one or two amino acids, an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or 21, or a sequence that differs from it by one or two amino acids, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, or a sequence that differs from it by one or two amino acids comprising a VH region, and (b) A heavy chain complementarity-determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 11 or 32, or a sequence differing therefrom by one or two amino acids, an LCDR2 having the amino acid sequence of SEQ ID NO: 12, or a sequence differing therefrom by one or two amino acids, and an LCDR3 having the amino acid sequence of SEQ ID NO: 13, or a sequence differing therefrom by one or two amino acids A VL region comprising

[0015] In some embodiments, the first antigen-binding domain comprises: (a) A VH region comprising an HCDR1 having the amino acid sequence of SEQ ID NO: 3, an HCDR2 having the amino acid sequence of SEQ ID NO: 4 or 21, and an HCDR3 having the amino acid sequence of SEQ ID NO: 5, and (b) A VL region comprising an LCDR1 having the amino acid sequence of SEQ ID NO: 11 or 32, an LCDR2 having the amino acid sequence of SEQ ID NO: 12, and an LCDR3 having the amino acid sequence of SEQ ID NO: 13.

[0016] In some embodiments, the first antigen-binding domain comprises: (a) (i) An HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively, or (ii) An HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively Comprising a VH region, and (b) (i) An LCDR1, an LCDR2, and an LCDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively, or (ii) An LCDR1, an LCDR2, and an LCDR3 having the amino acid sequences of SEQ ID NOs: 32, 12, and 13, respectively Comprising a VL region.

[0017] In some embodiments, the first antigen-binding domain comprises: (a) A VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, 20, 23, 25, 27, or 29, and (b) A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 10, 31, 36, or 38.

[0018] In some embodiments, the first antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 2, 20, 23, 25, 27, or 29, and (b) A VL region having the amino acid sequence of SEQ ID NO: 10, 31, 36, or 38.

[0019] In some embodiments, the first antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 2 and a VL region having the amino acid sequence of SEQ ID NO: 10, (b) A VH region having the amino acid sequence of SEQ ID NO: 23 and a VL region having the amino acid sequence of SEQ ID NO: 38, (c) A VH region having the amino acid sequence of SEQ ID NO: 25 and a VL region having the amino acid sequence of SEQ ID NO: 38, (d) A VH region having the amino acid sequence of SEQ ID NO: 27 and a VL region having the amino acid sequence of SEQ ID NO: 38, or (e) A VH region having the amino acid sequence of SEQ ID NO: 29 and a VL region having the amino acid sequence of SEQ ID NO: 38.

[0020] In some embodiments, the first antigen-binding domain comprises: a VH region having the amino acid sequence of SEQ ID NO: 25 and a VL region having the amino acid sequence of SEQ ID NO: 38.

[0021] In some embodiments, the first antigen-binding domain comprises: (a) An HCDR1 having the amino acid sequence of SEQ ID NO: 122, or a sequence that differs from it by one or two amino acids, an HCDR2 having the amino acid sequence of SEQ ID NO: 123, or a sequence that differs from it by one or two amino acids, and an HCDR3 having the amino acid sequence of SEQ ID NO: 124, or a sequence that differs from it by one or two amino acids comprising a VH region, and (b) an LCDR1 having the amino acid sequence of SEQ ID NO: 127, or a sequence that differs from it by one or two amino acids, an LCDR2 having the amino acid sequence of SEQ ID NO: 128, or a sequence that differs from it by one or two amino acids, and an LCDR3 having the amino acid sequence of SEQ ID NO: 129, or a sequence that differs from it by one or two amino acids comprising a VL region.

[0022] In some embodiments, the first antigen-binding domain comprises: (a) an HCDR1 having the amino acid sequence of SEQ ID NO: 122, an HCDR2 having the amino acid sequence of SEQ ID NO: 123, and an HCDR3 having the amino acid sequence of SEQ ID NO: 124 comprising a VH region, and (b) an LCDR1 having the amino acid sequence of SEQ ID NO: 127, an LCDR2 having the amino acid sequence of SEQ ID NO: 128, and an LCDR3 having the amino acid sequence of SEQ ID NO: 139 comprising a VL region.

[0023] In some embodiments, the first antigen-binding domain comprises: (a) a VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 121, and (b) A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 126.

[0024] In some embodiments, the first antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 121, and (b) A VL region having the amino acid sequence of SEQ ID NO: 126.

[0025] In some embodiments, the first antigen-binding domain comprises: (a) An HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141, or 149, or a sequence that differs from it by one or two amino acids, An HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150, or 160, or a sequence that differs from it by one or two amino acids, and An HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143, or 151, or a sequence that differs from it by one or two amino acids comprising a VH region, and (b) An LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153, or 165, or a sequence that differs from it by one or two amino acids, An LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146, or 154, or a sequence that differs from it by one or two amino acids, and An LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147, or 155, or a sequence that differs from it by one or two amino acids comprising a VL region.

[0026] In some embodiments, the first antigen-binding domain comprises: (a) An HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141, or 149, An HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150, or 160, and an HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143, or 151 comprising a VH region, and (b) an LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153, or 165, an LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146, or 154, and an LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147, or 155 comprising a VL region.

[0027] In some embodiments, the first antigen-binding domain comprises: (a) (i) An HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 133, 134, and 135, respectively, (ii) An HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 141, 142, and 143, respectively, (iii) An HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 149, 150, and 151, respectively, or (iv) An HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 149, 160, and 151, respectively comprising a VH region, and (b) (i) An LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 137, 138, and 139, respectively, (ii) An LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 145, 146, and 147, respectively, (iii) An LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 153, 154, and 155, respectively, or (iv) An LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 165, 154, and 155, respectively The VL region containing

[0028] In some embodiments, the first antigen-binding domain comprises: (a) A VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 132, 140, 148, 159, or 162, and (b) A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 136, 144, 152, 164, 167, or 169.

[0029] In some embodiments, the first antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 132, 140, 148, 159, or 162, and (b) A VL region having the amino acid sequence of SEQ ID NO: 136, 144, 152, 164, 167, or 169.

[0030] In some embodiments, the first antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 132 and a VL region having the amino acid sequence of SEQ ID NO: 139, (b) A VH region having the amino acid sequence of SEQ ID NO: 140 and a VL region having the amino acid sequence of SEQ ID NO: 144, or (c) A VH region having the amino acid sequence of SEQ ID NO: 148 and a VL region having the amino acid sequence of SEQ ID NO: 152.

[0031] In some embodiments, the first antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 159 and a VL region having the amino acid sequence of SEQ ID NO: 164, (b) A VH region having the amino acid sequence of SEQ ID NO: 162 and a VL region having the amino acid sequence of SEQ ID NO: 167, or (c) A VH region having the amino acid sequence of SEQ ID NO: 162 and a VL region having the amino acid sequence of SEQ ID NO: 169.

[0032] In some embodiments, the second antigen-binding domain comprises: (a) An HCDR1 having the amino acid sequence of SEQ ID NO: 41, or a sequence differing therefrom by one or two amino acids, An HCDR2 having the amino acid sequence of SEQ ID NO: 42 or 53, or a sequence differing therefrom by one or two amino acids, and An HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence differing therefrom by one or two amino acids comprising a VH region, and (b) An LCDR1 having the amino acid sequence of SEQ ID NO: 46, or a sequence differing therefrom by one or two amino acids, An LCDR2 having the amino acid sequence of SEQ ID NO: 47, or a sequence differing therefrom by one or two amino acids, and An LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence differing therefrom by one or two amino acids comprising a VL region.

[0033] In some embodiments, the second antigen-binding domain comprises: (a) An HCDR1 having the amino acid sequence of SEQ ID NO: 41, An HCDR2 having the amino acid sequence of SEQ ID NO: 42 or 53, and An HCDR3 having the amino acid sequence of SEQ ID NO: 43 comprising a VH region, and (b) An LCDR1 having the amino acid sequence of SEQ ID NO: 46, An LCDR2 having the amino acid sequence of SEQ ID NO: 47, and An LCDR3 having the amino acid sequence of SEQ ID NO: 48 comprising a VL region.

[0034] In some embodiments, the second antigen-binding domain comprises a VH region comprising: (a) an HCDR1 having the amino acid sequence of SEQ ID NO: 60, 61, or 62, (b) an HCDR2 having the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67, or 68, and (c) an HCDR3 having the amino acid sequence of SEQ ID NO: 69, 70, 71, or 72.

[0035] In some embodiments, the second antigen-binding domain comprises a VH region comprising: (a) an LCDR1 having the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94, or 95, (b) an LCDR2 having the amino acid sequence of SEQ ID NO: 96 or 97, and (c) an LCDR3 having the amino acid sequence of SEQ ID NO: 98, 99, 100, 101, 102, 103, or 104.

[0036] In some embodiments, the second antigen-binding domain comprises: (a) (i) an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 42, and 43, respectively, (ii) an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 43, respectively, (iii) an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 60, 53, and 43, respectively, (iv) an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 61, 53, and 43, respectively, (v) an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 62, 53, and 43, respectively, (vi) an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 63, and 43, respectively, (vii) an HCDR1, an HCDR2, and an HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 64, and 43, respectively, (viii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 65, and 43, respectively, (ix) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 66, and 43, respectively, (x) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 67, and 43, respectively, (xi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 68, and 43, respectively, (xii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 69, respectively, (xiii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 70, respectively, (xiv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 71, respectively, or (xv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 72, respectively comprising a VH region, and (b) (i) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively, (ii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 90, 47, and 48, respectively, (iii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 91, 47, and 48, respectively, (iv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 92, 47, and 48, respectively, (v) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 93, 47, and 48, respectively, (vi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 94, 47, and 48, respectively, (vii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 95, 47, and 48, respectively, (viii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 96, and 48, respectively, (ix) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 97, and 48, respectively, (x) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 47, and 98, respectively, (xi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 47, and 99, respectively, (xii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 47, and 100, respectively, (xiii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 47, and 101, respectively, (xiv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 47, and 102, respectively, (xv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 47, and 103, respectively, or (xvi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NO: 46, 47, and 104, respectively A VL region comprising

[0037] In some embodiments, the second antigen-binding domain comprises: (a) A VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 40, 52, 55, 57, 59, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85, and (b) A VL region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 45, 87, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

[0038] In some embodiments, the second antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 40, 52, 55, 57, 59, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85, and (b) A VL region having the amino acid sequence of SEQ ID NO: 45, 87, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

[0039] In some embodiments, the second antigen-binding domain comprises: (a) A VH region having the amino acid sequence of SEQ ID NO: 40 and a VL region having the amino acid sequence of SEQ ID NO: 45, (b) A VH region having the amino acid sequence of SEQ ID NO: 52 and a VL region having the amino acid sequence of SEQ ID NO: 87, (c) A VH region having the amino acid sequence of SEQ ID NO: 55 and a VL region having the amino acid sequence of SEQ ID NO: 87, (d) A VH region having the amino acid sequence of SEQ ID NO: 57 and a VL region having the amino acid sequence of SEQ ID NO: 87, (e) A VH region having the amino acid sequence of SEQ ID NO: 59 and a VL region having the amino acid sequence of SEQ ID NO: 87, (f) A VH region having the amino acid sequence of SEQ ID NO: 52 and a VL region having the amino acid sequence of SEQ ID NO: 89, or (g) A VH region having the amino acid sequence of SEQ ID NO: 55 and a VL region having the amino acid sequence of SEQ ID NO: 89.

[0040] In some embodiments, the second antigen-binding domain comprises: a VH region having the amino acid sequence of SEQ ID NO: 52, and a VL region having the amino acid sequence of SEQ ID NO: 87.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain comprising a VH region of the first antigen-binding domain and a first heavy chain constant (CH) region or a fragment thereof, (b) a first light chain comprising a VL region of the first antigen-binding domain and a first light chain constant (CL) region or a fragment thereof, (c) a second heavy chain comprising a VH region of the second antigen-binding domain and a second CH region or a fragment thereof, and (d) a second light chain comprising a VL region of the second antigen-binding domain and a second CL region or a fragment thereof.

[0042] In some embodiments, each of the first CH region and the second CH region comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 130.

[0043] In some embodiments, the first heavy chain and the second heavy chain form a heterodimer, and optionally, one of the first heavy chain and the second heavy chain comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407, and the other heavy chain comprises a tryptophan residue at position 366, where the numbering of the constant region follows the EU index.

[0044] In some embodiments, the first CH region and / or the second CH region comprise one or more mutations that reduce or inhibit effector function. In some embodiments, the first CH region and / or the second CH region are human IgG1 CHs that comprise one of the following mutations, with the constant region numbering according to the EU index: (a) L234A and / or L235A, (b) A327G, A330S, and / or P331S, (c) E233P, L234V, L235A, and / or G236del, (d) E233P, L234V, and / or L235A, (e) E233P, L234V, L235A, G236del, A327G, A330S, and / or P331S, (f) E233P, L234V, L235A, A327G, A330S, and / or P331S, (g) N297A, (h) N297G, (i) N297Q, (j) L242C, N297C, and / or K334C, (k) A287C, N297G, and / or L306C, (l) R292C, N297G, and / or V302C, (m) N297G, V323C, and / or I332C, (n) V259C, N297G, and / or L306C, (o) L234F, L235Q, K322Q, M252Y, S254T, and / or T256E, (p) L234A, L235A, and / or P329G, or (q) L234A, L235Q, and K322Q. In some embodiments, the first CH region and / or the second CH region are human IgG2 CHs that comprise one of the following mutations, with the constant region numbering according to the EU index: (a) A330S and / or P331S, (b) V234A, G237A, P238S, H268A, V309L, A330S, and / or P331S, or (c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and / or T256E.In some embodiments, the first CH region and / or the second CH region is a human IgG4 CH containing one of the following mutations, and the numbering of the constant region follows the EU index: (a) E233P, F234V, L235A, and / or G236del, (b) E233P, F234V, and / or L235A, (c) S228P and / or L235E, or (d) S228P and / or L235A.

[0045] In some embodiments, the first CH region and / or the second CH region contains one or more mutations that enhance effector function. In some embodiments, the first CH region and / or the second CH region is a human IgG1 CH containing one of the following mutations, and the numbering of the constant region follows the EU index: (a) F243L, R292P, Y300L, V305I, and / or P396L, (b) S239D and / or I332E, (c) S239D, I332E, and / or A330L, (d) S298A, E333A, and / or K334A, (e) G236A, S239D, and / or I332E, (f) K326W and / or E333S, (g) S267E, H268F, and / or S324T, or (h) E345R, E430G, and / or S440Y.

[0046] In some embodiments, at least one of the first heavy chain and the second heavy chain is non-fucosylated. In some embodiments, the antibody or its antigen-binding fragment is produced in (a) a cell line having an alpha-1,6-fucosyltransferase (Fut8) knockout, or (b) a cell line that overexpresses beta1,4-N-acetylglucosaminyltransferase III (GnT-III) and optionally overexpresses Golgi mu-mannosidase II (ManII).

[0047] In some embodiments, the first CL region and / or the second CL region is a human kappa constant region.

[0048] In some embodiments, (a)(i) The first antigen-binding domain comprises a VH region containing HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively, and a VL region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 32, 12, and 13, respectively, and and (ii) The second antigen-binding domain comprises (ii) a VH region containing HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 43, respectively, and a VL region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively, or or (b)(i) The first antigen-binding domain comprises (b)(i) a VH region containing HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 122, 123, and 124, respectively, and a VL region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 127, 128, and 129, respectively, and and (ii) The second antigen-binding domain comprises (ii) a VH region containing HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 43, respectively, and a VL region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively. In some embodiments, (a) the first antigen-binding domain comprises a VH region having the amino acid sequence of SEQ ID NO: 25 and a VL region having the amino acid sequence of SEQ ID NO: 38, and the second antigen-binding domain comprises a VH region having the amino acid sequence of SEQ ID NO: 52 and a VL region having the amino acid sequence of SEQ ID NO: 87, or

[0049] (b) The first antigen-binding domain comprises a VH region having the amino acid sequence of SEQ ID NO: 121 and a VL region having the amino acid sequence of SEQ ID NO: 126, and the second antigen-binding domain comprises a VH region having the amino acid sequence of SEQ ID NO: 52 and a VL region having the amino acid sequence of SEQ ID NO: 87.

[0050] In some embodiments, the antigen-binding fragment is a Fab, Fab’, Fab2, F(ab’)2, Fv, single-chain Fv (scFv), or diabody.

[0051] In some embodiments, the antibody or its antigen-binding fragment is conjugated to an agent. In some embodiments, the agent is a cytotoxic agent or a label.

[0052] In another aspect, a composition comprising the above-described antibody or its antigen-binding fragment is provided. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region and an N-glycosidically linked sugar chain linked to the Fc region, and optionally, less than 50% of the N-glycosidically linked sugar chains contain fucose residues. In some embodiments, substantially none of the N-glycosidically linked sugar chains contain fucose residues.

[0053] In another aspect, an antibody or its antigen-binding fragment capable of binding to siglec-6 is provided, the antibody or its antigen-binding fragment comprising: (a) A heavy-chain complementarity-determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 133, 141, or 149, or a sequence differing therefrom by one or two amino acids, An HCDR² having the amino acid sequence of SEQ ID NO: 134, 142, 150, or 160, or a sequence differing therefrom by one or two amino acids, and An HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143, or 151, or a sequence differing therefrom by one or two amino acids Comprising a heavy-chain variable (VH) region, and (b) A light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 137, 145, 153, or 165, or a sequence differing therefrom by one or two amino acids, an LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146, or 154, or a sequence differing therefrom by one or two amino acids, and, an LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147, or 155, or a sequence differing therefrom by one or two amino acids comprising a light chain variable (VL) region.

[0054] In some embodiments, the antibody or antigen-binding fragment comprises: (a) an HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141, or 149, an HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150, or 160, and an HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143, or 151 comprising a VH region, and (b) an LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153, or 165, an LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146, or 154, and an LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147, or 155 comprising a VL region.

