Antibodies targeting c-Kit and / or siglecc and uses thereof

Abstract

Undesirable mast cell activity is associated with various diseases or conditions, such as allergy, inflammatory diseases, autoimmune diseases, and cancer. New and improved therapeutic agents that can target c-Kit and / or siglec-6 (e.g., for mast cell related diseases or disorders) still have been desired. Disclosed herein are molecules, such as antibodies and antibody fragments, capable of binding to c-Kit and / or siglec-6. Disclosed herein are molecules, such as antibodies and antibody fragments, capable of binding to c-Kit and / or siglec-6.

Description

[0001] Related Applications

[0002] 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 provisional patent application are hereby incorporated by reference for all purposes. Background Art

[0003] Mast cells (MC) are innate immune cells that play a key role in the inflammatory process. When MC is activated, it secretes various immune mediators, such as cytokines, leukotrienes, and a large number of proteases into the environment. The differentiation, growth, and survival of mast cells are strongly regulated by local tissue environmental factors. Stem cell factor (SCF) and IL-3 are among the best characterized factors. Undesirable mast cell activity is associated with various diseases or conditions, such as allergies, inflammatory diseases, autoimmune diseases, and cancer.

[0004] c-Kit is a type III receptor tyrosine kinase and is a well-known cell surface receptor that binds to its physiological ligand, SCF. c-Kit participates in various intracellular signaling pathways and has multiple functions during embryogenesis and adulthood. c-Kit is highly expressed in MCs; after full differentiation, MCs rely on c-Kit-dependent signaling for survival, function, and growth. Antibodies to c-Kit have been developed, such as CDX-0158.

[0005] Siglec-6 is a member of the CD33-related subfamily of sialic acid-binding immunoglobulin-like lectins (siglecs). Siglec-6 is present on human mast cells, some B cells, and the cytotrophoblast and syncytiotrophoblast of the placenta. Siglec-6 has been shown to have inhibitory activity on both IgE-mediated and non-IgE-mediated mast cell responses.

[0006] There remains a need for new and improved therapeutic agents that can target c-Kit and / or siglec-6 (eg, for mast cell-related diseases or disorders). Summary of the invention

[0007] The present application provides an antigen binding site capable of binding to c-Kit and / or siglec-6.

[0008] In one aspect, an antibody or 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.

[0009] In some embodiments, the first antigen binding domain is capable of binding siglec-6 with a higher affinity than the second antigen binding domain is capable of binding c-Kit. In some embodiments, the first antigen binding domain is capable of binding siglec-6 with a higher affinity than the second antigen binding domain is capable of binding c-Kit. D In some embodiments, the first antigen binding domain can bind to siglec-6 with an affinity characterized by a K D The affinity of the peptides or peptides may be between about 1 nM and about 900 uM, between about 5 nM and about 500 uM, between about 10 nM and about 100 uM, between about 20 nM and about 10 10 uM, or between about 25 and about 500 nM to bind c-Kit.

[0010] In some embodiments, the antibody or antigen-binding fragment is capable of binding to cells expressing silect-6.

[0011] In some embodiments, the antibody or antigen binding fragment is capable of binding to mast cells. In some embodiments, the antibody or antigen binding fragment preferentially binds to mast cells rather than another immune cell. In some embodiments, the immune cell is a T cell, a dendritic cell, a macrophage, a neutrophil, or a basophil.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof does not induce mast cell degranulation.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof has a comparable or lower internalization rate than a reference antibody capable of binding to silect-6. In some embodiments, the reference antibody capable of binding to siglect-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.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof has comparable or lower toxicity than a reference antibody capable of binding to c-Kit. In some embodiments, the reference antibody capable of binding 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.

[0015] In some embodiments, the first antigen binding domain comprises:

[0016] (a) a VH region comprising: a heavy chain complementary determining region 1 (HCDR1) having an amino acid sequence of SEQ ID NO: 3, or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having an amino acid sequence of SEQ ID NO: 4 or 21, or a sequence having 1 or 2 amino acid differences therefrom, and a HCDR3 having an amino acid sequence of SEQ ID NO: 5, or a sequence having 1 or 2 amino acid differences therefrom; and

[0017] (b) a VL region comprising: a heavy chain complementary determining region 1 (LCDR1) having an amino acid sequence of SEQ ID NO: 11 or 32, or a sequence having 1 or 2 amino acid differences therefrom, a LCDR2 having an amino acid sequence of SEQ ID NO: 12, or a sequence having 1 or 2 amino acid differences therefrom, and a LCDR3 having an amino acid sequence of SEQ ID NO: 13, or a sequence having 1 or 2 amino acid differences therefrom.