[0055] In some embodiments, the antibody or antigen-binding fragment comprises: (a)(i) an HCDR1 having the amino acid sequence of SEQ ID NO: 133, or a sequence differing therefrom by one or two amino acids, an HCDR2 having the amino acid sequence of SEQ ID NO: 134, or a sequence differing therefrom by one or two amino acids, and an HCDR3 having the amino acid sequence of SEQ ID NO: 135, or a sequence differing therefrom by one or two amino acids a VH region comprising, and (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 137, or a sequence differing therefrom by one or two amino acids, an LCDR2 having the amino acid sequence of SEQ ID NO: 138, or a sequence differing therefrom by one or two amino acids, and an LCDR3 having the amino acid sequence of SEQ ID NO: 139, or a sequence differing therefrom by one or two amino acids a VL region comprising, or (b)(i) an HCDR1 having the amino acid sequence of SEQ ID NO: 141, or a sequence differing therefrom by one or two amino acids, an HCDR2 having the amino acid sequence of SEQ ID NO: 142, or a sequence differing therefrom by one or two amino acids, and an HCDR3 having the amino acid sequence of SEQ ID NO: 143, or a sequence differing therefrom by one or two amino acids a VH region comprising, and (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 145, or a sequence differing therefrom by one or two amino acids, an LCDR2 having the amino acid sequence of SEQ ID NO: 146, or a sequence differing therefrom by one or two amino acids, and an LCDR3 having the amino acid sequence of SEQ ID NO: 147, or a sequence differing therefrom by one or two amino acids a VL region comprising, or (c)(i) an HCDR1 having the amino acid sequence of SEQ ID NO: 149, or a sequence differing therefrom by one or two amino acids, an HCDR2 having the amino acid sequence of SEQ ID NO: 150 or 160, or a sequence differing therefrom by one or two amino acids, and an HCDR3 having the amino acid sequence of SEQ ID NO: 151, or a sequence differing therefrom by one or two amino acids a VH region comprising, and (ii) An LCDR1 having the amino acid sequence of SEQ ID NO: 153 or 165, or a sequence differing therefrom by one or two amino acids, an LCDR2 having the amino acid sequence of SEQ ID NO: 154, or a sequence differing therefrom by one or two amino acids, and an LCDR3 having the amino acid sequence of SEQ ID NO: 155, or a sequence differing therefrom by one or two amino acids comprising a VL region.

[0056] In some embodiments, the antibody or antigen-binding fragment comprises: (a)(i) an HCDR1 having the amino acid sequence of SEQ ID NO: 133, an HCDR2 having the amino acid sequence of SEQ ID NO: 134, and an HCDR3 having the amino acid sequence of SEQ ID NO: 135 comprising a VH region, and (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 137, an LCDR2 having the amino acid sequence of SEQ ID NO: 138, and an LCDR3 having the amino acid sequence of SEQ ID NO: 139 comprising a VL region, or (b)(i) an HCDR1 having the amino acid sequence of SEQ ID NO: 141, an HCDR2 having the amino acid sequence of SEQ ID NO: 142, and an HCDR3 having the amino acid sequence of SEQ ID NO: 143 comprising a VH region, and (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 145, an LCDR2 having the amino acid sequence of SEQ ID NO: 146, and an LCDR3 having the amino acid sequence of SEQ ID NO: 147 comprising a VL region, or (c)(i) an HCDR1 having the amino acid sequence of SEQ ID NO: 149, An HCDR2 having the amino acid sequence of SEQ ID NO: 150 or 160, and an HCDR3 having the amino acid sequence of SEQ ID NO: 151 comprising a VH region, and (ii) an LCDR1 having the amino acid sequence of SEQ ID NO: 153 or 165, an LCDR2 having the amino acid sequence of SEQ ID NO: 154, and an LCDR3 having the amino acid sequence of SEQ ID NO: 155 comprising a VL region.

[0057] In some embodiments, the antibody or antigen-binding fragment comprises: (a) (i) an HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 133, 134, and 135, respectively, (ii) an HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 141, 142, and 143, respectively, (iii) an HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 149, 150, and 151, respectively, or (iv) an HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 149, 160, and 151, respectively comprising a VH region, and (b) (i) an LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 137, 138, and 139, respectively, (ii) an LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 145, 146, and 147, respectively, (iii) an LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 153, 154, and 155, respectively, or (iv) an LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 165, 154, and 155, respectively comprising a VL region.

[0058] In some embodiments, the VH region comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 132, 140, 148, 159, or 162, and the VL region comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 136, 144, 152, 164, 167, or 169.

[0059] In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 132, 140, 148, 159, or 162, and the VL region comprises the amino acid sequence of SEQ ID NO: 136, 144, 152, 164, 167, or 169.

[0060] In some embodiments, (a) the VH region comprises the amino acid sequence of SEQ ID NO: 132 and the VL region comprises the amino acid sequence of SEQ ID NO: 139, (b) the VH region comprises the amino acid sequence of SEQ ID NO: 140 and the VL region comprises the amino acid sequence of SEQ ID NO: 144, or (c) the VH region comprises the amino acid sequence of SEQ ID NO: 148 and the VL region comprises the amino acid sequence of SEQ ID NO: 152.

[0061] In some embodiments, (a) the VH region comprises the amino acid sequence of SEQ ID NO: 159 and the VL region comprises the amino acid sequence of SEQ ID NO: 164, (b) the VH region comprises the amino acid sequence of SEQ ID NO: 162 and the VL region comprises the amino acid sequence of SEQ ID NO: 167, or (c) the VH region comprises the amino acid sequence of SEQ ID NO: 162 and the VL region comprises the amino acid sequence of SEQ ID NO: 169.

[0062] In some embodiments, the antibody is a humanized antibody.

[0063] In another aspect, there is provided an antibody or an antigen-binding fragment thereof that can bind to siglec-6, the antibody or the antigen-binding fragment thereof comprising: (a) Heavy chain complementarity-determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 21, and HCDR3 having the amino acid sequence of SEQ ID NO: 5 A heavy chain variable domain (VH) comprising, and (a) Light chain complementarity-determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 32, LCDR2 having the amino acid sequence of SEQ ID NO: 12, and LCDR3 having the amino acid sequence of SEQ ID NO: 13 A light chain variable domain (VL) comprising.

[0064] In some embodiments, the VH region comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 20, 23, 25, 27, or 29, and the VL region comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 31, 36, or 38.

[0065] In some embodiments, the antibody or antigen-binding fragment comprises a VH region comprising the amino acid sequence of SEQ ID NO: 20, 23, 25, 27, or 29, and a VL region comprising the amino acid sequence of SEQ ID NO: 31, 36, or 38.

[0066] In some embodiments, (a) the VH region comprises the amino acid sequence of SEQ ID NO: 23 and the VL region comprises the amino acid sequence of SEQ ID NO: 38, (b) the VH region comprises the amino acid sequence of SEQ ID NO: 25 and the VL region comprises the amino acid sequence of SEQ ID NO: 38, (c) the VH region comprises the amino acid sequence of SEQ ID NO: 27 and the VL region comprises the amino acid sequence of SEQ ID NO: 38, or (d) the VH region comprises the amino acid sequence of SEQ ID NO: 29 and the VL region comprises the amino acid sequence of SEQ ID NO: 38.

[0067] In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NO: 25, and the VL region comprises the amino acid sequence of SEQ ID NO: 38.

[0068] In some embodiments, the antibody or antigen-binding fragment can bind to siglec-6 with a K D value of up to about 10 nM, up to about 5 nM, or up to about 1 nM. In some embodiments, the antibody or antigen-binding fragment thereof can (a) bind to human siglec-6, (b) bind to cynomolgus siglec-6, (c) optionally bind to mast cells preferentially over another immune cell (e.g., a T cell, dendritic cell, macrophage, neutrophil, or basophil), (d) not induce mast cell degranulation, and / or (e) have an equivalent or lower internalization rate compared to a reference antibody that can bind to siglec-⑥ Optionally, the reference antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 2 and a VL region comprising the amino acid sequence of SEQ ID NO: 10.

[0069] In another aspect, there is provided a humanized antibody or antigen-binding fragment thereof that can bind to c-Kit, the antibody or antigen-binding fragment thereof comprising: (a) a heavy chain complementarity-determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 41, or a sequence that differs from it by one amino acid, an HCDR2 having the amino acid sequence of SEQ ID NO: 53, or a sequence that differs from it by one amino acid, and an HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence that differs from it by one amino acid comprising a heavy chain variable domain (VH), and (a) a light chain complementarity-determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 46, or a sequence that differs from it by one amino acid, an LCDR2 having the amino acid sequence of SEQ ID NO: 47, or a sequence that differs from it by one amino acid, and An LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence that differs from it by one amino acid A light chain variable domain (VL) comprising the same

[0070] In some embodiments, the antibody or antigen-binding fragment comprises: (a) An HCDR1 having the amino acid sequence of SEQ ID NO: 41, An HCDR2 having the amino acid sequence of SEQ ID NO: 53, and An HCDR3 having the amino acid sequence of SEQ ID NO: 43 Comprising a VH region, and (b) An LCDR1 having the amino acid sequence of SEQ ID NO: 46, An LCDR2 having the amino acid sequence of SEQ ID NO: 47, and An LCDR3 having the amino acid sequence of SEQ ID NO: 48 Comprising a VL region

[0071] In some embodiments, VH comprises: (a) An HCDR1 having the amino acid sequence of SEQ ID NO: 60, 61, or 62, (b) An HCDR2 having the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67, or 68, and (c) An HCDR3 having the amino acid sequence of SEQ ID NO: 69, 70, 71, or 72

[0072] In some embodiments, VL comprises: (a) An LCDR1 having the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94, or 95, (b) An LCDR2 having the amino acid sequence of SEQ ID NO: 96 or 97, and (c) An LCDR3 having the amino acid sequence of SEQ ID NO: 98, 99, 100, 101, 102, 103, or 104

[0073] In some embodiments, (a) The VH region is as follows: (i) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 43, respectively, (ii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 60, 53, and 43, respectively, (iii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 61, 53, and 43, respectively, (iv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 62, 53, and 43, respectively, (v) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 63, and 43, respectively, (vi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 64, and 43, respectively, (vii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 65, and 43, respectively, (viii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 66, and 43, respectively, (ix) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 67, and 43, respectively, (x) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 68, and 43, respectively, (xi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 69, respectively, (xii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 70, respectively, (xiii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 71, respectively, or (xiv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53, and 72, respectively comprising, (b) The VL region is as follows: (i) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively, (ii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 90, 47, and 48, respectively, (iii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 91, 47, and 48, respectively, (iv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 92, 47, and 48, respectively, (v) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 93, 47, and 48, respectively, (vi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 94, 47, and 48, respectively, (vii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 95, 47, and 48, respectively, (viii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 96, and 48, respectively, (ix) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 97, and 48, respectively, (x) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 98, respectively, (xi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 99, respectively, (xii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 100, respectively, (xiii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 101, respectively, (xiv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 102, respectively, (xv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 103, respectively, or (xvi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47, and 104, respectively comprising.

[0074] In some embodiments, the VH region comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NOs: 52, 55, 57, 59, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85, and the VL region comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity to the amino acid sequence of SEQ ID NOs: 87, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

[0075] In some embodiments, the VH region comprises the amino acid sequence of SEQ ID NOs: 52, 55, 57, 59, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85, and the VL region comprises the amino acid sequence of SEQ ID NOs: 87, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

[0076] In some embodiments, (a) the VH region comprises the amino acid sequence of SEQ ID NO: 40 and the VL region comprises the amino acid sequence of SEQ ID NO: 45, (b) the VH region comprises the amino acid sequence of SEQ ID NO: 52 and the VL region comprises the amino acid sequence of SEQ ID NO: 87, (c) the VH region comprises the amino acid sequence of SEQ ID NO: 55 and the VL region comprises the amino acid sequence of SEQ ID NO: 87, (d) the VH region comprises the amino acid sequence of SEQ ID NO: 57 and the VL region comprises the amino acid sequence of SEQ ID NO: 87, (e) the VH region comprises the amino acid sequence of SEQ ID NO: 59 and the VL region comprises the amino acid sequence of SEQ ID NO: 87, (f) The VH region contains the amino acid sequence of SEQ ID NO: 52, and the VL region contains the amino acid sequence of SEQ ID NO: 89, or (g) The VH region contains the amino acid sequence of SEQ ID NO: 55, and the VL region contains the amino acid sequence of SEQ ID NO: 89.

[0077] In some embodiments, the VH region contains the amino acid sequence of SEQ ID NO: 52, and the VL region contains the amino acid sequence of SEQ ID NO: 87.

[0078] In some embodiments, the antibody or antigen-binding fragment thereof can bind to siglec-6 with a K D value of about 10 to about 500 nM, about 20 to about 100 nM, or about 25 to about 80 nM. In some embodiments, the antibody or antigen-binding fragment thereof can (a) bind to human c-Kit, (b) bind to cynomolgus c-Kit, and / or (c) have equivalent or lower toxicity compared to a reference antibody that can bind to c-Kit, optionally, the reference antibody comprises a VH region containing the amino acid sequence of SEQ ID NO: 40 and a VL region containing the amino acid sequence of SEQ ID NO: 45.

[0079] In some embodiments, the antigen-binding fragment described above is a Fab, F(ab’)2, Fab’, single-chain Fv (scFv), Fv fragment, Fd fragment, or diabody.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof contains an antibody heavy chain constant region, optionally, the constant region is a human IgG heavy chain constant region, optionally, the human IgG heavy chain constant region is an IgG1 heavy chain constant region. In some embodiments, the constant region contains one or more mutations that reduce or suppress effector function. In some embodiments, the constant region contains one or more mutations that enhance effector function. In some embodiments, at least one of the first and second heavy chains is defucosylated (e.g., at least 75% is defucosylated).

[0081] In some embodiments, the antibodies or antigen-binding fragments described herein are conjugated to a therapeutic agent or a label.

[0082] In another aspect, a polynucleotide encoding the antibody or antigen-binding fragment thereof described herein is provided. In another aspect, an expression vector comprising the polynucleotide is provided. In another aspect, a host cell comprising the polynucleotide or the expression vector is provided. In some embodiments, the host cell is a mammalian cell or an insect cell. In some embodiments, the host cell comprises (a) a Fut8 knockout and / or (b) overexpresses GnT-III and optionally ManII.

[0083] In another aspect, a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein is provided.

[0084] In another aspect, a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof described herein or the pharmaceutical composition described herein, is provided. In some embodiments, the disease or disorder is a mast cell-related disease or disorder. In some embodiments, the disease or disorder is an autoimmune disease, allergy, inflammation, pruritus, or cancer. In some embodiments, the disease or disorder is related to the expression of siglec-6 and / or c-Kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0085]

Figure 1

[0086]

Figure 2

[0087]

Figure 3

BRIEF DESCRIPTION OF THE DRAWINGS

[0088] DETAILED DESCRIPTION DEFINITIONS As used herein, the terms "a" and "an" mean "one or more" and include plural unless the context is inappropriate.

[0089] As used herein, the terms "about," "approximately," and "comparable to" when used herein with respect to a value refer to a value similar to the referenced value in the context in which the referenced value is used. Generally, one of ordinary skill in the art with knowledge of the context will understand the relevant degree of variation subsumed in "about," "approximately," or "comparable to" in that context. For example, in some embodiments, the terms "about," "approximately," and "comparable to" may include values that fall within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0090] As used herein, the terms "antagonist," "neutralize," or "block," when used with respect to an antibody or an antigen-binding fragment thereof, are intended to refer to an antibody or fragment thereof whose binding to a target results in inhibition of at least a portion of the biological activity of that target.

[0091] As used herein, "antibody" refers to a polypeptide, or a fragment thereof, having an amino acid sequence that specifically binds to a designated antigen, including immunoglobulins and fragments thereof. Antibodies according to the present invention can be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). Those skilled in the art will understand that characteristic sequences or portions of an antibody can include amino acids found in one or more regions of the antibody (e.g., variable regions, hypervariable regions, constant regions, heavy chains, light chains, and combinations thereof). Further, those skilled in the art will understand that characteristic sequences or portions of an antibody may include one or more polypeptide chains and may include sequence elements found in the same or different polypeptide chains.

[0092] As used herein, "antibody mimetic" refers to any molecule that can mimic the ability of an antibody to bind an antigen, but is not limited to an antibody structure. Examples of antibody mimetics include, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, centyrins, DARPins, finomers, monobodies, and nanoclamps.

[0093] An "antigen-binding fragment" of an antibody comprises a portion of an intact antibody that is still capable of antigen binding. In certain embodiments, papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments and a remaining "Fc" fragment, a designation that reflects the ability to crystallize readily. The Fab fragment consists of the variable domain of the heavy chain (V H ) together with the entire light chain and the first constant domain of one heavy chain (C HIt consists of 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. In certain embodiments, pepsin treatment of an antibody yields a single large F(ab’)2 fragment, which is approximately equivalent to two Fab fragments with different antigen-binding activities disulfide-bonded together and can still cross-link to an antigen. The Fab’ fragment differs from the Fab fragment in having several additional residues at the carboxy terminus of the C H 1 domain. Fab’-SH refers to a Fab’ in which the cysteine residue of the constant domain has a free thiol group. F(ab’)2 antibody fragments were originally produced as pairs of Fab’ fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

[0094] As used herein, the term “antigen-binding site” refers to any molecule or any part of a molecule capable of binding an antigen. In a human antibody, the antigen-binding site is formed by the amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly branched extension regions within the V regions of the heavy and light chains are called “hypervariable regions” and are inserted between more conserved side-chain extension regions known as “framework regions” or “FRs”. Thus, the term “FR” refers to the amino acid sequences found naturally between and adjacent to hypervariable regions within an immunoglobulin. In a human antibody, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and each of the three hypervariable regions of the heavy and light chains is called a “complementary determining region” or “CDR”. In certain animals such as camels and cartilaginous fish, the antigen-binding site may be formed by a single antibody chain that provides a “single-domain antibody”. Also, the antigen-binding site can be an antibody mimetic or can be present within an antibody mimetic.

[0095] CDRs can be determined by the methods described in Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological Interest. (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996). CDRs determined based on these definitions typically also include an overlap or subset of amino acid residues when compared to each other. In certain embodiments, the term "CDR" is a CDR as defined by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A., Protein Sequence and Structure Analysis of Antibody Variable Domains, in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991). In certain embodiments, the heavy chain CDRs and light chain CDRs of an antibody are defined using different rules. For example, in certain embodiments, the heavy chain CDRs are defined according to MacCallum (supra) and the light chain CDRs are defined according to Kabat (supra). CDRH1, CDRH2, and CDRH3 denote the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 denote the light chain CDRs.

[0096] As used herein, the "binding affinity" of a molecule of the disclosure (e.g., an antigen-binding site) for a given target (e.g., FCER1 or c-Kit) can be determined by a number of methods known to those of skill in the art. Such methods include, but are not limited to, fluorescence titration, ELISA (enzyme-linked immunosorbent assay), calorimetry, e.g., isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR).

[0097] As used herein, "bispecific" refers to a molecule that can specifically bind to at least two distinct targets. Typically, a bispecific molecule comprises two antigen-binding sites, each of which is specific for a different target. In some embodiments, the bispecific molecule can bind to two targets simultaneously.

[0098] As used herein, unless otherwise specified, "c-Kit" refers to human c-Kit as defined by UniProt P10721 and includes variants, isoforms, and interspecies homologs of human c-Kit. c-Kit is also known interchangeably as "KIT", "CD117", "mast / stem cell growth factor receptor Kit", "SCFR", "proto-oncogene c-Kit", or "tyrosine protein kinase Kit".

[0099] As used herein, "detectable affinity" typically refers to the ability to bind to a given target with an affinity constant of up to about 10 D or EC 50 M or less, as measured by K -5 or EC D or EC 50 (the lower the K

[0100] or EC

[0100] value, the better the binding ability). The importance of lower affinities that are no longer measurable by common methods such as ELISA (enzyme-linked immunosorbent assay) is secondary.As used herein, the term "epitope" is an antigenic determinant that is known as a paratope and interacts with a specific antigen-binding site within the variable region of an antibody molecule, which is composed of six complementarity-determining regions of the antibody. A single antigen may have multiple epitopes. Epitopes can be conformational or linear. Conformational epitopes are composed of amino acids that are spatially juxtaposed and derived from different segments of a linear polypeptide chain. Linear epitopes are composed of adjacent amino acid residues within a polypeptide chain.