[0018] In some embodiments, the first antigen binding domain comprises:

[0019] (a) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 3, a HCDR2 having the amino acid sequence of SEQ ID NO: 4 or 21, and a HCDR3 having the amino acid sequence of SEQ ID NO: 5; and

[0020] (b) a VL region comprising: a LCDR1 having an amino acid sequence of SEQ ID NO:11 or 32, a LCDR2 having an amino acid sequence of SEQ ID NO:12, and a LCDR3 having an amino acid sequence of SEQ ID NO:13.

[0021] In some embodiments, the first antigen binding domain comprises:

[0022] (a) a VH region comprising:

[0023] (i) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively; or

[0024] (ii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 21 and 5, respectively; and

[0025] (b) a VL region comprising:

[0026] (i) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; or

[0027] (ii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 32, 12 and 13, respectively.

[0028] In some embodiments, the first antigen binding domain comprises:

[0029] (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

[0030] (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.

[0031] In some embodiments, the first antigen binding domain comprises:

[0032] (a) a VH region having an amino acid sequence of SEQ ID NO: 2, 20, 23, 25, 27 or 29; and

[0033] (b) a VL region having the amino acid sequence of SEQ ID NO: 10, 31, 36 or 38.

[0034] In some embodiments, the first antigen binding domain comprises:

[0035] (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;

[0036] (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;

[0037] (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;

[0038] (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

[0039] (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.

[0040] 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.

[0041] In some embodiments, the first antigen binding domain comprises:

[0042] (a) a VH region comprising:

[0043] A HCDR1 having the amino acid sequence of SEQ ID NO: 122 or a sequence having 1 or 2 amino acid differences therefrom,

[0044] a HCDR2 having the amino acid sequence of SEQ ID NO: 123, or a sequence having 1 or 2 amino acid differences therefrom, and

[0045] A HCDR3 having the amino acid sequence of SEQ ID NO: 124, or a sequence having 1 or 2 amino acid differences therefrom; and

[0046] (b) a VL region comprising:

[0047] LCDR1 having the amino acid sequence of SEQ ID NO: 127 or a sequence having 1 or 2 amino acid differences therefrom,

[0048] LCDR2 having the amino acid sequence of SEQ ID NO: 128 or a sequence having 1 or 2 amino acid differences therefrom, and

[0049] A LCDR3 having the amino acid sequence of SEQ ID NO: 129, or a sequence having 1 or 2 amino acid differences therefrom.

[0050] In some embodiments, the first antigen binding domain comprises:

[0051] (a) a VH region comprising:

[0052] a HCDR1 having the amino acid sequence of SEQ ID NO: 122,

[0053] a HCDR2 having the amino acid sequence of SEQ ID NO: 123, and

[0054] a HCDR3 having the amino acid sequence of SEQ ID NO: 124; and

[0055] (b) a VL region comprising:

[0056] LCDR1 having the amino acid sequence of SEQ ID NO: 127,

[0057] LCDR2 having the amino acid sequence of SEQ ID NO: 128, and

[0058] LCDR3 having the amino acid sequence of SEQ ID NO:139.

[0059] In some embodiments, the first antigen binding domain comprises:

[0060] (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

[0061] (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.

[0062] In some embodiments, the first antigen binding domain comprises:

[0063] (a) a VH region having the amino acid sequence of SEQ ID NO: 121; and

[0064] (b) A VL region having the amino acid sequence of SEQ ID NO: 126.

[0065] In some embodiments, the first antigen binding domain comprises:

[0066] (a) a VH region comprising:

[0067] A HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141 or 149, or a sequence having 1 or 2 amino acid differences therefrom,

[0068] a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, or a sequence having 1 or 2 amino acid differences therefrom, and

[0069] A HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151, or a sequence having 1 or 2 amino acid differences therefrom; and

[0070] (b) a VL region comprising:

[0071] LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165, or a sequence having 1 or 2 amino acid differences therefrom,

[0072] LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, or a sequence having 1 or 2 amino acid differences therefrom, and

[0073] A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155, or a sequence having 1 or 2 amino acid differences therefrom.

[0074] In some embodiments, the first antigen binding domain comprises:

[0075] (a) a VH region comprising:

[0076] a HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141 or 149,

[0077] a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, and

[0078] a HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151; and

[0079] (b) a VL region comprising:

[0080] LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165,

[0081] LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, and

[0082] A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155.

[0083] In some embodiments, the first antigen binding domain comprises:

[0084] (a) a VH region comprising:

[0085] (i) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 133, 134 and 135, respectively;

[0086] (ii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 141, 142 and 143, respectively;

[0087] (iii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 149, 150 and 151, respectively; or

[0088] (iv) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 149, 160 and 151, respectively; and

[0089] (b) a VL region comprising:

[0090] (i) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively;

[0091] (ii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 145, 146 and 147, respectively;

[0092] (iii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 153, 154 and 155, respectively; or

[0093] (iv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 165, 154 and 155, respectively.

[0094] In some embodiments, the first antigen binding domain comprises:

[0095] (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

[0096] (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.

[0097] In some embodiments, the first antigen binding domain comprises:

[0098] (a) a VH region having an amino acid sequence of SEQ ID NO: 132, 140, 148, 159 or 162; and

[0099] (b) a VL region having the amino acid sequence of SEQ ID NO: 136, 144, 152, 164, 167 or 169.

[0100] In some embodiments, the first antigen binding domain comprises:

[0101] (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;

[0102] (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

[0103] (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.

[0104] In some embodiments, the first antigen binding domain comprises:

[0105] (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;

[0106] (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

[0107] (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.

[0108] In some embodiments, the second antigen binding domain comprises:

[0109] (a) a VH region comprising:

[0110] A HCDR1 having the amino acid sequence of SEQ ID NO: 41 or a sequence having 1 or 2 amino acid differences therefrom,

[0111] a HCDR2 having the amino acid sequence of SEQ ID NO: 42 or 53, or a sequence having 1 or 2 amino acid differences therefrom, and

[0112] A HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence having 1 or 2 amino acid differences therefrom; and

[0113] (b) a VL region comprising:

[0114] LCDR1 having the amino acid sequence of SEQ ID NO: 46 or a sequence having 1 or 2 amino acid differences therefrom,

[0115] LCDR2 having the amino acid sequence of SEQ ID NO: 47 or a sequence having 1 or 2 amino acid differences therefrom, and

[0116] A LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence having 1 or 2 amino acid differences therefrom.

[0117] In some embodiments, the second antigen binding domain comprises:

[0118] (a) a VH region comprising:

[0119] a HCDR1 having the amino acid sequence of SEQ ID NO:41,

[0120] a HCDR2 having the amino acid sequence of SEQ ID NO: 42 or 53, and

[0121] a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and

[0122] (b) a VL region comprising:

[0123] LCDR1 having the amino acid sequence of SEQ ID NO:46,

[0124] LCDR2 having the amino acid sequence of SEQ ID NO:47, and

[0125] LCDR3 having the amino acid sequence of SEQ ID NO:48.

[0126] In some embodiments, the second antigen binding domain comprises a VH region comprising:

[0127] (a) a HCDR1 having the amino acid sequence of SEQ ID NO: 60, 61 or 62,

[0128] (b) a HCDR2 having the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67 or 68, and

[0129] (c) a HCDR3 having the amino acid sequence of SEQ ID NO: 69, 70, 71 or 72. In some embodiments, the second antigen binding domain comprises a VH region comprising:

[0130] (a) LCDR1 having the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94 or 95,

[0131] (b) LCDR2 having the amino acid sequence of SEQ ID NO: 96 or 97, and

[0132] (c) LCDR3 having the amino acid sequence of SEQ ID NO: 98, 99, 100, 101, 102, 103 or 104.