[0101] The "Fc" fragment contains the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0102] A "fragment" with respect to an antigen (e.g., siglec-6 or c-Kit) refers to a truncated N-terminus and / or C-terminus of the antigen or a protein domain. In some embodiments, the fragment of the antigen retains the ability to be recognized and / or bound by an antibody or an antigen-binding fragment thereof of the present disclosure.

[0103] As used herein, "fucosylation" or "fucosylated" refers to the presence of fucose residues within an oligosaccharide attached to the peptide backbone of an antibody. Specifically, a fucosylated antibody contains an α(1,6)-linked fucose on one or both of the innermost N-acetylglucosamine (GlcNAc) residues of the N-linked oligosaccharides attached to the antibody Fc region. "Non-fucosylated", "defucosylated", or "fucose-deficient" antibodies refer to glycosylated antibody variants that include an Fc region, and the sugar structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, a non-fucosylated antibody or a fucose-deficient antibody has reduced fucose compared to the amount of fucose in the same antibody produced in a cell line. In some embodiments, an antibody with reduced or absent fucose has improved ADCC function.

[0104] The "degree of fucosylation" is the percentage of fucosylated oligosaccharides relative to all oligosaccharides, which can be identified by methods known in the art and can be identified, for example, in N-glycosidase F-treated antibody compositions evaluated by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In a composition of "fully fucosylated antibodies", essentially all oligosaccharides contain a fucose residue, i.e., are fucosylated. In some embodiments, a composition of fully fucosylated antibodies has a degree of fucosylation of at least about 90%. In contrast, in a composition of "fully non-fucosylated antibodies", none of the oligosaccharides are essentially fucosylated. In some embodiments, a composition of fully non-fucosylated antibodies has a degree of fucosylation of less than about 10%. In a composition of "partially fucosylated antibodies", only some of the oligosaccharides contain fucose. In such a composition, an individual antibody may or may not contain a fucose residue in the N-linked oligosaccharide within the Fc region, or may contain it on one or both sides, provided that the composition essentially does not contain any individual antibodies that lack a fucose residue in the N-linked oligosaccharides within the Fc region, nor essentially all individual antibodies that contain fucose residues in both N-linked oligosaccharides within the Fc region. In one embodiment, a composition of partially fucosylated antibodies has a degree of fucosylation of about 10% to about 80% (e.g., about 50% to about 80%, about 60% to about 80%, or about 70% to about 80%). Assays for measuring the degree of fucosylation, as well as methods and cell lines for producing antibodies in which fucosylation has changed, reduced, or disappeared, are known in the art (see, for example, WO2023 / 044390A1).

[0105] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. For example, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Further, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on an antigen. The modifier "monoclonal" indicates the characteristic of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be made using the hybridoma method or by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries.

[0106] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier that renders the composition substantially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0107] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of the compounds described in this application and can provide the compounds described in this application or their active metabolites or residues upon administration to a subject. As is known to those skilled in the art, the "salts" of the compounds described in this application can be derived from inorganic or organic acids and bases.

[0108] Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids such as oxalic acid, although not pharmaceutically acceptable per se, can be used in the preparation of salts useful as intermediates in obtaining the compounds described in this application and their pharmaceutically acceptable acid addition salts.

[0109] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of the formula NW4 + (wherein W is C 1~4 alkyl).

[0110] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include, for example, those formed with suitable cations such as Na + , NH4 + , and NW4 + (wherein W is C 1~4 alkyl) and containing anions of the compounds described in this application.

[0111] In the case of therapeutic use, salts of the compounds described in this application are intended to be pharmaceutically acceptable. However, salts of acids and bases that are not pharmaceutically acceptable may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0112] As used herein, unless otherwise specified, "siglec-6" means human siglec-6 as defined by UniProt O43699, and includes variants, isoforms, and interspecies homologs of human siglec-6. Siglec-6 is also known interchangeably as "SIGLEC6", "sialic acid-binding Ig-like lectin 6", "CD33 antigen-like 1", "CD33L", "CD33L1", "CD33L2", "CD327", "CDw327", "obesity-binding protein 1", or "OB-BP1".

[0113] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably humans.

[0114] As used herein, the phrases "therapeutically effective amount" and "effective amount" are used interchangeably and refer to an amount effective at the dosages and for the periods necessary to achieve the desired therapeutic effect. The therapeutically effective amount may vary depending on factors such as the type of disease (e.g., cancer), the condition, the age, sex, and / or weight of the individual, and the ability of the immunoconjugate (or its pharmaceutical composition) to elicit the desired response in the individual. The effective amount may also be an amount at which any toxic or detrimental effects of the immunoconjugate or its pharmaceutical composition are outweighed by the therapeutically beneficial effects.

[0115] As used herein, "treating" a symptom (e.g., a symptom described herein such as cancer) or "treatment" of a symptom is a procedure for obtaining a beneficial or desired result, e.g., a clinical result. Beneficial or desired results include, but are not limited to, reduction or amelioration of one or more signs or symptoms, whether detectable or undetectable, decrease in the degree of a disease, disorder, or symptom, the disease, disorder, or symptom being in a stable (i.e., not worsening) state, prevention of spread of a disease, disorder, or symptom (e.g., primary cancer or secondary metastasis), delay or slowing of progression of a disease, disorder, or symptom, improvement or alleviation of a disease, disorder, or symptom, and (partial or complete) remission.

[0116] antigen-binding site an antigen-binding site capable of binding to c-Kit In another aspect, the present application provides an antigen-binding site capable of binding to c-Kit.

[0117] In some embodiments, an antigen-binding site capable of binding to c-Kit and present as an antibody mimetic is provided. In some embodiments, a variant of an antigen-binding site capable of binding to c-Kit and present as an antibody mimetic described herein is provided, and such antigen-binding site is capable of binding to c-Kit and comprises an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the antigen-binding site present as an antibody mimetic described herein.

[0118] In some embodiments, an antigen-binding site that can bind to c-Kit is provided, which exists as an antibody (e.g., a bispecific antibody). In some embodiments, a variant of the antigen-binding site that can bind to c-Kit is provided, which exists as the antibody described herein, and such an antigen-binding site can bind to c-Kit and has an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence contained in the antigen-binding site that exists as the antibody described herein.

[0119] In some embodiments, the provided antigen-binding site that can bind to c-Kit comprises a heavy-chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and / or a light-chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3. In some embodiments, the provided antigen-binding site that can bind to c-Kit comprises VH and VL, where VH comprises CDR-H1, CDR-H2, and CDR-H3, and VL comprises CDR-L1, CDR-L2, and CDR-L3. Here, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are each independently selected from those of the VH or VL described in Table 10. The CDRs of VH and VL are determined by the Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those described in Table 10. In some embodiments, there is provided an antigen-binding site that is a variant of the antigen-binding site capable of binding to c-Kit described herein, such an antigen-binding site having CDR sequences that each differ by no more than two amino acid residues (e.g., two or one amino acid residue) from the CDR sequences described in Table 10. In some embodiments, there is provided an antigen-binding site that is a variant of the antigen-binding site capable of binding to c-Kit described herein, such an antigen-binding site having a set of six CDRs that collectively differ by no more than two amino acid residues (e.g., two or one amino acid residue) from the set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 10. In some embodiments, there is provided an antigen-binding site that is a variant of the antigen-binding site capable of binding to c-Kit described herein, such an antigen-binding site having a set of six CDRs that collectively differ by no more than two amino acid residues (e.g., two or one amino acid residue) from the set of six CDRs of an anti-c-Kit having a set of VH and VL selected from Table 10.

[0120] In some embodiments, the provided antigen-binding site that can bind to c-Kit comprises a VH sequence as set forth in Table 10 and a VL sequence as set forth in Table 10. In some embodiments, there is provided an antigen-binding site that is a variant of an antigen-binding site capable of binding to c-Kit as described herein, such as an anti-c-Kit having a set of VH and VL selected from Table 10, and such an antigen-binding site has (1) a VH comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VH set forth in Table 10, and (2) a VL comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL set forth in Table 10.

[0121] In some embodiments, the provided antigen-binding site that can bind to c-Kit comprises a heavy chain sequence as set forth in Table 10 and a light chain sequence as set forth in Table 10. In some embodiments, provided is an antigen-binding site that is a variant of an antigen-binding site that can bind to c-Kit as described herein, such as an anti-c-Kit having a set of heavy and light chains selected from Table 10, wherein such an antigen-binding site has (1) a heavy chain comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the heavy chain set forth in Table 10, and (2) a light chain comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the light chain set forth in Table 10.

[0122] In certain embodiments, the antigen-binding sites described in this application are derived from murine SR1. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 40, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 45. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 40 and 45, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH includes CDR-H1, CDR-H2, and CDR-H3, which include the amino acid sequences of SEQ ID NOs: 41, 42, and 43, respectively. In certain embodiments, the VL includes CDR-L1, CDR-L2, and CDR-L3, which include the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively. In certain embodiments, the antigen-binding site includes (a) a VH comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 41, 42, and 43, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively.

[0123] In certain embodiments, the antigen-binding sites described in this application are derived from humanized SR1-1. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 52, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 87. In certain embodiments, the VH comprises the amino acid sequence of SEQ ID NO: 52, 55, 57, 59, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85. In certain embodiments, the VL comprises the amino acid sequence of SEQ ID NO: 87, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 52 and 87, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 41, 53, and 43, respectively. In certain embodiments, the VL comprises CDR-LS, CDR-L2, and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR-H1, CDR-H2, and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 41, 53, and 43, respectively, and (b) a VL comprising CDR-L1, CDR-L2, and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 46, 47, and 48, respectively.

[0124] In certain embodiments, the humanized SR1-1 variant comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and the sequence of each of these differs from the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 52 and 87, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art, by no more than one amino acid. In certain embodiments, VH comprises CDR-H1, CDR-H2, and CDR-H3, and the sequence of each of these differs from SEQ ID NOs: 41, 53, and 43, respectively, by no more than one amino acid. In certain embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3, and the sequence of each of these differs from SEQ ID NOs: 46, 47, and 48, respectively, by no more than one amino acid. In certain embodiments, the humanized SR1-1 variant comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and these sequences differ collectively from the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 52 and 87, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art, by at least one (e.g., one, two, or three) amino acid residues. In certain embodiments, VH comprises CDR-H1, CDR-H2, and CDR-H3, and these sequences differ collectively from SEQ ID NOs: 41, 53, and 43, respectively, by at least one amino acid residue. In certain embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3, and these sequences differ collectively from SEQ ID NOs: 46, 47, and 48, respectively, by at least one amino acid residue. In certain embodiments, CDR-H1 comprises the amino acid sequence of SEQ ID NO: 60, 61, or 62. In certain embodiments, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67, or 68. In certain embodiments, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 69, 70, 71, or 72.In certain embodiments, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94, or 95. In certain embodiments, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 96 or 97. In certain embodiments, CDR-L3 comprises the amino acid sequence of SEQ ID NO: 98, 99, 100, 101, 102, 103, or 104.

[0125] In certain embodiments, the antigen-binding site described in this application is derived from humanized SR1-2. For example, in certain embodiments, the antigen-binding site described in this application comprises a VH having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 55, and a VL having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 87. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 55 and 87, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0126] In certain embodiments, the antigen-binding sites described in this application are derived from humanized SR1-3. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 57, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 87. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 57 and 87, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0127] In certain embodiments, the antigen-binding sites described in this application are derived from humanized SR1-4. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 59, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 87. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 59 and 87, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0128] In certain embodiments, the antigen-binding sites described in this application are derived from humanized SR1-5. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 52, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 89. In certain embodiments, the antigen-binding sites include CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 52 and 89, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0129] In certain embodiments, the antigen-binding sites described in this application are derived from humanized SR1-6. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 55, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 89. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 5 and 89, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0130] In each of the foregoing embodiments, the VH and / or VL sequences that bind to c-Kit may include amino acid changes (e.g., at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions) in the framework regions of the VH and / or VL without affecting their ability to bind to c-Kit. For example, the VH and VL sequences that bind to c-Kit can include cysteine heterodimerization mutations, which are contemplated herein to promote the formation of disulfide bridges between VH and VL to form an scFv.

[0131] In certain embodiments, the antigen-binding sites disclosed herein bind to human c-Kit with a K D value (affinity greater than these numerical values). In certain embodiments, the antigen-binding sites of the present application have a K D value (affinity less than these numerical values) for binding to human c-Kit. In certain embodiments, the antigen-binding sites of the present application have a K DIt binds to human c-Kit with a value. In certain embodiments, the antigen-binding site of the present application is from about 1 pM to about 900 μM, 1 pM to about 500 μM, 1 pM to about 100 μM, 1 pM to about 50 μM, 1 pM to about 10 μM, 1 pM to about 5 μM, 1 pM to about 1 μM, 1 pM to about 500 nM, 1 pM to about 100 nM, 1 pM to about 50 nM, 1 pM to about 10 nM, 1 pM to about 5 nM, 1 pM to about 1 nM, 1 pM to about 500 pM, 1 pM to about 100 pM, 1 pM to about 50 pM, 1 pM to about 10 pM, 1 pM to about 5 pM, 5 pM to about 900 μM, 5 pM to about 500 μM, 5 pM to about 100 μM, 5 pM to about 50 μM, 5 pM to about 10 μM, 5 pM to about 5 μM, 5 pM to about 1 μM, 5 pM to about 500 nM, 5 pM to about 100 nM, 5 pM to about 50 nM, 5 pM to about 10 nM, 5 pM to about 5 nM, 5 pM to about 1 nM, 5 pM to about 500 pM, 5 pM to about 100 pM, 5 pM to about 50 pM, 5 pM to about 10 pM, 10 pM to about 900 μM, 10 pM to about 500 μM, 10 pM to about 100 μM, 10 pM to about 50 μM, 10 pM to about 10 μM, 10 pM to about 5 μM, 10 pM to about 1 μM, 10 pM to about 500 nM, 10 pM to about 100 nM, 10 pM to about 50 nM, 10 pM to about 10 nM, 10 pM to about 5 nM, 10 pM to about 1 nM, 10 pM to about 500 pM, 10 pM to about 100 pM, 10 pM to about 50 pM, 50 pM to about 900 μM, 50 pM to about 500 μM, 50 pM to about 100 μM, 50 pM to about 50 μM, 50 pM to about 10 μM, 50 pM to about 5 μM, 50 pM to about 1 μM, 50 pM to about 500 nM, 50 pM to about 100 nM, 50 pM to about 50 nM, 50 pM to about 10 nM, 50 pM to about 5 nM, 50 pM to about 1 nM, 50 pM to about 500 pM, 50 pM to about 100 pM, 100 pM to about 900 μM, 100 pM to about 500 μM, 100 pM to about 100 μM, 100 pM to about 50 μM, 100 pM to about 10 μM, 100 pM to about 5 μM, 100 pM to about 1 μM, 100 pM to about 500 nM, 100 pM to about 100 nM, 100 pM to about 50 nM, 100 pM to about 10 nM, 100 pM to about 5 nM, 100 pM to about 1 nM, 100 pM to about 500 pM, 500 pM to about 500 μM, 500 pM to about 100 μM, 500 pM to about 50 μM, 500 pM to about 10 μM, 500 pM to about 5 μM, 500 pM to about 1 μM, 500 pM to about 500 nM,K in the range of 500 pM to about 100 nM, 500 pM to about 50 nM, 500 pM to about 10 nM, 500 pM to about 5 nM, 500 pM to about 1 nM, 1 nM to about 500 μM, 1 nM to about 100 μM, 1 nM to about 50 μM, 1 nM to about 10 μM, 1 nM to about 5 μM, 1 nM to about 1 μM, 1 nM to about 500 nM, 1 nM to about 100 nM, 1 nM to about 50 nM, 1 nM to about 10 nM, 1 nM to about 5 nM, 5 nM to about 100 μM, 5 nM to about 50 μM, 5 nM to about 10 μM, 5 nM to about 5 μM, 5 nM to about 1 μM, 5 nM to about 500 nM, 5 nM to about 100 nM, 5 nM to about 50 nM, 5 nM to about 10 nM, 10 nM to about 10 μM, 10 nM to about 5 μM, 10 nM to about 1 μM, 10 nM to about 500 nM, 10 nM to about 100 nM, 10 nM to about 50 nM, 50 nM to about 10 μM, 50 nM to about 5 μM, 50 nM to about 1 μM, 50 nM to about 500 nM, 50 nM to about 100 nM, 100 nM to about 10 μM, 100 nM to about 5 μM, 100 nM to about 1 μM, 100 nM to about 500 nM, 500 nM to about 10 μM, 500 nM to about 5 μM, 500 nM to about 1 μM, 1 μM to about 10 μM, 1 μM to about 5 μM, or 5 μM to about 10 μM, D binds to human c-Kit at a value. In some embodiments, K D values are measured using standard binding assays, such as biolayer interferometry.

[0132] In some embodiments, the antigen-binding sites disclosed herein bind to cynomolgus c-Kit with an affinity (K D value) equivalent to that which binds to human c-Kit. In some embodiments, the affinity of the antigen-binding site for cynomolgus c-Kit is within 2-fold, 3-fold, 5-fold, or 10-fold of its affinity for human c-Kit as measured in the same assay (either the affinity for human c-Kit is higher or the affinity for cynomolgus c-Kit is higher).

[0133] In some embodiments, the antigen-binding sites disclosed herein bind to c-Kit expressed on the cell surface.

[0134] In some embodiments, the antigen-binding sites disclosed herein do not significantly bind to other c-Kit family members. In some embodiments, the antigen-binding sites disclosed herein do not significantly bind to PDGFRa, PDGFRb, CSF1R, or FLT3.

[0135] An antigen-binding site capable of binding to siglec-6 In another aspect, the present application provides an antigen-binding site capable of binding to siglec-6.

[0136] In some embodiments, an antigen-binding site capable of binding to siglec-6 and present as an antibody mimetic is provided. In some embodiments, a variant of the antigen-binding site capable of binding to siglec-6 and present as the antibody mimetic described herein is provided, and such an antigen-binding site is capable of binding to siglec-6 and has an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the antigen-binding site present as the antibody mimetic described herein.

[0137] In some embodiments, an antigen-binding site capable of binding to siglec-6 and present as an antibody (e.g., a bispecific antibody) is provided. In some embodiments, a variant of the antigen-binding site capable of binding to siglec-6 and present as the antibody described herein is provided, and such an antigen-binding site is capable of binding to siglec-6 and has an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence contained in the antigen-binding site present as the antibody described herein.

[0138] In some embodiments, the provided antigen-binding site that can bind to siglec-6 comprises a heavy-chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and / or a light-chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3.

[0139] In some embodiments, the provided antigen-binding site capable of binding to siglec-6 comprises a VH and a VL, where VH comprises CDR-H1, CDR-H2, and CDR-H3, and VL comprises CDR-L1, CDR-L2, and CDR-L3. Here, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are each independently selected from those of the VH or VL described in Table 9. The CDRs of VH and VL are determined by the Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each independently selected from those described in Table 9. In some embodiments, an antigen-binding site is provided that is a variant of the antigen-binding site capable of binding to siglec-6 described herein, such antigen-binding site having CDR sequences that differ by no more than two amino acid residues (e.g., two or one amino acid residue) from the CDR sequences described in Table 9. In some embodiments, an antigen-binding site is provided that is a variant of the antigen-binding site capable of binding to siglec-6 described herein, such antigen-binding site having a set of six CDRs that differ collectively by no more than two amino acid residues (e.g., two or one amino acid residue) from the set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 9. In some embodiments, an antigen-binding site is provided that is a variant of the antigen-binding site capable of binding to siglec-6 described herein, such antigen-binding site having a set of six CDRs that differ collectively by no more than two amino acid residues (e.g., two or one amino acid residue) from the set of six CDRs of an anti-siglec-6 having a set of VH and VL selected from Table 9.

[0140] In some embodiments, the provided antigen-binding site that can bind to siglec-6 comprises a VH sequence as described in Table 9 and a VL sequence as described in Table 9. In some embodiments, there is provided an antigen-binding site that is a variant of an antigen-binding site that can bind to siglec-6 as described herein, such as an anti-siglec-6 having a set of VH and VL selected from Table 9, such antigen-binding site having (1) a VH comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VH described in Table 9, and (2) a VL domain comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the VL described in Table 9.

[0141] In some embodiments, the provided antigen-binding site that can bind to siglec-6 comprises a heavy chain sequence as set forth in Table 9 and a light chain sequence as set forth in Table 9. In some embodiments, provided is an antigen-binding site that is a variant of an antigen-binding site that can bind to siglec-6 as described herein, such as an anti-siglec-6 having a set of heavy and light chains selected from Table 9, such antigen-binding site having (1) a heavy chain comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the heavy chain set forth in Table 9, and (2) a light chain comprising an amino acid sequence that is at least 85%, at least 87.5%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of the light chain set forth in Table 9.

[0142] In certain embodiments, the antigen-binding sites described in this application are derived from murine RND. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 2, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 10. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 2 and 10, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH includes CDR-H1, CDR-H2, and CDR-H3, which include the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively. In certain embodiments, the VL includes CDR-L1, CDR-L2, and CDR-L3, which include the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively. In certain embodiments, the antigen-binding site includes (a) a VH comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively.

[0143] In certain embodiments, the antigen-binding sites described in this application are derived from humanized RND-2. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 23, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 38. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 23 and 38, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, VH includes CDR-H1, CDR-H2, and CDR-H3, which include the amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively. In certain embodiments, VL includes CDR-L1, CDR-L2, and CDR-L3, which include the amino acid sequences of SEQ ID NOs: 32, 12, and 13, respectively. In certain embodiments, the antigen-binding site includes (a) a VH comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 32, 12, and 13, respectively.

[0144] In certain embodiments, the antigen-binding sites described in this application are derived from humanized RND-3. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 25, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 38. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 25 and 38, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, VH includes CDR-H1, CDR-H2, and CDR-H3, which include the amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively.

[0145] In certain embodiments, the antigen-binding sites described in this application are derived from humanized RND-4. For example, in certain embodiments, the antigen-binding sites described in this application include a VH having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 27, and a VL having an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 38. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 27 and 38, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH includes CDR-H1, CDR-H2, and CDR-H3, which include the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively. In certain embodiments, the VL includes CDR-L1, CDR-L2, and CDR-L3, which include the amino acid sequences of SEQ ID NOs: 32, 12, and 13, respectively. In certain embodiments, the antigen-binding site includes (a) a VH including CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively, and (b) a VL including CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 32, 12, and 13, respectively.

[0146] In certain embodiments, the antigen-binding sites described in this application are derived from humanized RND-5. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 29, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 38. In certain embodiments, the antigen-binding sites include CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 29 and 38, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0147] In certain embodiments, the antigen-binding sites described in this application are derived from JML-1. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 121, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 126. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 121 and 126, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 122, 123, and 124, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 127, 128, and 129, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3, comprising the amino acid sequences of SEQ ID NOs: 122, 123, and 124, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, comprising the amino acid sequences of SEQ ID NOs: 127, 128, and 129, respectively.

[0148] In certain embodiments, the antigen-binding sites described in this application are derived from mouse M2. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 132, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 136. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 132 and 136, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH includes CDR-H1, CDR-H2, and CDR-H3, which include the amino acid sequences of SEQ ID NOs: 133, 134, and 135, respectively. In certain embodiments, the VL includes CDR-L1, CDR-L2, and CDR-L3, which include the amino acid sequences of SEQ ID NOs: 137, 138, and 139, respectively. In certain embodiments, the antigen-binding site includes (a) a VH comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 133, 134, and 135, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 137, 138, and 139, respectively.

[0149] In certain embodiments, the antigen-binding sites described in this application are derived from mouse M7. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 140, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 144. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 140 and 144, respectively, determined by the Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH includes CDR-H1, CDR-H2, and CDR-H3, which include the amino acid sequences of SEQ ID NOs: 141, 142, and 143, respectively. In certain embodiments, the VL includes CDR-L1, CDR-L2, and CDR-L3, which include the amino acid sequences of SEQ ID NOs: 145, 146, and 147, respectively. In certain embodiments, the antigen-binding site includes (a) a VH comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 141, 142, and 143, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, which include the amino acid sequences of SEQ ID NOs: 145, 146, and 147, respectively.

[0150] In certain embodiments, the antigen-binding site described in this application is derived from mouse M11. For example, in certain embodiments, the antigen-binding site described in this application comprises a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 148, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 152. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 148 and 152, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 149, 150, and 151, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 153, 154, and 155, respectively. In certain embodiments, the antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3, comprising the amino acid sequences of SEQ ID NOs: 149, 150, and 151, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, comprising the amino acid sequences of SEQ ID NOs: 153, 154, and 155, respectively.

[0151] In certain embodiments, the antigen-binding sites described in this application are derived from humanized M11-1. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 159, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 164. In certain embodiments, the antigen-binding site includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 159 and 164, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, the VH includes CDR-H1, CDR-H2, and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 149, 160, and 151, respectively. In certain embodiments, the VL includes CDR-L1, CDR-L2, and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 165, 154, and 155, respectively. In certain embodiments, the antigen-binding site includes (a) a VH comprising CDR1, CDR2, and CDR3, comprising the amino acid sequences of SEQ ID NOs: 149, 160, and 151, respectively, and (b) a VL comprising CDR1, CDR2, and CDR3, comprising the amino acid sequences of SEQ ID NOs: 165, 154, and 155, respectively.

[0152] In certain embodiments, the antigen-binding site described in this application is derived from humanized M11-2. For example, in certain embodiments, the antigen-binding site described in this application comprises a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 162, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 167. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 162 and 167, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0153] In certain embodiments, the antigen-binding sites described in this application are derived from humanized M11-3. For example, in certain embodiments, the antigen-binding sites described in this application include a VH comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the amino acid sequence of SEQ ID NO: 162, and a VL comprising an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 169. In certain embodiments, the antigen-binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the VH and VL sequences of SEQ ID NOs: 162 and 169, respectively, determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0154] In certain embodiments, the antigen-binding sites disclosed herein have a K of 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 0.9 pM, 0.8 pM, 0.7 pM, 0.6 pM, 0.5 pM, 0.4 pM, 0.3 pM, 0.2 pM, 0.1 pM, 90 fM, 80 fM, 70 fM, 60 fM, 50 fM, 40 fM, 30 fM, 20 fM, 10 fM, 9 fM, 8 fM, 7 fM, 6 fM, 5 fM, 4 fM, 3 fM, 2 fM, or 1 fM or less. DBind to human siglec-6 with a value (affinity above those numerical values). In certain embodiments, the antigen-binding sites disclosed herein have a K of 10 nM or less D Bind to human siglec-6 with a value (affinity above that numerical value). In certain embodiments, the antigen-binding sites disclosed herein have a K of 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM, 2.0 nM, 2.1 nM, 2.2 nM, 2.3 nM, 2.4 nM, 2.5 nM, 2.6 nM, 2.7 nM, 2.8 nM, 2.9 nM, 3.0 nM, 3.1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, 4.0 nM, 4.1 nM, 4.2 nM, 4.3 nM, 4.4 nM, 4.5 nM, 4.6 nM, 4.7 nM, 4.8 nM, 4.9 nM, or 5.0 nM or less D Bind to human siglec-6 with a value (affinity above those numerical values). In certain embodiments, the antigen-binding sites of the present application have a K in the femtomolar to nanomolar range DIt binds to human siglec-6 with a value. In certain embodiments, the antigen-binding site of the present application is from about 1 fM to about 900 nM, 1 fM to about 500 nM, 1 fM to about 100 nM, 1 fM to about 50 nM, 1 fM to about 10 nM, 1 fM to about 5 nM, 1 fM to about 1 nM, 1 fM to about 500 pM, 1 fM to about 100 pM, 1 fM to about 50 pM, 1 fM to about 10 pM, 1 fM to about 5 pM, 1 fM to about 1 pM, 1 fM to about 500 fM, 1 fM to about 100 fM, 1 fM to about 50 fM, 1 fM to about 10 fM, 1 fM to about 5 fM, 5 fM to about 900 nM, 5 fM to about 500 nM, 5 fM to about 100 nM, 5 fM to about 50 nM, 5 fM to about 10 nM, 5 fM to about 5 fM, 5 fM to about 1 nM, 5 fM to about 500 pM, 5 fM to about 100 pM, 5 fM to about 50 pM, 5 fM to about 10 pM, 5 fM to about 5 pM, 5 fM to about 1 pM, 5 fM to about 500 fM, 5 fM to about 100 fM, 5 fM to about 50 fM, 5 fM to about 10 fM, 10 fM to about 900 nM, 10 fM to about 500 nM, 10 fM to about 100 nM, 10 fM to about 50 nM, 10 fM to about 10 nM, 10 fM to about 5 nM, 10 fM to about 1 nM, 10 fM to about 500 pM, 10 fM to about 100 pM, 10 fM to about 50 nM, 10 fM to about 10 pM, 10 fM to about 5 pM, 10 fM to about 1 pM, 10 fM to about 500 fM, 10 fM to about 100 fM, 10 fM to about 50 fM, 50 fM to about 900 nM, 50 fM to about 500 nM, 50 fM to about 100 nM, 50 fM to about 50 nM, 50 fM to about 10 nM, 50 fM to about 5 nM, 50 fM to about 1 nM, 50 fM to about 500 pM, 50 fM to about 100 pM, 50 fM to about 50 pM, 50 fM to about 10 pM, 50 fM to about 5 pM, 50 fM to about 1 pM, 50 fM to about 500 fM, 50 fM to about 100 fM, 100 fM to about 900 nM, 100 fM to about 500 nM, 100 fM to about 100 nM, 100 fM to about 50 nM, 100 fM to about 10 nM, 100 fM to about 5 nM, 100 fM to about 1 nM, 100 fM to about 500 pM, 100 fM to about 100 pM, 100 fM to about 50 pM, 100 fM to about 10 pM, 100 fM to about 5 pM, 100 fM to about 1 pM, 100 fM to about 500 fM, 500 fM to about 900 nM, 500 fM to about 500 nM, 500 fM to about 100 nM, 500 fM to about 50 nM, 500 fM to about 10 nM, 500 fM to about 5 nM, 500 fM to about 1 nM,K ranges from 500 fM to about 500 pM, 500 fM to about 100 pM, 500 fM to about 50 pM, 500 fM to about 10 pM, 500 fM to about 5 pM, 500 fM to about 1 pM, 1 pM to about 900 nM, 1 pM to about 500 nM, 1 pM to about 100 nM, 1 pM to about 50 nM, 1 pM to about 10 nM, 1 pM to about 5 nM, 1 pM to about 1 nM, 1 pM to about 500 pM, 1 pM to about 100 pM, 1 pM to about 50 pM, 1 pM to about 10 pM, 1 pM to about 5 pM, 5 pM to about 10 nM, 5 pM to about 5 nM, 5 pM to about 1 nM, 5 pM to about 500 pM, 5 pM to about 100 pM, 5 pM to about 50 pM, 5 pM to about 10 pM, 10 pM to about 10 nM, 10 pM to about 5 nM, 10 pM to about 1 nM, 10 pM to about 500 pM, 10 pM to about 100 pM, 10 pM to about 50 pM, 50 pM to about 10 nM, 50 pM to about 5 nM, 50 pM to about 1 nM, 50 pM to about 500 pM, 50 pM to about 100 pM, 100 pM to about 10 nM, 100 pM to about 5 nM, 100 pM to about 1 nM, 100 pM to about 500 pM, 500 pM to about 10 nM, 500 pM to about 5 nM, 500 pM to about 1 nM, 1 nM to about 10 nM, 1 nM to about 5 nM, or 5 nM to about 10 nM, D binds to human siglec-6 at a K D value. In some embodiments, the K

[0155] value is measured using a standard binding assay, such as biolayer interferometry. In some embodiments, the antigen-binding sites disclosed herein bind to cynomolgus siglec-6 with an affinity (K D value) equivalent to that which binds to human siglec-6. In some embodiments, the affinity of the antigen-binding site for cynomolgus siglec-6 is within 2-fold, 3-fold, 5-fold, or 10-fold of its affinity for human siglec-6, as measured in the same assay (either the affinity for human siglec-6 is stronger or the affinity for cynomolgus siglec-6 is stronger).

[0156] In some embodiments, the antigen-binding sites disclosed herein bind to siglec-6 expressed on the cell surface. In some embodiments, the antigen-binding sites disclosed herein bind to mast cells. In some embodiments, the antigen-binding sites disclosed herein bind to mast cells associated with other immune cells (e.g., T cells, dendritic cells, macrophages, neutrophils, or basophils).

[0157] In some embodiments, the antigen-binding sites disclosed herein do not significantly bind to other siglec-6 family members. In some embodiments, the antigen-binding sites disclosed herein do not significantly bind to siglec1-5, 7-12, 14, or 15.

[0158] In some embodiments, the antigen-binding sites disclosed herein do not induce massive cell degranulation.

[0159] Molecules comprising an antigen-binding site Similarly, provided herein are molecules comprising the disclosed antigen-binding sites. Such molecules may be, but are not limited to, antibodies or antigen-binding fragments thereof, antibody fragments, nanobodies, antibody mimetics, and the like.

[0160] In some embodiments, an antibody or antigen-binding fragment thereof comprising the disclosed antigen-binding site is provided. In some embodiments, the antibody or antigen-binding fragment thereof is multispecific, e.g., bispecific. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is non-fucosylated (defucosylated). In some embodiments, the antibody is up to about 90%, up to about 80%, up to about 70%, up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 25%, up to about 20%, up to about 15%, up to about 10%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1% fucosylated. In some embodiments, the antibody is defucosylated. In some embodiments, the antibody is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% defucosylated.

[0161] In some embodiments, the antibody or antigen-binding fragment thereof can bind to c-Kit. In some embodiments, the antibody or antigen-binding fragment thereof can bind to siglec-6. In some embodiments, the antibody or antigen-binding fragment thereof can bind to both c-Kit and siglec-6.

[0162] In some embodiments, provided are antibody fragments comprising the disclosed antigen-binding sites. For example, the fragments may be, but are not limited to, Fab, scFab (single-chain Fab), F(ab’)2, Fab’, single-chain Fv (scFv), Fv fragments, or diabodies (dimers of scFv). In some embodiments, the antibody fragments are multispecific, e.g., bispecific. In some embodiments, the antibody fragments can bind to c-Kit. In some embodiments, the antibody fragments can bind to siglec-6. In some embodiments, the antibody fragments can bind to both c-Kit and siglec-6.

[0163] In some embodiments, provided are antibody mimetics comprising the disclosed antigen-binding sites. For example, the fragments may be, but are not limited to, affibodies, affilins, affimers, affitins, alphabodies, anticalins, avimers, centyrins, DARPins, finomers, monobodies, or nanoclamps. In some embodiments, the antibody mimetics are multispecific, e.g., bispecific. In some embodiments, the antibody mimetics can bind to c-Kit. In some embodiments, the antibody mimetics can bind to siglec-6. In some embodiments, the antibody mimetics can bind to both c-Kit and siglec-6.

[0164] In some embodiments, provided are bispecific molecules comprising two antigen-binding sites disclosed herein.

[0165] In some embodiments, the bispecific molecules comprise an antigen-binding site that can bind to c-Kit as described herein. In some embodiments, the bispecific molecules comprise an antigen-binding site that can bind to siglec-6 as described herein. In some embodiments, the bispecific molecules comprise an antigen-binding site that can bind to c-Kit as described herein and an antigen-binding site that can bind to siglec-6 as described herein.

[0166] In some embodiments, a bispecific molecule is provided that comprises (i) an antigen-binding site capable of binding to siglec-6 as described herein, and (ii) an antigen-binding site capable of binding to c-Kit as described herein. In some embodiments, the bispecific molecule comprises a first antigen-binding site comprising a VH region and a VL region of an antigen-binding site capable of binding to siglec-6 as described herein, and a second antigen-binding site comprising a VH region and a VL region of an antigen-binding site capable of binding to c-Kit as described herein. In some embodiments, the first antigen-binding site (capable of binding to siglec-6) comprises a VH region and a VL region, which are, or are derived from, the VH / VL regions of murine RND, humanized RND, or JML-1, as described herein, and the second antigen-binding domain (capable of binding to c-Kit) comprises a VH region and a VL region, which are, or are derived from, the VH / VL regions of mutant SR1 or humanized SR1, as described herein.

[0167] In some embodiments, the bispecific molecule is capable of binding to cells that express siglec-6. In some embodiments, the bispecific molecule is capable of binding to cells that express c-Kit. In some embodiments, the bispecific molecule is capable of binding to mast cells. In some embodiments, the bispecific molecule binds to mast cells preferentially over other immune cells (e.g., T cells, dendritic cells, macrophages, neutrophils, or basophils). In some embodiments, the bispecific molecule has an internalization rate equal to or lower than that of a reference antigen-binding site capable of binding to siglec-6 (e.g., an antibody comprising VH and VL of SEQ ID NOs: 2 and 10, respectively). In some embodiments, the bispecific molecule has a toxicity equal to or lower than that of a reference antigen-binding site capable of binding to c-Kit (e.g., an antibody comprising VH and VL of SEQ ID NOs: 40 and 45, respectively).

[0168] Various forms and uses of bispecific molecules are known in the art. The bispecific molecules according to the present invention are not limited to any particular bispecific form or method of its production.