[0133] In some embodiments, the second antigen binding domain comprises:

[0134] (a) a VH region comprising:

[0135] (i) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively;

[0136] (ii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 43, respectively;

[0137] (iii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 60, 53 and 43, respectively;

[0138] (iv) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 61, 53 and 43, respectively;

[0139] (v) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 62, 53 and 43, respectively;

[0140] (vi) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 63 and 43, respectively;

[0141] (vii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 64 and 43, respectively;

[0142] (viii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 65 and 43, respectively;

[0143] (ix) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 66 and 43, respectively;

[0144] (x) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 67 and 43, respectively;

[0145] (xi) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 68 and 43, respectively;

[0146] (xii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 69, respectively;

[0147] (xiii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 70, respectively;

[0148] (xiv) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 71, respectively; or

[0149] (xv) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 72, respectively; and

[0150] (b) a VL region comprising:

[0151] (i) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively;

[0152] (ii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 90, 47 and 48, respectively;

[0153] (iii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 91, 47 and 48, respectively;

[0154] (iv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 92, 47 and 48, respectively;

[0155] (v) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 93, 47 and 48, respectively;

[0156] (vi) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 94, 47 and 48, respectively;

[0157] (vii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 95, 47 and 48, respectively;

[0158] (viii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 96 and 48, respectively;

[0159] (ix) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 97 and 48, respectively;

[0160] (x) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 98, respectively;

[0161] (xi) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 99, respectively;

[0162] (xii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 100, respectively;

[0163] (xiii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 101, respectively;

[0164] (xiv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 102, respectively;

[0165] (xv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 103, respectively; or

[0166] (xvi) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 104, respectively.

[0167] In some embodiments, the second antigen binding domain comprises:

[0168] (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

[0169] (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.

[0170] In some embodiments, the second antigen binding domain comprises:

[0171] (a) a VH region having an 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

[0172] (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.

[0173] In some embodiments, the second antigen binding domain comprises:

[0174] (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;

[0175] (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;

[0176] (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;

[0177] (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;

[0178] (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;

[0179] (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

[0180] (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.

[0181] 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.

[0182] In some embodiments, the antibody or antigen-binding fragment thereof comprises:

[0183] (a) a first heavy chain comprising the VH region of a first antigen binding domain and a first heavy chain constant (CH) region or a fragment thereof;

[0184] (b) a first light chain comprising the VL region of the first antigen binding domain and a first light chain constant (CL) region or a fragment thereof;

[0185] (c) a second heavy chain comprising a VH region of a second antigen-binding domain and a second CH region or a fragment thereof; and

[0186] (d) a second light chain comprising the VL region of the second antigen-binding domain and a second CL region or a fragment thereof.

[0187] In some embodiments, the first and second CH regions each comprise 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.

[0188] In some embodiments, the first and second heavy chains form a heterodimer, optionally wherein one of the first and second heavy chains 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, wherein the numbering of the constant regions is according to the EU index.

[0189] In some embodiments, the first and / or second CH region comprises one or more mutations that reduce or eliminate effector function. In some embodiments, the first and / or second CH region is a human IgG1 CH comprising one of the following mutations, wherein the numbering of the constant region is 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) N 297G; (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 and / or second CH region is a human IgG2 CH comprising one of the following mutations, wherein the numbering of the constant region is 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 and / or second CH region is a human IgG4 CH comprising one of the following mutations, wherein the numbering of the constant region is 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.

[0190] In some embodiments, the first and / or second CH region comprises one or more mutations that enhance effector function. In some embodiments, the first and / or second CH region is a human IgG1 CH comprising one of the following mutations, wherein the constant region is 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.

[0191] In some embodiments, at least one of the first and second heavy chains is non-fucosylated. In some embodiments, the antibody or antigen-binding fragment thereof is produced in the following cell lines: (a) a cell line with a knockout of α-1,6-fucosyltransferase (Fut8); or (b) a cell line overexpressing β1,4-N-acetylglucosaminyltransferase III (GnT-III) and optionally overexpressing Golgi μ-mannosidase II (ManII).

[0192] In some embodiments, the first and / or second CL region is a human kappa constant region.