[0169] In some embodiments, the bispecific molecule can be, but is not limited to, a bispecific antibody or a fragment or derivative thereof comprising: (i) a single antibody having two arms each comprising a different antigen-binding site, (ii) a bispecific scFv (e.g., via two scFvs linked by a peptide linker), (iii) a dual variable domain antibody (DVD-Ig) in which each light and heavy chain comprises two variable domains in series via a short peptide bond, (iv) a bispecific (Fab’)2 fragment, (v) a diabody, (vi) a tandem diabody (TandAb) which is a fusion of two diabodies, and (vii) a trivalent bispecific binding protein “dock and lock (DNL)-Fab3” comprising two identical Fab fragments linked to different Fab fragments, (viii) an IgG-like molecule having an Fc engineered to dimerize heterologously, (ix) a recombinant IgG-like bispecific targeting molecule in which both sides of the molecule each comprise at least two Fab fragments or portions of Fab fragments of different antibodies, (x) an IgG fusion molecule in which a full-length IgG antibody is fused to an additional Fab fragment or portion of a Fab fragment, (xi) an Fc fusion molecule, (v) a Fab fusion molecule, and (vi) an scFv, diabody, or heavy chain antibody (e.g., a domain antibody, nanobody) in which different scFvs, diabodies, or heavy chain antibodies are fused to each other or to a carrier molecule which is fused to another protein or a heavy chain constant domain, Fc region or portion thereof.

[0170] Examples of IgG-like molecules with engineered Fc include, but are not limited to, triomab, knobs-into-holes (kih) molecules (e.g., kih IgG with a common light chain), CrossMAb, orthoFab IgG molecules, electrostatically adapted molecules, LUZ-Y molecules, DIG-bodies and PIG-bodies molecules, strand-exchange engineered domain body (SEEDbody) molecules, bionics molecules, FcΔAdp molecules, bispecific IgG1 and IgG2 molecules, Azymetric scaffold molecules, and DuoBody molecules. Examples of recombinant IgG-like bispecific molecules include, but are not limited to, bispecific (DT)-Ig molecules, one-to-one antibodies, mAb 2 , and Zybody. Examples of IgG fusion molecules include, but are not limited to, DVD-Ig molecules and IgG-scFv. Examples of Fc fusion molecules include, but are not limited to, scFv / Fc fusions, SCORPION molecules, and Fc-DART molecules. Examples of Fab fusion bispecific antibodies include, but are not limited to, F(ab)2 molecules, DNL molecules, and Fab-Fv molecules. Examples of scFv-based and diabody-based antibodies and domain antibodies include, but are not limited to, bispecific T cell engager (BiTE) molecules, tandem diabody molecules (TandAb), dual affinity retargeting technology (DART) molecules, single-chain diabody molecules, TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scfv fusions, COMBODY molecules, bispecific nanobodies, and bispecific heavy chain-only domain antibodies.

[0171] In certain embodiments, the disclosed bispecific molecules can be fusion proteins that include one or more antibody mimetics. In certain embodiments, the disclosed bispecific molecules can be fusion proteins that include one or more antibodies or antigen-binding fragments thereof. In some embodiments, the disclosed bispecific molecules can be fusion proteins that include one or more antibody mimetics and one or more antibodies or antigen-binding fragments thereof.

[0172] In some embodiments, the molecule comprising the disclosed antigen-binding site further comprises a constant region of an antibody or a fragment or variant thereof. In some embodiments, the constant region of the antibody may be, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, and in particular, for example, selected from the heavy chain constant regions of (for example, human) IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the constant region of the antibody or a fragment or variant thereof has an amino acid sequence that is at least 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, or IgE, preferably, for example, the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the constant region of the antibody may be a light chain constant region selected from, for example, (for example, human) kappa or lambda light chain constant regions. The constant region can be altered, for example, mutated, to modify the properties of the antibody (for example, to enhance or reduce one or more of Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, and / or complement function). In one embodiment, the antibody has effector function and / or can fix complement. In other embodiments, the antibody does not recruit effector cells and does not fix complement. In another embodiment, the antibody has a reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other variant that does not support binding to Fc receptors, for example, it has a mutagenized or deleted Fc receptor binding region.

[0173] In some embodiments, the constant region comprises one or more mutations that reduce effector function. For example, in certain embodiments, the constant region comprises a heavy chain constant region of IgG1 that includes one or more of the following mutations, numbered according to the EU index: (a) L234A and / or L235A, (b) A327G, A330S, and / or P331S, (c) E233P, L234V, L235A, and / or G236del, (d) E233P, L234V, and / or L235A, (e) E233P, L234V, L235A, G236del, A327G, A330S, and / or P331S, (f) E233P, L234V, L235A, A327G, A330S, and / or P331S, (g) N297A, (h) N297G, (i) N297Q, (j) L242C, N297C, and / or K334C, (k) A287C, N297G, and / or L306C, (l) R292C, N297G, and / or V302C, (m) N297G, V323C, and / or I332C, (n) V259C, N297G, and / or L306C, (o) L234F, L235Q, K322Q, M252Y, S254T, and / or T256E, (p) L234A, L235A, and / or P329G, or (q) L234A, L235Q, and K322Q. In certain embodiments, the constant region comprises a heavy chain constant region of IgG2 that includes one or more of the following mutations, numbered according to the EU index: (a) A330S and / or P331S, (b) V234A, G237A, P238S, H268A, V309L, A330S, and / or P331S, or (c) V234A, G237A, H268Q, V309L, A330S, P331S, C232S, C233S, S267E, L328F, M252Y, S254T, and / or T256E. In certain embodiments, the constant region comprises a heavy chain constant region of IgG4 that includes one or more of the following mutations, numbered according to the EU index: (a) E233P, F234V, L235A, and / or G236del, (b) E233P, F234V, and / or L235A, (c) S228P and / or L235E, or (d) S228P and / or L235A.

[0174] In some embodiments, the constant region comprises one or more mutations that enhance effector function. For example, in certain embodiments, the constant region comprises a heavy chain constant region of IgG1 that includes one or more of the following mutations, numbered according to the EU index: (a) F243L, R292P, Y300L, V305I, and / or P396L, (b) S239D and / or I332E, (c) S239D, I332E, and / or A330L, (d) S298A, E333A, and / or K334A, (e) G236A, S239D, and / or I332E, (f) K326W and / or E333S, (g) S267E, H268F, and / or S324T, or (h) E345R, E430G, and / or S440Y.

[0175] In some embodiments, the disclosed antigen-binding site is linked to the constant region of the antibody or a fragment or variant thereof. In certain embodiments, the antigen-binding site is linked to an IgG constant region that includes a hinge, CH2, and CH3 domains, with or without a CH1 domain.

[0176] In some embodiments, the molecule comprising the disclosed antigen-binding site further comprises a non-fucosylated Fc region. In some embodiments, the Fc region is the Fc region of non-fucosylated IgG1.

[0177] Amino acid sequence modification Amino acid sequence modifications of the antigen-binding sites and molecules containing the antigen-binding sites (e.g., antibodies or antigen-binding fragments thereof) disclosed herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of an antibody or antigen-binding fragment. Amino acid sequence variants may be prepared, for example, by introducing appropriate nucleotide changes into the nucleic acid sequence encoding the antigen-binding site or the molecule containing the antigen-binding site, or by peptide synthesis. Such modifications can include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made, provided that the antigen-binding site or the molecule containing the antigen-binding site retains the desired properties and / or functions. In some embodiments, amino acid changes are introduced to alter post-translational processing, for example, to change the number or location of glycosylation sites.

[0178] A particularly useful method for identifying certain residues or regions that are preferred sites for mutagenesis is called "alanine scanning mutagenesis." In this method, residues or groups of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction between the amino acid and the antigen. The amino acid positions that demonstrate functional sensitivity to the substitution are then further defined by introducing additional or other variants at or instead of the substitution site. Thus, while the sites for introducing amino acid sequence variations are predetermined, the nature of the variations themselves need not be predetermined. For example, ala scanning or random mutagenesis may be performed at the target codon or region to analyze the performance of mutations at a given site, and the expressed variants may be screened for the desired activity.

[0179] Examples of amino acid sequence insertions include, but are not limited to, amino-terminal and / or carboxyl-terminal fusions encompassing a length range of polypeptides from one residue to over 100 residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include, but are not limited to, an N-terminal methionyl residue.

[0180] In some embodiments, the antigen-binding site or a molecule comprising the antigen-binding site is fused at one terminus to another polypeptide, e.g., a cytotoxic polypeptide, an enzyme, or a polypeptide that increases the serum half-life of an antibody or antigen-binding fragment.

[0181] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue of the amino acid sequence of the molecule replaced with a different residue. The sites of greatest interest with respect to substitution mutagenesis are typically the hypervariable regions, although framework region alterations are also contemplated. Examples of conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". More substantial changes may be introduced under the heading "Exemplary Substitutions" in Table 1 or as further described below with respect to amino acid classes, and the resulting antibodies or antigen-binding fragments may be screened.

[0182] [Table 1]

[0183] Substantial modification of the biological properties of the antibody can be achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the substituted region, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are typically classified according to general side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile, (2) Neutral hydrophilic: Cys, Ser, Thr, (3) Acidic: Asp, Glu, (4) Basic: Asn, Gln, His, Lys, Arg, (5) Residues affecting chain orientation: Gly, Pro, and (6) Aromatic: Trp, Tyr, Phe.

[0184] Non-conservative substitutions can involve the exchange of a member of one of these classes for another.

[0185] In addition, or alternatively, cysteine residues that are not involved in maintaining the appropriate conformation of the antibody or antigen-binding fragment are generally substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can be added to the antibody to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

[0186] In some embodiments, the substituted variant contains substitutions within one or more hypervariable region residues of the parent antibody (e.g., a human antibody). Generally, the resulting variants having improved biological properties compared to the parent antibody from which they are generated are selected for further development.

[0187] Methods for generating such substituted variants involve affinity maturation using phage display. In an example of such a method, several hypervariable region sites (e.g., 6 - 7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner, for example, from filamentous phage particles, as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity).

[0188] To identify candidate hypervariable region sites for modification, an alanine scanning mutagenesis method may be performed to identify hypervariable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody or antigen-binding fragment and the antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, a panel of variants is subjected to screening, and antibodies having excellent properties in one or more relevant assays can be selected for further development.

[0189] In some embodiments, the original glycosylation pattern of the parental antibody is altered. Such alterations can include the deletion of one or more sugar moieties found in the antibody and / or the addition of one or more glycosylation sites not present in the antibody.

[0190] Antibody glycosylation is typically either N-linked or O-linked. N-linked refers to the attachment of a sugar moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of a sugar moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences within a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0191] The addition of a glycosylation site to an antibody can be achieved by altering the amino acid sequence of the antibody or antigen-binding fragment to include one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be done by the addition or substitution of one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0192] Nucleic acid molecules encoding amino acid sequence variants of antibodies or antigen-binding fragments can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of pre-prepared variants or non-variant versions of the antibody or its antigen-binding fragment.

[0193] In some embodiments, modifications that increase serum half-life are used. For example, salvage receptor binding epitopes can be incorporated into molecules comprising the disclosed antigen-binding site, as described, for example, in U.S. Patent No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that contributes to an increase in the in vivo serum half-life of the IgG molecule.

[0194] Methods for making antigen-binding sites and molecules comprising antigen-binding sites The proteins described herein can be made using recombinant DNA techniques well known to those of skill in the art.

[0195] For example, one or more nucleic acid sequences encoding a protein comprising the disclosed antigen-binding site can be cloned into one or more expression vectors. The expression vectors can be stably transfected into a host cell capable of expressing the gene. After transfection, a single clone can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix. The clone can be cultured under conditions suitable for scale-up in a bioreactor and maintain the expression of the protein comprising the antigen-binding site disclosed herein. The protein can be isolated and purified using methods known in the art, such as centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography.

[0196] Accordingly, provided herein are isolated nucleic acids encoding an antigen-binding site and a molecule (e.g., a protein) comprising the antigen-binding site, vectors and host cells comprising the nucleic acid, and recombinant techniques for production.

[0197] In certain embodiments, provided are one or more isolated nucleic acids comprising a sequence encoding the variable region of the immunoglobulin heavy chain and / or the immunoglobulin light chain of any antibody disclosed herein, and one or more expression vectors expressing the variable region of the immunoglobulin heavy chain and / or the immunoglobulin light chain of any antibody disclosed herein. Similarly, provided are host cells comprising one or more of the foregoing expression vectors and / or isolated nucleic acids.

[0198] In some embodiments, the antigen-binding sites and molecules (e.g., proteins) comprising the antigen-binding sites of the present disclosure may be fused to another agent, such as another therapeutic agent. Construction of fusion proteins is within the scope of those skilled in the art.

[0199] Monoclonal antibody Monoclonal antibodies can be produced initially using the hybridoma method or by recombinant DNA methods (U.S. Patent No. 4,816,567).

[0200] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized to elicit lymphocytes that produce or can produce antibodies that specifically bind to the protein used for immunization. Alternatively, or in addition, lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusogen such as polyethylene glycol to form hybridoma cells.

[0201] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium. The medium preferably contains one or more substances that inhibit the growth or survival of unfused parental myeloma cells (also referred to as fusion partners). For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances prevent the growth of HGPRT-deficient cells.

[0202] Examples of suitable fusion partners include, but are not limited to, myeloma cells that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a selective medium that selects against unfused parental cells. Examples of suitable myeloma cell lines include, but are not limited to, mouse myeloma lines such as the mouse myeloma lines derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 and derivatives such as X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md., USA. Human myelomas and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

[0203] According to the hybridoma method, the culture medium in which hybridoma cells grow is assayed for the production of monoclonal antibodies directed against an antigen. For example, the binding specificity of a monoclonal antibody produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

[0204] The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis.

[0205] Once hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned, for example, by limiting dilution procedures, and grown by standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites in an animal, for example, by i.p. injecting the cells into a mouse.

[0206] Monoclonal antibodies secreted by subclones are preferably separated from the culture medium, ascites fluid, or serum by conventional antibody purification techniques such as affinity chromatography (e.g., using protein A or protein G-Sepharose®), ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0207] DNA encoding a monoclonal antibody can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of a mouse antibody). Hybridoma cells can function as a suitable source of such DNA. Once isolated, the DNA may be placed within an expression vector, which is then transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce the antibody protein, to obtain a synthetic monoclonal antibody within the recombinant host cell.

[0208] In certain embodiments, monoclonal antibodies or antibody fragments are isolated from an antibody phage library. High-affinity (nM range) human antibodies can be produced, for example, by chain shuffling. Combinatorial infection and in vivo recombination can provide strategies for constructing very large phage libraries. These techniques are viable alternatives to the conventional monoclonal antibody hybridoma technology for isolating monoclonal antibodies.

[0209] DNA encoding an antibody can be modified, for example, by replacing the human heavy and light chain constant domain (CH and CL) sequences with homologous mouse sequences (e.g., U.S. Patent No. 4,816,567), or by fusing the immunoglobulin encoding sequences with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide), to produce a chimeric antibody polypeptide or a fusion antibody polypeptide.

[0210] Human Antibodies and Phage Display Methods Human antibodies can be produced by methods known in the art, including the methods described herein. For example, it is possible to produce transgenic animals (e.g., mice) that, upon immunization, can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain joining (JH) gene in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transplantation of a human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO 97 / 17852.

[0211] Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from a repertoire of immunoglobulin variable (V) domain genes from non-immunized donors. According to this technique, antibody V domain genes are cloned in-frame into the major or minor coat protein genes of filamentous bacteriophages such as M13 or fd and displayed as functional antibody fragments on the surface of phage particles. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of the genes encoding the antibodies that exhibit those properties. Thus, phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats. Some sources of V gene segments can be used for phage display from random combinatorial libraries of V genes, such as libraries derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed, and antibodies against a wide variety of antigens (including autoantigens) can be essentially isolated according to the following methods described in the art. See, e.g., U.S. Pat. Nos. 5,565,332 and 5,573,905.

[0212] In addition, human antibodies can be generated by in vitro activated B cells (see, for example, U.S. Patent Nos. 5,567,610 and 5,229,275).

[0213] Competitive assays for determining whether an antibody binds to the same epitope as the disclosed antibody or competes with the disclosed antibody for binding are known in the art. Exemplary competitive assays include immunoassays (e.g., ELISA assay, RIA assay), surface plasmon resonance (e.g., BIAcore analysis), biolayer interferometry, and flow cytometry.

[0214] Typically, a competitive assay involves the use of an antigen bound to a solid phase surface or expressed on the cell surface, a test antibody, and a reference antibody. The reference antibody is labeled and the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of labeled reference antibody binding to the solid phase surface or cell in the presence of the test antibody. Usually, the test antibody is present in excess (e.g., 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold). Antibodies identified by a competitive assay (e.g., competing antibodies) include antibodies that bind to the same epitope or a similar (e.g., overlapping) epitope as the reference antibody and antibodies that bind to an adjacent epitope that is sufficiently close to the epitope bound by the reference antibody such that steric hindrance occurs.

[0215] If the competitive assay can be performed in both directions, it can be ensured that the presence of the label does not interfere with or inhibit binding. For example, in a first direction, the reference antibody is labeled and the test antibody is unlabeled, and in a second direction, the test antibody is labeled and the reference antibody is unlabeled.

[0216] A test antibody competes with a reference antibody for specific binding to an antigen if an excess (e.g., 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold) of one antibody inhibits the binding of the other antibody by, for example, at least 50%, 75%, 90%, 95%, or 99% as measured in a competitive binding assay.

[0217] Two antibodies can be determined to bind to the same epitope if essentially all amino acid mutations of the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies can be determined to bind to overlapping epitopes if only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0218] The antibodies disclosed herein may be further optimized (e.g., affinity matured) to improve biochemical properties including affinity and / or specificity, to improve biophysical properties including aggregation, stability, precipitation and / or non-specific interactions, and / or to reduce immunogenicity. Affinity maturation procedures are within the skill of the art. For example, diversity can be introduced into immunoglobulin heavy and / or immunoglobulin light chains by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and / or site-directed mutagenesis.

[0219] In certain embodiments, the isolated human antibody comprises one or more somatic mutations. In these cases, the antibody can be optimized by modifying the antibody to a human germline sequence (e.g., by a process called germlining).

[0220] Generally, an optimized antibody has at least the same or substantially the same affinity for its antigen as the non-optimized (or parent) antibody from which the antibody is derived. For example, in certain embodiments, the optimized antibody has a higher affinity for the antigen compared to the parent antibody.

[0221] Pharmaceutical composition In certain embodiments, the provided molecule comprising the disclosed antigen-binding site is incorporated into a pharmaceutical composition suitable for administration to a subject, together with one or more pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carrier" refers to any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof.

[0222] In some embodiments, the pharmaceutical composition comprises one or more isotonic agents or stabilizers. Non-limiting examples of such isotonic agents or stabilizers include saccharides (e.g., sucrose), polyalcohols (e.g., mannitol or sorbitol), and sodium chloride.

[0223] In some embodiments, the pharmaceutical composition comprises one or more fillers and / or cryoprotectants (e.g., mannitol or glycine), buffers (e.g., phosphate, acetate, or histidine buffers), surfactants (e.g., polysorbate), antioxidants (e.g., methionine), and / or metal ions or chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0224] In some embodiments, the pharmaceutical composition comprises one or more adjunct substances such as wetting or emulsifying agents, preservatives (e.g., benzyl alcohol), or buffers, which can enhance the shelf life and / or effectiveness of the immunoconjugates disclosed herein.

[0225] The pharmaceutical composition can be provided in any of a variety of forms. These include, for example, liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories, among others, including liquid, semi-solid, and solid dosage forms. The suitability of a particular form can depend on the intended mode of administration and therapeutic use.

[0226] In some embodiments, the pharmaceutical composition is in the form of an injection solution or an infusion solution.

[0227] The pharmaceutical composition is typically sterile and stable under the conditions of manufacture, transport, and storage. The pharmaceutical composition can be formulated, for example, as a solution, microemulsion, dispersion, liposome, or other ordered structure. In some embodiments, the pharmaceutical composition is formulated as a structure particularly suitable for high drug concentrations. For example, a sterile injection solution can be prepared by incorporating a therapeutic agent (e.g., an immunoconjugate) in a desired amount in a suitable solvent by one or a combination of the components listed herein, followed optionally by sterilization (e.g., filtration sterilization). Generally, a dispersion can be prepared by incorporating an immunoconjugate in a sterile vehicle containing a basic dispersion medium and other components, such as additional components mentioned herein. In the case of sterile powders for the preparation of sterile injection solutions, examples of preparation methods include vacuum drying and lyophilization to obtain a powder of the immunoconjugate and any additional desired components, for example, from its pre-sterilized filtered solution.

[0228] The appropriate fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a specific particle size (e.g., in the case of a dispersion), and / or by the use of a surfactant. Sustained absorption of the injection composition can be brought about, for example, by including an agent in the composition that delays absorption (e.g., monostearate and gelatin).

[0229] Treatment method This application provides a method for treating a disease or disorder using a molecule (e.g., a protein such as an antibody or an antigen-binding fragment thereof) or a pharmaceutical composition described herein. The treatment methods disclosed herein generally include administering a therapeutically effective amount of the disclosed molecule comprising an antigen-binding site or a pharmaceutical composition thereof to a subject in need thereof (e.g., a human subject).

[0230] In some embodiments, the disease or disorder is a mast cell-related disease or disorder. In some embodiments, the mast cell-related disease or disorder is associated with undesirable mast cell activity, such as excessive proliferation of mast cells. Non-limiting examples of mast cell-related diseases or disorders include upper airway diseases such as allergic rhinitis and sinusitis, foreign body aspiration, glottic stenosis, tracheal stenosis, laryngotracheomalacia, vascular ring, chronic obstructive pulmonary disease (COPD), and congestive heart failure, eosinophilic bronchitis, polychondritis, sarcoidosis, papillomatosis, arthritis (e.g., rheumatoid arthritis), Wegener's granulomatosis, primary cirrhosis, allergy reactive to oral immunotherapy, secondary sclerosing cholangitis due to mast cell cholangiopathy, itching, pruritus or itching associated with chronic kidney disease due to or associated with primary biliary cirrhosis, bronchiolitis obliterans syndrome after lung transplantation or hematopoietic stem cell transplantation, inflammatory bowel syndrome, functional diseases, bronchopulmonary dysplasia in infants, cerebral ischemia, seizures, pneumonia due to influenza, sepsis, scleroderma, cluster headache, cardiac fibrosis, primary sclerosing cholangitis (PSC), Morquio disease, aspirin-intolerant asthma, vulvodynia, chronic pelvic pain syndrome (prostatitis), and cancer (e.g., lung cancer).

[0231] In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the disease or disorder is an allergy. In some embodiments, the disease or disorder is an inflammation. In some embodiments, the disease or disorder is an itching. In some embodiments, the disease or disorder is a cancer.

[0232] The therapeutically effective amount can be administered via a single dose or multiple doses (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 doses). When administered via multiple doses, any of a variety of suitable treatment regimens can be used, including administration at regular intervals (e.g., once a day, once every 3 days, once every 4 days, once every 5 days, 3 times a week, 2 times a week, once a week, once every 2 weeks, once every 3 weeks, etc.).

[0233] Effective dosage regimens in a treatment method (e.g., the amount of each therapeutic agent, the relative timing of treatment, etc.) may depend on the severity of the disease or symptom, as well as the weight and general condition of the subject. For example, the therapeutically effective amount of a specific composition containing a therapeutic agent applied to a mammal (e.g., a human) can be determined by those skilled in the art considering the age, weight, and individual differences in symptoms of the mammal. The therapeutically effective amount and / or the optimal amount may also be determined empirically by those skilled in the art.

[0234] A molecule containing an antigen-binding site or its pharmaceutical composition may be administered by any of various suitable routes including, but not limited to, parenteral (e.g., intravenous or subcutaneous) or enteral routes.

[0235] List of exemplary embodiments The present invention is further illustrated by the following non-limiting exemplary embodiments. Embodiment 1. An antigen-binding site capable of binding to FCER1A, FCER1B, and / or FCER1G. Embodiment 2. The antigen-binding site according to Embodiment 1, capable of binding to FCER1A. Embodiment 3. The antigen-binding site according to Embodiment 1, capable of binding to FCER1B. Embodiment 4. The antigen-binding site according to Embodiment 1, capable of binding to FCER1G. Embodiment 5. An antigen-binding site capable of binding to c-Kit. Embodiment 6. An antigen-binding site capable of binding to siglec-6. Embodiment 7. An antigen-binding site capable of binding to siglec-8. Embodiment 8. The antigen-binding site according to any one of Embodiments 1 to 7, present as a single-chain variable fragment (scFv). The antigen-binding site according to any one of Embodiments 1 to 7, which exists as an antibody mimetic (for example, an affibody, an affilin, an affimer, an aftin, an alphabody, an anticalin, an avimer, a centyrin, a DARPin, a finomer, a monobody, or a nanoclamp). Embodiment 10. (a) Complementary determining region 1 (CDR1), CDR2, and / or CDR3 of the heavy chain (HC) shown in any one of SEQ ID NOs: 39, 49, 51, 54, 56, or 58, and / or (b) CDR1, CDR2, and / or CDR3 of the light chain (LC) shown in any one of SEQ ID NOs: 44, 50, 86, or 88 The antigen-binding site according to any one of Embodiments 5 and 8 to 9, which contains the above. Embodiment 11. (a) Complementary determining region: CDR-H1 containing the amino acid sequence: SYNMH (SEQ ID NO: 41), CDR-H2 containing the amino acid sequence: VIYSGNGDTSYNQKFKG (SEQ ID NO: 42) or IYSGNGDTSYNQKFKGK (SEQ ID NO: 33), and CDR-H3 containing the amino acid sequence: ERDTRFGN (SEQ ID NO: 43) The heavy chain variable domain (VH) containing the above, and / or (b) Complementary determining region: CDR-L1 containing the amino acid sequence: RASESVDIYGNSFMH (SEQ ID NO: 46), CDR-L2 containing the amino acid sequence: LASNLES (SEQ ID NO: 47), and CDR-L3 containing the amino acid sequence: QQNNEDPYT (SEQ ID NO: 48) The light chain variable domain (VL) containing the above The antigen-binding site according to any one of Embodiments 5 and 8 to 10, which contains the above. Embodiment 12. (a) A VH containing an amino acid sequence having at least 85% identity to the VH of the HC shown in SEQ ID NO: 39, and / or A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown by SEQ ID NO: 44, or (b) A VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown by SEQ ID NO: 49, and / or A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown by SEQ ID NO: 50, or (c) A VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown by any one of SEQ ID NOs: 51, 54, 56, and 58, and / or A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown by SEQ ID NO: 86 or 88, The antigen-binding site according to any one of Embodiments 5 and 8 to 11, comprising Embodiment 13. (a) An HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC shown by SEQ ID NO: 39 and having at least 85% amino acid sequence identity to SEQ ID NO: 39, and / or An LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC shown by SEQ ID NO: 44 and having at least 85% amino acid sequence identity to SEQ ID NO: 44, or (b) An HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC shown by SEQ ID NO: 49 and having at least 85% amino acid sequence identity to SEQ ID NO: 49, and / or An LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC shown by SEQ ID NO: 50 and having at least 85% amino acid sequence identity to SEQ ID NO: 50, or (c) An HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC shown by any one of SEQ ID NOs: 51, 54, 56, and 58 and having at least 85% amino acid sequence identity thereto, and / or An LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC shown by SEQ ID NO: 86 or 88 and having at least 85% amino acid sequence identity thereto, The antigen-binding site according to any one of Embodiments 5 and 8 to 12, comprising Embodiment 14. The antigen-binding site according to Embodiment 12 or 13, wherein the sequence identity is at least 90%, 95%, 96%, 97%, 98%, or 99%. Embodiment 15. (a) VH of HC shown in SEQ ID NO: 39 and / or VL of LC shown in SEQ ID NO: 44, or (b) VH of HC shown in SEQ ID NO: 49 and / or VL of LC shown in SEQ ID NO: 50, or (c) VH of HC shown in any one of SEQ ID NOs: 51, 54, 56, and 58 and / or VL of LC shown in SEQ ID NO: 86 or 88 The antigen-binding site according to any one of Embodiments 5 and 8 to 14, comprising Embodiment 16. (a) CDR1, CDR2, and / or CDR3 of HC shown in any one of SEQ ID NOs: 1, 17, 19, 22, 24, 26, or 28, and / or (b) CDR1, CDR2, and / or CDR3 of LC shown in any one of SEQ ID NOs: 9, 18, 30, 35, or 37, the antigen-binding site according to any one of Embodiments 6 and 8 to 9. Embodiment 17. (a) Complementary determining region: CDR-H1 comprising the amino acid sequence: SYWMH (SEQ ID NO: 3), CDR-L2 comprising the amino acid sequence: EIYPTNGGTTYNEKFKR (SEQ ID NO: 4), and CDR-H3 comprising the amino acid sequence: EDFYAMDY (SEQ ID NO: 5) VH comprising, and / or (b) Complementary determining region: CDR-L1 comprising the amino acid sequence: KSSQSLLDSDGKTCLN (SEQ ID NO: 11), CDR-L2 comprising the amino acid sequence: LVSKLDS (SEQ ID NO: 12), and CDR-L3 comprising the amino acid sequence: WQGTHFPYT (SEQ ID NO: 13) VL comprising An antigen-binding site according to any one of Embodiments 6, 8 to 9, and 16, comprising Embodiment 18. (a) Complementary determining region: A CDR-H1 comprising the amino acid sequence: GYAFTSYWMH (SEQ ID NO: 6), A CDR-H2 comprising the amino acid sequence: EIYPTNGGTT (SEQ ID NO: 7), and A CDR-H3 comprising the amino acid sequence: EDFYAMDY (SEQ ID NO: 8) comprising VH, and / or (b) Complementary determining region: A CDR-L1 comprising the amino acid sequence: KSSQSLLDSDGKTCLN (SEQ ID NO: 14), A CDR-L2 comprising the amino acid sequence: LVSKLDS (SEQ ID NO: 15), and A CDR-L3 comprising the amino acid sequence: WQGTHFPYT (SEQ ID NO: 16) comprising VL An antigen-binding site according to any one of Embodiments 6, 8 to 9, and 16, comprising Embodiment 19. (a) A VH comprising an amino acid sequence having at least 85% identity to the VH of HC shown in SEQ ID NO: 1, and / or A VL comprising an amino acid sequence having at least 85% identity to the VL of LC shown in SEQ ID NO: 9, or (b) A VH comprising an amino acid sequence having at least 85% identity to the VH of HC shown in SEQ ID NO: 17, and / or A VL comprising an amino acid sequence having at least 85% identity to the VL of LC shown in SEQ ID NO: 18, or (c) A VH comprising an amino acid sequence having at least 85% identity to the VH of HC shown in any one of SEQ ID NOs: 19, 22, 24, 26, and 28, and / or A VL comprising an amino acid sequence having at least 85% identity to the VL of LC shown in any one of SEQ ID NOs: 30, 35, and 37 The antigen-binding site according to any one of Embodiments 6, 8 to 9, and 16 to 18, which comprises Embodiment 20. (a) An HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC shown in SEQ ID NO: 1 and having at least 85% amino acid sequence identity to SEQ ID NO: 1, and / or An LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC shown in SEQ ID NO: 9 and having at least 85% amino acid sequence identity to SEQ ID NO: 9, or (b) An HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC shown in SEQ ID NO: 17 and having at least 85% amino acid sequence identity to SEQ ID NO: 17, and / or An LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC shown in SEQ ID NO: 18 and having at least 85% amino acid sequence identity to SEQ ID NO: 18, or (c) An HC comprising CDR-H1, CDR-H2, and CDR-H3 of the HC shown in any one of SEQ ID NOs: 19, 22, 24, 26, and 28 and having at least 85% amino acid sequence identity thereto, and / or An LC comprising CDR-L1, CDR-L2, and CDR-L3 of the LC shown in any one of SEQ ID NOs: 30, 35, and 37 and having at least 85% amino acid sequence identity thereto, The antigen-binding site according to any one of Embodiments 6, 8 to 9, and 16 to 19, which comprises Embodiment 21. The antigen-binding site according to Embodiment 19 or 20, wherein the sequence identity is at least 90%, 95%, 96%, 97%, 98%, or 99%. Embodiment 22. (a) VH of the HC shown in SEQ ID NO: 1 and / or VL of the LC shown in SEQ ID NO: 9, or (b) VH of the HC shown in SEQ ID NO: 17 and / or VL of the LC shown in SEQ ID NO: 18, or (c) The VH of HC shown in any one of SEQ ID NOs: 19, 22, 24, 26, and 28, and / or the VL of LC shown in any one of SEQ ID NOs: 30, 35, and 37 The antigen-binding site according to any one of Embodiments 6, 8-9, and 16-21, comprising . Embodiment 23. (a) CDR1, CDR2, and / or CDR3 of VH shown in SEQ ID NO: 121, and / or (b) CDR1, CDR2, and / or CDR3 of LC shown in SEQ ID NO: 126 The antigen-binding site according to any one of Embodiments 6-9, comprising . Embodiment 24. (a) Complementary determining region: CDR-H1 comprising the amino acid sequence: DYGMH (SEQ ID NO: 122), CDR-H2 comprising the amino acid sequence: GISWNSGSIGYADSVKG (SEQ ID NO: 123), and CDR-H3 comprising the amino acid sequence: GGQTIDI (SEQ ID NO: 124) The VH comprising , and / or (b) Complementary determining region: CDR-L1 comprising the amino acid sequence: RASQSISSYLN (SEQ ID NO: 127), CDR-L2 comprising the amino acid sequence: AASSLQS (SEQ ID NO: 128), and CDR-L3 comprising the amino acid sequence: QQSYSTPFT (SEQ ID NO: 129) The VL comprising The antigen-binding site according to any one of Embodiments 6-9 and 23, comprising . Embodiment 25. A VH comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 121, and / or A VL comprising an amino acid sequence having at least 85% identity to SEQ ID NO: 126, the antigen-binding site according to any one of Embodiments 6-9, and 23-24, comprising . Embodiment 26. An HC comprising the CDR-H1, CDR-H2, and CDR-H3 of the HC shown in SEQ ID NO: 120 and having at least 85% amino acid sequence identity to SEQ ID NO: 120, and / or An LC comprising the CDR-L1, CDR-L2, and CDR-L3 of the LC shown in SEQ ID NO: 125 and having at least 85% amino acid sequence identity to SEQ ID NO: 125 The antigen-binding site according to any one of Embodiments 6 to 9 and 23 to 25, comprising the same. Embodiment 27. The antigen-binding site according to Embodiment 25 or 26, wherein the sequence identity is at least 90%, 95%, 96%, 97%, 98%, or 99%. Embodiment 28. The antigen-binding site according to any one of Embodiments 6 to 9 and 23 to 27, comprising a VH comprising the amino acid sequence shown in SEQ ID NO: 121 and / or a VL comprising the amino acid sequence shown in SEQ ID NO: 126. Embodiment 29. An antibody or an antigen-binding fragment thereof comprising the antigen-binding site according to any one of Embodiments 1 to 4. Embodiment 30. An antibody or an antigen-binding fragment thereof comprising the antigen-binding site according to any one of Embodiments 5 and 10 to 15. Embodiment 31. The antibody or an antigen-binding fragment thereof according to Embodiment 30, which can block c-Kit signal transduction. Embodiment 32. An antibody or an antigen-binding fragment thereof comprising the antigen-binding site according to any one of Embodiments 6 and 16 to 22. Embodiment 33. The antibody or an antigen-binding fragment thereof according to Embodiment 32, which can stimulate siglec-6. Embodiment 34. An antibody or an antigen-binding fragment thereof comprising the antigen-binding site according to Embodiment 7. Embodiment 35. An antibody or an antigen-binding fragment thereof comprising the antigen-binding site according to any one of Embodiments 23 to 28. Embodiment 36. The antibody or an antigen-binding fragment thereof according to any one of Embodiments 29 to 35, wherein the antibody is a humanized antibody. Embodiment 37. The antibody or an antigen-binding fragment thereof according to any one of Embodiments 29 to 35, wherein the antibody is a human antibody. Embodiment 38. The antibody or antigen-binding fragment thereof according to any one of Embodiments 29 to 35, wherein the antibody is a multispecific antibody. Embodiment 39. The antibody or antigen-binding fragment thereof according to any one of Embodiments 29 to 35, wherein the antibody is a bispecific antibody. Embodiment 40. The antibody or antigen-binding fragment thereof according to any one of Embodiments 29 to 35, wherein the antigen-binding fragment is a Fab, F(ab’)2, Fab’, scFv, Fv fragment, Fd fragment, or diabody. Embodiment 41. The antibody or antigen-binding fragment thereof according to any one of Embodiments 29 to 35, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region. Embodiment 42. The antibody or antigen-binding fragment thereof according to Embodiment 41, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region. Embodiment 43. The antibody or antigen-binding fragment thereof according to any one of Embodiments 29 to 42, which has a reduced effector function or a null effector function. Embodiment 44. The antibody or antigen-binding fragment thereof according to any one of Embodiments 29 to 43, which is defucosylated. Embodiment 45. The antibody or antigen-binding fragment thereof according to Embodiment 44, which is at least about 75% defucosylated. Embodiment 46. The antibody or antigen-binding fragment thereof according to Embodiment 44 or 45, which can deplete mast cells. Embodiment 47. A protein comprising the antigen-binding site according to any one of Embodiments 1 to 4. Embodiment 48. A protein comprising the antigen-binding site according to any one of Embodiments 5 and 10 to 15. Embodiment 49. A protein comprising the antigen-binding site according to any one of Embodiments 6 and 16 to 22. Embodiment 50. A protein comprising the antigen-binding site according to Embodiment 7. Embodiment 51. A protein comprising the antigen-binding site according to any one of Embodiments 23 to 28. Protein comprising two or more antigen-binding sites selected from: (i) the antigen-binding site according to any one of Embodiments 1 to 4; (ii) the antigen-binding site according to any one of Embodiments 5 and 10 to 15; (iii) the antigen-binding site according to any one of Embodiments 6 and 16 to 22; (iv) the antigen-binding site according to Embodiment 7; and (v) the antigen-binding site according to any one of Embodiments 23 to 28. Protein comprising two antigen-binding sites selected from: (i) the antigen-binding site according to any one of Embodiments 1 to 4; (ii) the antigen-binding site according to any one of Embodiments 5 and 10 to 15; (iii) the antigen-binding site according to any one of Embodiments 6 and 16 to 22; (iv) the antigen-binding site according to Embodiment 7; and (v) the antigen-binding site according to any one of Embodiments 23 to 28. Embodiment 54. (i) The antigen-binding site according to any one of Embodiments 5 and 10 to 15, and (ii) The antigen-binding site selected from (a) the antigen-binding site according to Embodiment 2, (b) the antigen-binding site according to Embodiment 3, or (c) the antigen-binding site according to any one of Embodiments 6 and 16 to 22 Protein. Protein according to any one of Embodiments 47 to 54, further comprising an antibody heavy chain constant region. Protein according to Embodiment 55, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region. Protein according to any one of Embodiments 47 to 56, having reduced effector function or null effector function. Protein according to any one of Embodiments 47 to 57, which is defucosylated. Protein according to Embodiment 58, at least about 75% of which is defucosylated. Protein according to Embodiment 58 or 59, which can deplete mast cells. Isolated nucleic acid encoding the antigen-binding site according to any one of Embodiments 1 to 28, an antibody or an antigen-binding fragment thereof according to any one of Embodiments 29 to 46, or a protein according to any one of Embodiments 47 to 60. Embodiment 62. An expression vector comprising the isolated nucleic acid according to Embodiment 61. Embodiment 63. A host cell comprising the isolated nucleic acid molecule according to Embodiment 61 or the expression vector according to Embodiment 62. Embodiment 64. A pharmaceutical composition comprising the antigen-binding site according to any one of Embodiments 1 to 28, an antibody or an antigen-binding fragment thereof according to any one of Embodiments 29 to 46, or a protein according to any one of Embodiments 47 to 60, and a pharmaceutically acceptable carrier. Embodiment 65. A method for treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the antigen-binding site according to any one of Embodiments 1 to 28, an antibody or an antigen-binding fragment thereof according to any one of Embodiments 29 to 46, a protein according to any one of Embodiments 47 to 60, or the pharmaceutical composition according to Embodiment 64. Embodiment 66. The method according to Embodiment 65, wherein the disease or disorder is a mast cell-related disease or disorder. Embodiment 67. The method according to Embodiment 65, wherein the disease or disorder is an autoimmune disease. Embodiment 68. The method according to Embodiment 65, wherein the disease or disorder is an allergy. Embodiment 69. The method according to Embodiment 65, wherein the disease or disorder is inflammation. Embodiment 70. The method according to Embodiment 65, wherein the disease or disorder is itching. Embodiment 71. The method according to Embodiment 65, wherein the disease or disorder is cancer. Embodiment 72. The antibody or an antigen-binding fragment thereof according to Embodiment 12 or 13, comprising an amino acid sequence selected from any one of SEQ ID NOs: 1, 9, 17 to 19, 22, 24, 26, 28, 30, 35, 37, 120, 121, 125, and 126, or a fragment thereof. Embodiment 73. An antibody or an antigen-binding fragment thereof according to Embodiment 12 or 13, comprising at least one amino acid sequence selected from any one of SEQ ID NOs: 1, 9, 17 to 19, 22, 24, 26, 28, 30, 35, 37, 120, 121, 125, and 126, or a fragment thereof. Embodiment 74. An antibody or an antigen-binding fragment thereof according to Embodiment 12 or 13, comprising at least one CDR sequence found in a sequence selected from any one of SEQ ID NOs: 1, 9, 17 to 19, 2, 24, 26, 28, 30, 35, 37, 120, 121, 125, and 126. Embodiment 75. An antibody or an antigen-binding fragment thereof according to Embodiment 12 or 13, comprising at least two CDR sequences found in a sequence selected from any one of SEQ ID NOs: 1, 9, 17 to 19, २२, २४, २६, २८, ३०, ३५, ३७, १२०, १२१, १२५, and १२६. Embodiment 76. An antibody or an antigen-binding fragment thereof according to Embodiment 12 or 13, comprising at least three CDR sequences found in a sequence selected from any one of SEQ ID NOs: 1, 9, <17> to <19>, <22>, <24>, <26>, <28>, <30>, <35>, <37>, <120>, <121>, <125>, and <126>. Embodiment 77. An antibody or an antigen-binding fragment thereof according to Embodiment 11, comprising an amino acid sequence selected from any one of SEQ ID NOs: 39, 44, 49 to 51, 54, 56, 58, 86, and 88, or a fragment thereof. Embodiment 78. An antibody or an antigen-binding fragment thereof according to Embodiment 11, comprising at least one amino acid sequence selected from any one of SEQ ID NOs: 39, 44, 49 to 51, 54, 56, 58, 86, and 88, or a fragment thereof. Embodiment 79. An antibody or an antigen-binding fragment thereof according to Embodiment 11, comprising at least two amino acid sequences selected from any one of SEQ ID NOs: 39, 44, 49 to 51, 54, 56, 58, 86, and 88, or a fragment thereof. Embodiment 80. The antibody or antigen-binding fragment thereof according to Embodiment 11, comprising at least one CDR found in a sequence selected from any one of SEQ ID NOs: 39, 44, 49-51, 54, 56, 58, 86, and 88, or a fragment thereof. Embodiment 81. The antibody or antigen-binding fragment thereof according to Embodiment 11, comprising at least two CDRs found in a sequence selected from any one of SEQ ID NOs: 39, 44, 49-51, 54, 56, 58, 86, and 88, or a fragment thereof. Embodiment 82. The antibody or antigen-binding fragment thereof according to Embodiment 11, comprising at least three CDRs found in a sequence selected from any one of SEQ ID NOs: 39, 44, 49-51, 54, 56, 58, 86, and 88, or a fragment thereof. Embodiment 83. An antibody or antigen-binding fragment comprising an amino acid sequence selected from any one of SEQ ID NOs: 1, 9, 17-19, 22, 24, 26, 28, 30, 35, 37, 39, 44, 49-51, 54, 56, 58, 86, 88, 120, 121, 125, and 126, or a fragment thereof.