[0193] In some embodiments,

[0194] (a)(i) the first antigen binding domain comprises:

[0195] A VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 21 and 5, respectively; and

[0196] A VL region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 32, 12 and 13, respectively; and

[0197] (ii) the second antigen binding domain comprises:

[0198] a VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 43, respectively; and

[0199] A VL region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively; or

[0200] (b)(i) the first antigen binding domain comprises:

[0201] A VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 122, 123 and 124, respectively; and

[0202] A VL region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 127, 128 and 129, respectively; and

[0203] (ii) the second antigen binding domain comprises:

[0204] a VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 43, respectively; and

[0205] A VL region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively; or

[0206] In some embodiments, (a) the first antigen binding domain comprises a VH region having an amino acid sequence of SEQ ID NO: 25 and a VL region having an amino acid sequence of SEQ ID NO: 38; and the second antigen binding domain comprises a VH region having an amino acid sequence of SEQ ID NO: 52 and a VL region having an amino acid sequence of SEQ ID NO: 87; or

[0207] (b) the first antigen-binding domain comprises a VH region having an amino acid sequence of SEQ ID NO: 121 and a VL region having an amino acid sequence of SEQ ID NO: 126; and the second antigen-binding domain comprises a VH region having an amino acid sequence of SEQ ID NO: 52 and a VL region having an amino acid sequence of SEQ ID NO: 87.

[0208] In some embodiments, the antigen binding fragment is a Fab, Fab', Fab2, F(ab')2, Fv, single-chain Fv (scFv), or a diabody.

[0209] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to an agent. In some embodiments, the agent is a cytotoxic agent or a label.

[0210] In another aspect, a composition comprising the above-mentioned antibody or antigen-binding fragment thereof is provided. In some embodiments, the antibody or antigen-binding fragment comprises an Fc region and an N-glycoside-linked carbohydrate chain connected to the Fc region, optionally wherein less than 50% of the N-glycoside-linked carbohydrate chains contain fucose residues. In some embodiments, substantially no N-glycoside-linked carbohydrate chains contain fucose residues.

[0211] In another aspect, an antibody or antigen-binding fragment thereof capable of binding to siglec-6 is provided, comprising:

[0212] (a) a heavy chain variable (VH) region comprising:

[0213] A heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 133, 141 or 149, or a sequence having 1 or 2 amino acid differences therefrom,

[0214] a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, or a sequence having 1 or 2 amino acid differences therefrom, and

[0215] A HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151, or a sequence having 1 or 2 amino acid differences therefrom; and

[0216] (b) a light chain variable (VL) region comprising:

[0217] A light chain complementary determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165, or a sequence having 1 or 2 amino acid differences therefrom,

[0218] LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, or a sequence having 1 or 2 amino acid differences therefrom, and

[0219] A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155, or a sequence having 1 or 2 amino acid differences therefrom.

[0220] In some embodiments, the antibody or antigen-binding fragment comprises:

[0221] (a) a VH region comprising:

[0222] a HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141 or 149,

[0223] a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, and

[0224] a HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151; and

[0225] (b) a VL region comprising:

[0226] LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165,

[0227] LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, and

[0228] A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155.

[0229] In some embodiments, the antibody or antigen-binding fragment comprises:

[0230] (a)(i) a VH region comprising:

[0231] A HCDR1 having the amino acid sequence of SEQ ID NO: 133 or a sequence having 1 or 2 amino acid differences therefrom,

[0232] a HCDR2 having the amino acid sequence of SEQ ID NO: 134 or a sequence having 1 or 2 amino acid differences therefrom, and

[0233] A HCDR3 having the amino acid sequence of SEQ ID NO: 135, or a sequence having 1 or 2 amino acid differences therefrom; and

[0234] (ii) a VL region comprising:

[0235] LCDR1 having the amino acid sequence of SEQ ID NO: 137 or a sequence having 1 or 2 amino acid differences therefrom,

[0236] LCDR2 having the amino acid sequence of SEQ ID NO: 138 or a sequence having 1 or 2 amino acid differences therefrom, and

[0237] LCDR3 having the amino acid sequence of SEQ ID NO: 139, or a sequence having 1 or 2 amino acid differences therefrom; or