Examples

[0236] Example 1. Antibody Generation Human antibodies targeting FCER1, c-Kit, and siglecs (e.g., siglec-6 and siglec8) are produced by methods known in the art.

[0237] In some embodiments, transgenic mice that express functional endogenous immunoglobulins but cannot express human immunoglobulin genes are used. In particular, the human heavy and light chain immunoglobulin gene complexes are introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to the human heavy and light chain genes, the human variable, constant, and diversity regions are introduced into mouse embryonic stem cells. The mouse heavy and light chain immunoglobulin genes are made non-functional separately or simultaneously from the introduction of the human immunoglobulin locus by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal way with a selected antigen, for example, all or part of an antigen (e.g., FCER1, c-Kit, or siglec). Monoclonal antibodies directed against this antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes carried by the transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic hypermutation.

[0238] In some other embodiments, FCER1, c-Kit, and siglec (e.g., siglec-6 and siglec8)-targeted antibodies are produced by screening yeast display libraries.

[0239] Furthermore, c-Kit / siglec-6 bispecific antibodies are produced by methods known in the art. In some embodiments, the c-Kit / siglec-6 bispecific antibodies are in the CrossMab format.

[0240] Example 2. Binding of antibodies to c-Kit The target binding of the c-Kit antibody to c-Kit-expressing cells was evaluated by flow cytometry. The cultured M-07e cells were resuspended in FACS blocking buffer (PBS + 10% normal human serum) for 30 minutes. After washing, the cells were incubated with the c-Kit antibody in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide) at 4°C for 30 minutes, washed, and then incubated with a PE-labeled anti-human secondary antibody. The cells were incubated in the dark at 4°C for 30 minutes. After washing, cell fluorescence measurement analysis was performed using a BD FACS Symphony A3 (BD Biosciences). The cells were classified by scatter characteristics and separated for single-cell analysis using side scatter width versus side scatter area. Antibody binding to the cells was quantified by measuring fluorescence emission at an excitation wavelength of 561 nm using a 586 / 15 nm filter. Data analysis was performed using FlowJo software and summarized in Table 2. All c-Kit antibodies tested were able to bind to cells expressing c-Kit.

[0241]

Table 2

[0242] Furthermore, the binding affinity of the c-Kit antibody for human c-Kit and cynomolgus monkey c-Kit was measured by biolayer interferometry (BLA) using an Octect device. Briefly, the capture sensor was loaded with a three-fold diluted test antibody. Association and dissociation rates were determined and used to calculate the equilibrium dissociation constant K D (Table 3). The affinity for c-Kit measured by the K D value was observed in the sub-nanomolar range for the humanized c-Kit antibodies (hSR-1-1 to hSR1-6) tested and in the single-digit nanomolar range for the chimeric c-Kit antibody (chSR1) tested.

[0243]

Table 3

[0244] Example 3. Cross-reactivity and non-specific binding of c-Kit antibodies analyzed by ELISA An ELISA-based assay was used to evaluate the cross-reactivity and non-specific binding of c-Kit antibodies to human c-Kit, cynomolgus c-Kit, and c-Kit family members.

[0245] His-tagged human c-Kit, His-tagged cynomolgus c-Kit, and His-tagged extracellular domains of PDGFRa, PDGFRb, CSF1R, and FLT3 were immobilized on 96-well ELISA plates. The plates were washed with PBST (phosphate-buffered saline + 0.05% Tween 20) and blocked with PBS containing 1% BSA for 1 hour. After washing with PBST, the mAb was added to the coated plates and incubated at room temperature for 2 hours. The plates were washed and incubated with an HRP-conjugated goat anti-human IgG secondary antibody at room temperature for 1 hour. After washing, antibody binding was detected by colorimetric measurement by adding 3,3’,5,5’-tetramethylbenzidine (TMB) and incubating at room temperature for 10 - 20 minutes, followed by the addition of 2.5N sulfuric acid stop solution. The absorbance at 450 nm was measured using a plate reader (PerkinElmer). The data were analyzed using GraphPad Prism.

[0246] The results for exemplary c-Kit antibodies are shown in Table 4. The c-Kit antibodies were able to bind to c-Kit in both human and cynomolgus, but did not cross-react with the c-Kit family members PDGFRa, PDGFRb, CSF1R, or FLT3.

[0247] [Table 4]

[0248] Example 4. Binding of antibodies to siglec-6 The target binding of the siglec-6 antibody to siglec-6-expressing cells was evaluated by flow cytometry. Cultured HMC1.2 cells were resuspended in FACS blocking buffer (PBS + 10% normal human serum) for 30 minutes. After washing, the cells were incubated with the siglec-6 antibody in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide) at 4°C for 30 minutes, washed, and then incubated with a PE-labeled anti-human secondary antibody or a PE-labeled anti-mouse secondary antibody. The cells were incubated in the dark at 4°C for 30 minutes. After washing, cell fluorescence measurement analysis was performed using a BD FACS Symphony A3 (BD Biosciences). The cells were classified according to their scattering characteristics and separated for single-cell analysis using side scatter width versus side scatter area. Antibody binding to the cells was quantified by measuring the fluorescence emission at an excitation wavelength of 561 nm using a 586 / 15 nm filter. Data analysis was performed using FlowJo software and summarized in Table 5. All siglec-6 antibodies tested were able to bind to cells expressing siglec-6.

[0249]

Table 5

[0250] Furthermore, the binding affinity of the siglec-6 antibody to human siglec-6 was measured by BLA using an Octect instrument. Briefly, the capture sensor was loaded with a three-fold diluted test antibody. The association and dissociation rates were determined and used to calculate the equilibrium dissociation constant K D (Table 6). The affinity to siglec-6 measured by the K D value was observed in the sub-nanomolar range for the humanized siglec-6 antibodies and chimeric siglec-6 antibodies tested.

[0251]

Table 6

[0252] Example 5. Cross-reactivity and non-specific binding of siglec-6 antibodies analyzed by ELISA An ELISA-based assay was used to evaluate the cross-reactivity and non-specific binding of siglec-6 antibodies to human siglec-6, cynomolgus monkey siglec-6, and siglec-6 family members siglec1-5, 7-12, 14, and 15.

[0253] Cultured HEK293 cells expressing human siglec-6, cynomolgus monkey siglec-6, or siglec6 family members were resuspended in FACS blocking buffer (PBS + 10% normal human serum) for 30 minutes. After washing, the cells were incubated with siglec-6 antibodies in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide), incubated at 4°C for 30 minutes, washed, and incubated with a PE-labeled anti-human secondary antibody. The cells were incubated in the dark at 4°C for 30 minutes. After washing, cell fluorescence measurement analysis was performed using a BD FACS Symphony A3 (BD Biosciences). The cells were classified by scatter characteristics and separated for single-cell analysis using side scatter width versus side scatter area. Antibody binding to the cells was quantified by measuring fluorescence emission at an excitation wavelength of 561 nm using a 586 / 15 nm filter. Data analysis was performed using FlowJo software.

[0254] The results for exemplary siglec-6 antibodies are shown in Table 7. The siglec-6 antibodies were able to bind to both human and cynomolgus monkey siglec-6, but did not cross-react with siglec1-5, 7-12, 14, or 15, which are siglec-6 family members.

[0255] [Table 7]

[0256] Example 6. Inhibition of cell proliferation by c-Kit antibodies The ability of c-Kit antibodies to inhibit stem cell factor (SCF)-dependent cell proliferation was evaluated.

[0257] M-07e cells were grown according to the vendor's recommendations. Cells were starved overnight (16 hours) with SCF. SCF-starved cells were seeded into a 96-well plate at a density of 15,000 cells per well in medium without SCF. c-Kit antibodies (SR1 antibody or CDX-0159) or a control were titrated onto the cells, and the plate was incubated at 37 °C and 5% CO2 for 2 hours. After 2 hours of incubation, SCF was added to a final concentration of 50 ng / mL, and the plate was incubated at 37 °C and 5% CO2 for 72 hours. Cell viability was measured using Cell Titer Glo (Promega). Cell Titer Glo was added to each well, and the plate was incubated for 2 minutes on a benchtop shaker protected from light. Luminescence was measured using a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0258] Exemplary results are shown in FIG. 1. Here, antibody concentration was plotted as a function of relative luminescence, and curve fitting was performed using a four-parameter non-linear regression algorithm. IC 50 values were determined and summarized in Table 8. The SR1 antibody tested inhibited SCF-dependent proliferation in M-07e cells.

[0259] [Table 8]

[0260] Example 7. Inhibition of cell proliferation by c-Kit / siglec-6 bispecific antibodies The ability of c-Kit / siglec-6 antibodies to inhibit SCF-dependent cell proliferation was evaluated.

[0261] M-07e cells were starved with SCF overnight (16 hours). The SCF-starved cells were seeded in a medium without SCF at a density of 15,000 cells per well of a 96-well plate. Anti-siglec-6 JML-1 and hRND-3, anti-c-Kit chSR1 and CDX-0159, and anti-siglec / anti-c-Kit bispecific JML-1 / hSR1-1 and hRND-3 / hSR1-1 were titrated onto the cells and incubated at 37 °C, 5% CO2 for 2 hours. After 2-hour incubation, SCF was added to a final concentration of 50 ng / mL, and the plates were incubated at 37 °C, 5% CO2 for 72 hours. Cell viability was measured using Cell Titer Glo (Promega). Cell Titer Glo was added to each well, and the plates were incubated for 2 minutes on a bench-top shaker while protected from light. Luminescence was measured using a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism, and curve fitting was performed using a four-parameter non-linear regression algorithm. Relative proliferation was calculated by comparing the mean relative fluorescence units (RFU) from each sample (cells + antibody + SCF) to the mean RFU of cells incubated with SCF alone.

[0262] The results are shown in Figure 2. The c-Kit / siglec-6 bispecific antibodies tested inhibited SCF-dependent proliferation in M-07e cells. The siglec-6 monospecific antibodies did not inhibit proliferation.

[0263] Example 8. Mast cell degranulation assay Experiments were conducted to evaluate whether the disclosed siglec-6 antibodies induce degranulation of mast cells.

[0264] CD34+ progenitor cells from whole blood were cultured in mast cell medium containing cytokines and growth factors. The cultures matured into mast cells 6 - 8 weeks from the start of the differentiation process.

[0265] To induce FcγRI expression in mast cells, the cells were treated with 100 UI / mL of IFN-γ for 72 hours. IFN-γ-treated mast cells and untreated mast cells were made cytokine-deficient for 4 hours. The cells were washed in Tyrode's buffer and seeded at 35,000 cells per well in a 96-well plate. The plate was incubated at 37 °C and 5% CO2 for 30 minutes. Normal human IgG1 Fc, effector-silent IgG1 Fc (containing the L234A / L235A / P329G mutation), or effector-enhanced IgG1 Fc (defucosylated) and siglec6 antibody with PMA / ionomycin were added to the appropriate wells together with SCF, and the plate was incubated at 37 °C for 30 minutes (in air). The medium from the treated mast cell cultures was added to a 96-well plate containing PNAG solution (0.35% p-nitrophenyl N-acetyl-β-D-glucosaminide) and incubated at 37 °C for 90 minutes (in air). Similarly, the remaining mast cells were lysed in 0.1% Triton X-100 and added to the PNAG solution for 90 minutes. Glycine buffer (400 mM, pH 10.7) was added to all wells, and the absorbance at 405 nm was read on a plate reader. β-Hexosaminidase was measured by calculating the percentage of β-hexosaminidase released from the β-hexosaminidase contained in the mast cells.

[0266] The results are shown in Figure 3. None of the three Siglec-6 hRND-3 antibodies containing modifications in the Fc domain induced degranulation of CD34-derived mast cells.

[0267] Sequence supplement In some sequences, the variable regions are shown in bold and the CDRs are underlined.

[0268]

Table 9

[0269]

Table 10

[0270]

Table 11

[0271] Incorporation by Reference Unless otherwise indicated, all disclosures of each of the patent documents and scientific papers referenced in this specification are incorporated by reference for all purposes.

[0272] Equivalents / Other Embodiments Those skilled in the art will be able to recognize or confirm many equivalents to the specific embodiments described herein by performing only routine experimentation. Such equivalents are intended to be covered by the appended claims.

Claims

1. An antibody or its antigen-binding fragment comprising a first antigen-binding domain capable of binding to siglc-6 and a second antigen-binding domain capable of binding to c-Kit.

2. The antibody or antigen-binding fragment according to claim 1, wherein the second antigen-binding domain comprises the following: (a) Heavy chain complementarity determination region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 41, or a sequence that differs from it by one or two amino acids, or optionally having the amino acid sequence of SEQ ID NO: 60, 61, or 62, HCDR2 having the amino acid sequence of SEQ ID NO: 53 or 42, or a sequence that differs from it by one or two amino acids, and optionally having the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67, or 68, and HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence that differs from it by one or two amino acids, or optionally having the amino acid sequence of SEQ ID NOs: 69, 70, 71, or 72. Heavy chain variable (VH) region including, (b) Heavy chain complementarity determination region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 46, or a sequence that differs from it by one or two amino acids, or optionally having the amino acid sequence of SEQ ID NOs: 90, 91, 92, 93, 94, or 95, LCDR2 having the amino acid sequence of SEQ ID NO: 47, or a sequence that differs from it by one or two amino acids, and optionally having the amino acid sequence of SEQ ID NO: 96 or 97, and LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence that differs from it by one or two amino acids, or optionally having the amino acid sequence of SEQ ID NOs: 98, 99, 100, 101, 102, 103, or 104. The light chain variable (VL) region, which includes [a specific component].

3. The antibody or antigen-binding fragment according to claim 2, wherein the second antigen-binding domain comprises the following: (a) (i) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 43, respectively (ii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 42, and 43, respectively (iii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 60, 53, and 43, respectively. (iv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 61, 53, and 43, respectively. (v) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 62, 53, and 43, respectively (vi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 63, and 43, respectively. (vii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 64, and 43, respectively. (viiii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 65, and 43, respectively. (ix) HCDR1, HCDR2, and HCDR3, each having the amino acid sequences of SEQ ID NOs. 41, 66, and 43, (x) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 67, and 43, respectively. (xi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 68, and 43, respectively. (xi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 69, respectively. (xiii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 70, respectively. (xiv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 71 respectively, or (xv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 72, respectively. The VH region including, (b) (i) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively (ii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 90, 47, and 48, respectively. (iii) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 91, 47, and 48, (iv) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 92, 47, and 48, (v) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 93, 47, and 48, respectively. (vi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 94, 47, and 48, respectively. (vii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 95, 47, and 48, respectively. (viiii) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 96, and 48, (ix) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 97, and 48, (x) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 98, respectively. (xi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 99, respectively. (xi) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 100, (xiiii) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 101, respectively. (xiv) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 102, respectively. (xv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 103 respectively, or (xvi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 104, respectively. The VL region, which includes this area.

4. The antibody or antigen-binding fragment according to claim 1, wherein the second antigen-binding domain comprises the following: A VH region containing an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 52, 40, 55, 57, 59, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85, and A VL region containing an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 87, 45, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

5. The antibody or antigen-binding fragment according to claim 4, wherein the second antigen-binding domain comprises the following: (a) A VH region having the amino acid sequence of SEQ ID NO: 52, and a VL region having the amino acid sequence of SEQ ID NO: 87, (b) A VH region having the amino acid sequence of SEQ ID NO: 40, and a VL region having the amino acid sequence of SEQ ID NO: 45 (c) VH region having the amino acid sequence of SEQ ID NO: 55, and VL region having the amino acid sequence of SEQ ID NO: 87, (d) A VH region having the amino acid sequence of SEQ ID NO: 57, and a VL region having the amino acid sequence of SEQ ID NO: 87, (e) A VH region having the amino acid sequence of SEQ ID NO: 59, and a VL region having the amino acid sequence of SEQ ID NO: 87, (f) A VH region having the amino acid sequence of SEQ ID NO: 52, and a VL region having the amino acid sequence of SEQ ID NO: 89, or (g) A VH region having the amino acid sequence of SEQ ID NO: 55, and a VL region having the amino acid sequence of SEQ ID NO:

89.

6. The antibody or antigen-binding fragment according to claim 1, wherein the second antigen-binding domain comprises the following: (a) A VH region containing HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 43 respectively, and a VL region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 48 respectively, or (b) A VH region having the amino acid sequence of SEQ ID NO: 52, and a VL region having the amino acid sequence of SEQ ID NO:

87.

7. The first antigen-binding domain described above is (a) HCDR1 having the amino acid sequence of SEQ ID NO: 3, or a sequence that differs from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 21 or 4, or a sequence that differs from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 5, or a sequence that differs from it by one or two amino acids. The VH region including, (b) LCDR1 having the amino acid sequence of SEQ ID NO: 32 or 11, or a sequence that differs from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 12, or a sequence that differs from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 13, or a sequence that differs from it by one or two amino acids. VL area including The antibody or antigen-binding fragment according to claim 1, comprising: Optionally, the antibody or its antigen-binding fragment wherein the first antigen-binding domain comprises the following: (a) (i) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively, or (ii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 4, and 5, respectively. The VH region including, (b) (i) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 32, 12, and 13, respectively, or (ii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively. The VL region, which includes this area.

8. The first antigen-binding domain described above is A VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 25, 2, 20, 23, 27, or 29, and VL region containing an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 38, 10, 31, or 36. The antibody or antigen-binding fragment according to claim 7, comprising: Optionally, the antibody or its antigen-binding fragment wherein the first antigen-binding domain comprises the following: (a) A VH region having the amino acid sequence of SEQ ID NO: 25, and a VL region having the amino acid sequence of SEQ ID NO: 38, (b) A VH region having the amino acid sequence of SEQ ID NO: 2, and a VL region having the amino acid sequence of SEQ ID NO: 10, (c) VH region having the amino acid sequence of SEQ ID NO: 23, and VL region having the amino acid sequence of SEQ ID NO: 38 (d) A VH region having the amino acid sequence of SEQ ID NO: 27, and a VL region having the amino acid sequence of SEQ ID NO: 38, or (e) A VH region having the amino acid sequence of SEQ ID NO: 29, and a VL region having the amino acid sequence of SEQ ID NO:

38.

9. The antibody or antigen-binding fragment according to claim 1, wherein the first antigen-binding domain comprises the following: (a) HCDR1 having the amino acid sequence of SEQ ID NO: 122, or a sequence that differs from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NO: 123, or a sequence that differs from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NO: 124, or a sequence that differs from it by one or two amino acids. The VH region including, (b) LCDR1 having the amino acid sequence of SEQ ID NO: 127, or a sequence that differs from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NO: 128, or a sequence that differs from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NO: 129, or a sequence that differs from it by one or two amino acids. The VL region, which includes this area.

10. The first antigen-binding domain described above is A VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 121, and A VL region containing an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

126. The antibody or antigen-binding fragment according to claim 9, comprising: Optionally, the antibody or its antigen-binding fragment wherein the first antigen-binding domain comprises the following: The VH region having the amino acid sequence of SEQ ID NO: 121, and The VL region having the amino acid sequence of SEQ ID NO:

126.

11. The first antigen-binding domain described above is (a) HCDR1 having the amino acid sequence of SEQ ID NOs. 133, 141, or 149, or a sequence that differs from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NOs. 134, 142, 150, or 160, or a sequence that differs from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NOs. 135, 143, or 151, or a sequence that differs from it by one or two amino acids. The VH region including, (b) LCDR1 having the amino acid sequence of SEQ ID NOs. 137, 145, 153, or 165, or a sequence that differs from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NOs. 138, 146, or 154, or a sequence that differs from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NOs. 139, 147, or 155, or a sequence that differs from it by one or two amino acids. VL area including The antibody or antigen-binding fragment according to claim 1, comprising: Optionally, the antibody or its antigen-binding fragment wherein the first antigen-binding domain comprises the following: (a) (i) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 133, 134, and 135, respectively (ii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 141, 142, and 143, respectively (iii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 149, 150, and 151, respectively, or (iv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 149, 160, and 151, respectively. The VH region including, (b) (i) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 137, 138, and 139, respectively (ii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 145, 146, and 147, respectively. (iii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 153, 154, and 155, respectively, or (iv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 165, 154, and 155, respectively. The VL region, which includes this area.

12. The first antigen-binding domain described above is A VH region comprising an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs: 132, 140, 148, 159, or 162, and VL region containing an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 136, 144, 152, 164, 167, or 169. The antibody or antigen-binding fragment according to claim 11, comprising: Optionally, the antibody or its antigen-binding fragment wherein the first antigen-binding domain comprises the following: (a) A VH region having the amino acid sequence of SEQ ID NO: 132, and a VL region having the amino acid sequence of SEQ ID NO: 136, (b) A VH region having the amino acid sequence of SEQ ID NO: 140, and a VL region having the amino acid sequence of SEQ ID NO: 144, (c) A VH region having the amino acid sequence of SEQ ID NO: 148, and a VL region having the amino acid sequence of SEQ ID NO: 152, (d) A VH region having the amino acid sequence of SEQ ID NO: 159, and a VL region having the amino acid sequence of SEQ ID NO: 164, (e) A VH region having the amino acid sequence of SEQ ID NO: 162, and a VL region having the amino acid sequence of SEQ ID NO: 167, or (f) A VH region having the amino acid sequence of SEQ ID NO: 162, and a VL region having the amino acid sequence of SEQ ID NO:

169.

13. The antibody or antigen-binding fragment according to claim 1, wherein the first antigen-binding domain comprises the following: (a) A VH region containing HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 3, 21, and 5 respectively, and a VL region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 32, 12, and 13 respectively, or (b) A VH region having the amino acid sequence of SEQ ID NO: 25, and a VL region having the amino acid sequence of SEQ ID NO:

38.

14. The antibody or antigen-binding fragment according to claim 1, wherein the first antigen-binding domain comprises the following: (a) A VH region containing HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 122, 123, and 124 respectively, and a VL region containing LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 127, 128, and 129 respectively, or (b) A VH region having the amino acid sequence of SEQ ID NO: 121, and a VL region having the amino acid sequence of SEQ ID NO:

126.

15. (a) (i) The first antigen-binding domain is The VH region includes HCDR1, HCDR2, and HCDR3, each having the amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively. The VL region includes LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 32, 12, and 13, respectively. including and (ii) The second antigen-binding domain, The VH region includes HCDR1, HCDR2, and HCDR3, each having the amino acid sequences of SEQ ID NOs. 41, 53, and 43, respectively. The VL region includes LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively. including, or (b) (i) The first antigen-binding domain is The VH region includes HCDR1, HCDR2, and HCDR3, each having the amino acid sequences of SEQ ID NOs. 122, 123, and 124, respectively. The VL region includes LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 127, 128, and 129, respectively. including and (ii) The second antigen-binding domain, The VH region includes HCDR1, HCDR2, and HCDR3, each having the amino acid sequences of SEQ ID NOs. 41, 53, and 43, respectively. The VL region includes LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively. including, The antibody or antigen-binding fragment according to claim 1.

16. (a) The first antigen-binding domain comprises a VH region having the amino acid sequence of SEQ ID NO: 25 and a VL region having the amino acid sequence of SEQ ID NO: 38, and The second antigen-binding domain includes a VH region having the amino acid sequence of SEQ ID NO: 52 and a VL region having the amino acid sequence of SEQ ID NO: 87, or (b) The first antigen-binding domain comprises a VH region having the amino acid sequence of SEQ ID NO: 121 and a VL region having the amino acid sequence of SEQ ID NO: 126, The second antigen-binding domain includes a VH region having the amino acid sequence of SEQ ID NO: 52 and a VL region having the amino acid sequence of SEQ ID NO:

87. The antibody or antigen-binding fragment according to claim 15.

17. (a) A first heavy chain comprising the VH region of the first antigen-binding domain and the first heavy chain constant (CH) region or a fragment thereof, (b) A first light chain comprising the VL region of the first antigen-binding domain and the first light chain constant (CL) region or a fragment thereof, (c) A second heavy chain comprising the VH region of the second antigen-binding domain and the second CH region or a fragment thereof, and (d) A second light chain comprising the VL region of the second antigen-binding domain and the second CL region or a fragment thereof. The antibody or antigen-binding fragment according to claim 1, comprising:

18. (a) The first CH region and the second CH region each contain an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO: 130, (b) The first heavy chain and the second heavy chain form a heterodimer. (c) The first CH region and / or the second CH region comprises one or more mutations that increase the serum half-life of the antibody or its antigen-binding fragment, (d) The first CH region and / or the second CH region include one or more mutations that reduce or suppress the effector function, or one or more mutations that enhance the effector function. (e) At least one of the first heavy chain and the second heavy chain is non-fucosylated, optionally, the antibody or its antigen-binding fragment is (i) a cell line having alpha-1,6-fucosyltransferase (Fut8) knockout, or (ii) a cell line overexpressing β1,4-N-acetylglucosamine transferase III (GnT-III) and optionally overexpressing Golgi μ-mannosidase II (ManII). Produced in and / or (f) The antibody or antigen-binding fragment according to claim 17, wherein the first CL region and / or the second CL region is a human kappa constant region.

19. (a) The first antigen-binding domain can bind to siglect-6 with a higher affinity than the second antigen-binding domain can bind to c-Kit, or (b) K is a molecule in which the first antigen-binding domain is less than about 100 uM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 100 pM, less than about 10 pM, less than about 1 pM, less than about 100 fM, or less than about 10 fM. Ｄ K is capable of binding to siglect-6 with an affinity characterized by a value, and the second antigen-binding domain is approximately 1 nM to approximately 900 μM, approximately 5 nM to approximately 500 μM, approximately 10 nM to approximately 100 μM, approximately 20 nM to approximately 10 μM, or approximately 25 to approximately 500 nM. Ｄ It can bind to c-Kit with affinity characterized by a value. The antibody or antigen-binding fragment according to claim 1.

20. (a) The antigen-binding fragment is Fab, Fab', Fab2, F(ab')2, Fv, single-chain Fv(scFv), or diabody, and / or (b) The antibody or its antigen-binding fragment is conjugated to the active substance, Optionally, the active substance is a cytotoxic active substance or label. The antibody or antigen-binding fragment according to claim 1.

21. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 20, wherein the antibody or antigen-binding fragment comprises an Fc region and an N-glycosidic-linked glycan linked to the Fc region, optionally comprising less than 50% of the N-glycosidic-linked glycan as fucose residues, and optionally substantially none of the N-glycosidic-linked glycan as fucose residues.

22. Humanized antibodies or antigen-binding fragments that can bind to c-Kit, including the following: (a) Heavy chain complementarity determination region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 41, or a sequence that differs from it by one amino acid, or optionally having the amino acid sequence of SEQ ID NO: 60, 61, or 62, HCDR2 having the amino acid sequence of SEQ ID NO: 53, or a sequence that differs from it by one amino acid, and optionally having the amino acid sequence of SEQ ID NOs: 63, 64, 65, 66, 67, or 68, and HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence that differs from it by one amino acid, or optionally having the amino acid sequence of SEQ ID NOs: 69, 70, 71, or 72. Heavy chain variable domain (VH) including, (b) Light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 46, or a sequence that differs from it by one amino acid, or optionally having the amino acid sequence of SEQ ID NOs: 90, 91, 92, 93, 94, or 95, LCDR2 having the amino acid sequence of SEQ ID NO: 47, or a sequence that differs from it by one amino acid, and optionally having the amino acid sequence of SEQ ID NO: 96 or 97, and LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence that differs from it by one amino acid, or optionally having the amino acid sequence of SEQ ID NOs: 98, 99, 100, 101, 102, 103, or 104. A light chain variable domain (VL) that includes this domain.

23. (a) The VH region is (i) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 43, respectively (ii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 60, 53, and 43, respectively (iii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 61, 53, and 43, respectively. (iv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 62, 53, and 43, respectively. (v) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 63, and 43, respectively (vi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 64, and 43, respectively. (vii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 65, and 43, respectively. (viiii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 66, and 43, respectively. (ix) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 67, and 43, respectively. (x) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 68, and 43, respectively. (xi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 69, respectively. (xi) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 70, respectively. (xiii) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 71 respectively, or (xiv) HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs. 41, 53, and 72, respectively. including and (b) The VL region is (i) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 48, respectively (ii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 90, 47, and 48, respectively. (iii) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 91, 47, and 48, (iv) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 92, 47, and 48, (v) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 93, 47, and 48, respectively. (vi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 94, 47, and 48, respectively. (vii) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 95, 47, and 48, respectively. (viiii) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 96, and 48, (ix) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 97, and 48, (x) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 98, respectively. (xi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 99, respectively. (xi) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 100, (xiiii) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 101, respectively. (xiv) LCDR1, LCDR2, and LCDR3, each having the amino acid sequences of SEQ ID NOs. 46, 47, and 102, respectively. (xv) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 103 respectively, or (xvi) LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs. 46, 47, and 104, respectively. including, The antibody or antigen-binding fragment according to claim 22.

24. The VH region contains an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 52, 40, 55, 57, 59, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85, and The VL region includes an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 87, 45, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119. The antibody or antigen-binding fragment according to claim 23.

25. Antibodies or antigen-binding fragments capable of binding to siglect-6, including the following: (a) Heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of sequence number 133, 141, or 149, or a sequence that differs from it by one or two amino acids, HCDR2 having the amino acid sequence of SEQ ID NOs. 134, 142, 150, or 160, or a sequence that differs from it by one or two amino acids, and HCDR3 having the amino acid sequence of SEQ ID NOs. 135, 143, or 151, or a sequence that differs from it by one or two amino acids. Heavy chain variable (VH) region including, (b) Light chain complementarity determining region 1 (LCDR1) having the amino acid sequence of sequence numbers 137, 145, 153, or 165, or a sequence that differs from it by one or two amino acids, LCDR2 having the amino acid sequence of SEQ ID NOs. 138, 146, or 154, or a sequence that differs from it by one or two amino acids, and LCDR3 having the amino acid sequence of SEQ ID NOs. 139, 147, or 155, or a sequence that differs from it by one or two amino acids. The light chain variable (VL) region, which includes [a specific component].

26. The VH region includes an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs: 132, 140, 148, 159, or 162, and The VL region includes an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs: 136, 144, 152, 164, 167, or 169. The antibody or antigen-binding fragment according to claim 25.

27. Humanized antibodies or antigen-binding fragments capable of binding to siglc-6, including the following: (a) Heavy chain complementarity determination region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 3, HCDR2 having the amino acid sequence of SEQ ID NO: 21, and HCDR3 having the amino acid sequence of SEQ ID NO: 5 Heavy chain variable domain (VH) including, (b) Light chain complementarity determination region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 32, LCDR2 having the amino acid sequence of SEQ ID NO: 12, and LCDR3 having the amino acid sequence of SEQ ID NO: 13 A light chain variable domain (VL) that includes this domain.

28. The VH region includes an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NOs. 20, 23, 25, 27, or 29, and The VL region includes an amino acid sequence having at least 70%, at least 80%, at least 90%, or at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 31, 36, or 38. The antibody or antigen-binding fragment according to claim 27.

29. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 20 and 22 to 28.

30. A host cell comprising the polynucleotide described in claim 29, which is optionally a mammalian cell or an insect cell, optionally comprising (a) Fut8 knockout and / or (b) GnT-III and optionally ManII overexpression.

31. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 20 and 22 to 28, and a pharmaceutically acceptable carrier.

32. A pharmaceutical composition according to claim 31 for use in treating or preventing a disease or disorder in a subject where such treatment is necessary, wherein the disease or disorder is a mast cell-related disease or disorder, or is related to the expression of siglec-6 and / or c-Kit.

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