[0238] (b)(i) a VH region comprising:

[0239] A HCDR1 having the amino acid sequence of SEQ ID NO: 141 or a sequence having 1 or 2 amino acid differences therefrom,

[0240] a HCDR2 having the amino acid sequence of SEQ ID NO: 142, or a sequence having 1 or 2 amino acid differences therefrom, and

[0241] A HCDR3 having the amino acid sequence of SEQ ID NO: 143, or a sequence having 1 or 2 amino acid differences therefrom; and

[0242] (ii) a VL region comprising:

[0243] LCDR1 having the amino acid sequence of SEQ ID NO: 145 or a sequence having 1 or 2 amino acid differences therefrom,

[0244] LCDR2 having the amino acid sequence of SEQ ID NO: 146 or a sequence having 1 or 2 amino acid differences therefrom, and

[0245] LCDR3 having the amino acid sequence of SEQ ID NO: 147, or a sequence having 1 or 2 amino acid differences therefrom; or

[0246] (c)(i) a VH region comprising:

[0247] A HCDR1 having the amino acid sequence of SEQ ID NO: 149 or a sequence having 1 or 2 amino acid differences therefrom,

[0248] a HCDR2 having the amino acid sequence of SEQ ID NO: 150 or 160, or a sequence having 1 or 2 amino acid differences therefrom, and

[0249] A HCDR3 having the amino acid sequence of SEQ ID NO: 151, or a sequence having 1 or 2 amino acid differences therefrom; and

[0250] (ii) a VL region comprising:

[0251] LCDR1 having the amino acid sequence of SEQ ID NO: 153 or 165, or a sequence having 1 or 2 amino acid differences therefrom,

[0252] LCDR2 having the amino acid sequence of SEQ ID NO: 154, or a sequence having 1 or 2 amino acid differences therefrom, and

[0253] A LCDR3 having the amino acid sequence of SEQ ID NO: 155, or a sequence having 1 or 2 amino acid differences therefrom.

[0254] In some embodiments, the antibody or antigen-binding fragment comprises:

[0255] (a)(i) a VH region comprising:

[0256] a HCDR1 having the amino acid sequence of SEQ ID NO: 133, a HCDR2 having the amino acid sequence of SEQ ID NO: 134, and a HCDR3 having the amino acid sequence of SEQ ID NO: 135; and (ii) a VL region comprising:

[0257] a LCDR1 having the amino acid sequence of SEQ ID NO: 137, a LCDR2 having the amino acid sequence of SEQ ID NO: 138, and a LCDR3 having the amino acid sequence of SEQ ID NO: 139; or (b)(i) a VH region comprising:

[0258] a HCDR1 having the amino acid sequence of SEQ ID NO: 141, a HCDR2 having the amino acid sequence of SEQ ID NO: 142, and a HCDR3 having the amino acid sequence of SEQ ID NO: 143; and (ii) a VL region comprising:

[0259] a LCDR1 having the amino acid sequence of SEQ ID NO: 145, a LCDR2 having the amino acid sequence of SEQ ID NO: 146, and a LCDR3 having the amino acid sequence of SEQ ID NO: 147; or (c)(i) a VH region comprising:

[0260] a HCDR1 having the amino acid sequence of SEQ ID NO: 149,

[0261] a HCDR2 having the amino acid sequence of SEQ ID NO: 150 or 160, and

[0262] a HCDR3 having the amino acid sequence of SEQ ID NO: 151; and

[0263] (ii) a VL region comprising:

[0264] LCDR1 having the amino acid sequence of SEQ ID NO: 153 or 165,

[0265] LCDR2 having the amino acid sequence of SEQ ID NO: 154, and

[0266] LCDR3 having the amino acid sequence of SEQ ID NO:155.

[0267] In some embodiments, the antibody or antigen-binding fragment comprises:

[0268] (a) a VH region comprising:

[0269] (i) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 133, 134 and 135, respectively;

[0270] (ii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 141, 142 and 143, respectively;

[0271] (iii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 149, 150 and 151, respectively; or

[0272] (iv) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 149, 160 and 151, respectively; and

[0273] (b) a VL region comprising:

[0274] (i) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively;

[0275] (ii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 145, 146 and 147, respectively;

[0276] (iii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 153, 154 and 155, respectively; or

[0277] (iv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 165, 154 and 155, respectively.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] In some embodiments, the antibody is a humanized antibody.

[0283] In another aspect, an antibody or antigen-binding fragment thereof capable of binding to siglec-6 is provided, comprising:

[0284] (a) a heavy chain variable domain (VH) comprising:

[0285] a heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 3,

[0286] a HCDR2 having the amino acid sequence of SEQ ID NO: 21, and

[0287] a HCDR3 having the amino acid sequence of SEQ ID NO: 5; and

[0288] (a) a light chain variable domain (VL) comprising:

[0289] a light chain complementary determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 32,

[0290] LCDR2 having the amino acid sequence of SEQ ID NO: 12, and

[0291] LCDR3 having the amino acid sequence of SEQ ID NO:13.

[0292] 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.

[0293] In some embodiments, the antibody or antigen-binding fragment comprises: a VH region comprising an amino acid sequence of SEQ ID NO: 20, 23, 25, 27, or 29; and a VL region comprising an amino acid sequence of SEQ ID NO: 31, 36, or 38.

[0294] 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;

[0295] (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;

[0296] (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

[0297] (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.

[0298] 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.

[0299] In some embodiments, the antibody or antigen-binding fragment can be expressed with a K of up to about 10 nM, up to about 5 nM, or up to about 1 nM. D Value binding to siglec-6. In some embodiments, the antibody or antigen-binding fragment thereof (a) is capable of binding to human siglec-6; (b) is capable of binding to cynomolgus monkey siglec-6; (c) is capable of binding to mast cells, optionally preferentially over another immune cell (e.g., T cells, dendritic cells, macrophages, neutrophils or basophils); (d) does not induce mast cell degranulation; and / or (e) has a comparable or lower internalization rate than a reference antibody capable of binding to siglec-6, optionally wherein 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.

[0300] In another aspect, a humanized antibody or antigen-binding fragment thereof capable of binding to c-Kit is provided, comprising:

[0301] (a) a heavy chain variable domain (VH) comprising:

[0302] A heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 41 or a sequence having 1 amino acid difference therefrom,

[0303] a HCDR2 having the amino acid sequence of SEQ ID NO: 53 or a sequence having 1 amino acid difference therefrom, and

[0304] A HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence having 1 amino acid difference therefrom; and

[0305] (a) a light chain variable domain (VL) comprising:

[0306] A light chain complementary determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 46 or a sequence having 1 amino acid difference therefrom,

[0307] LCDR2 having the amino acid sequence of SEQ ID NO: 47 or a sequence having 1 amino acid difference therefrom, and

[0308] A LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence having 1 amino acid difference therefrom.

[0309] In some embodiments, the antibody or antigen-binding fragment comprises:

[0310] (a) a VH region comprising:

[0311] a HCDR1 having the amino acid sequence of SEQ ID NO:41,

[0312] a HCDR2 having the amino acid sequence of SEQ ID NO:53, and

[0313] a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and

[0314] (b) a VL region comprising:

[0315] LCDR1 having the amino acid sequence of SEQ ID NO:46,

[0316] LCDR2 having the amino acid sequence of SEQ ID NO:47, and

[0317] LCDR3 having the amino acid sequence of SEQ ID NO:48.

[0318] In some embodiments, VH comprises:

[0319] (a) a HCDR1 having the amino acid sequence of SEQ ID NO: 60, 61 or 62,

[0320] (b) a HCDR2 having the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67 or 68, and

[0321] (c) a HCDR3 having the amino acid sequence of SEQ ID NO: 69, 70, 71 or 72.

[0322] In some embodiments, VL comprises:

[0323] (a) LCDR1 having the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94 or 95,

[0324] (b) LCDR2 having the amino acid sequence of SEQ ID NO: 96 or 97, and

[0325] (c) LCDR3 having the amino acid sequence of SEQ ID NO: 98, 99, 100, 101, 102, 103 or 104.

[0326] In some embodiments,

[0327] (a) The VH region comprises:

[0328] (i) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 43, respectively;

[0329] (ii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 60, 53 and 43, respectively;

[0330] (iii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 61, 53 and 43, respectively;

[0331] (iv) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 62, 53 and 43, respectively;

[0332] (v) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 63 and 43, respectively;

[0333] (vi) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 64 and 43, respectively;

[0334] (vii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 65 and 43, respectively;

[0335] (viii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 66 and 43, respectively;

[0336] (ix) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 67 and 43, respectively;

[0337] (x) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 68 and 43, respectively;

[0338] (xi) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 69, respectively;

[0339] (xii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 70, respectively;

[0340] (xiii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 71, respectively; or

[0341] (xiv) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 72, respectively; and

[0342] (b) The VL region comprises:

[0343] (i) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively;

[0344] (ii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 90, 47 and 48, respectively;

[0345] (iii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 91, 47 and 48, respectively;

[0346] (iv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 92, 47 and 48, respectively;

[0347] (v) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 93, 47 and 48, respectively;

[0348] (vi) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 94, 47 and 48, respectively;

[0349] (vii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 95, 47 and 48, respectively;

[0350] (viii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 96 and 48, respectively;

[0351] (ix) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 97 and 48, respectively;

[0352] (x) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 98, respectively;

[0353] (xi) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 99, respectively;

[0354] (xii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 100, respectively;

[0355] (xiii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 101, respectively;

[0356] (xiv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 102, respectively;

[0357] (xv) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 103, respectively; or

[0358] (xvi) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 104, respectively.

[0359] 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: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 NO:87, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119.

[0360] In some embodiments, the VH region 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; and the VL region 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.

[0361] In some embodiments,

[0362] (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;

[0363] (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;

[0364] (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;

[0365] (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;

[0366] (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;

[0367] (f) 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: 89; or

[0368] (g) 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:89.

[0369] In some embodiments, 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.

[0370] In some embodiments, the antibody or antigen-binding fragment thereof can be expressed with a K between about 10 and about 500 nM, between about 20 and about 100 nM; or between about 25 and about 80 nM. D In some embodiments, the antibody or antigen-binding fragment thereof (a) is capable of binding to human c-Kit; (b) is capable of binding to cynomolgus monkey c-Kit; and / or (c) has comparable or lower toxicity than a reference antibody capable of binding to c-Kit, optionally wherein the reference antibody 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.

[0371] In some embodiments, the antigen-binding fragment is Fab, F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, Fd fragment or diabody.

[0372] In some embodiments, the antibody or its antigen-binding fragment comprises an antibody heavy chain constant region, optionally wherein the constant region is a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region is an IgG1 heavy chain constant region. In some embodiments, the constant region comprises one or more mutations that reduce or eliminate effector function. In some embodiments, the constant region comprises one or more mutations that enhance effector function. In some embodiments, at least one of the first and second heavy chains is afucosylated (e.g., at least 75% afucosylated).

[0373] In some embodiments, the antibodies or antigen-binding fragments described herein are conjugated to a therapeutic agent or a label.

[0374] In another aspect, polynucleotides encoding antibodies or antigen-binding fragments thereof described herein are provided. In another aspect, expression vectors comprising the polynucleotides are provided. In another aspect, host cells comprising the polynucleotides or the expression vectors are provided. In some embodiments, the host cell is a mammalian or insect cell. In some embodiments, the host cell (a) comprises a Fut8 knockout; and / or (b) overexpresses GnT-III and optionally overexpresses ManII.

[0375] In another aspect, provided is a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein.

[0376] In another aspect, a method for treating a disease or condition in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof described herein or a pharmaceutical composition described herein. In some embodiments, the disease or condition is a mast cell-related disease or condition. In some embodiments, the disease or condition is an autoimmune disease, allergy, inflammation, itching, or cancer. In some embodiments, the disease or condition is associated with the expression of siglec-6 and / or c-Kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0377] Figure 1 Depicted is the inhibition of stem cell factor (SCF)-dependent cell proliferation in M-07e cells by SR1 c-Kit antibody.

[0378] Figure 2 Depicted is the inhibition of SCF-dependent cell proliferation in M-07e cells by c-Kit / siglec 6 bispecific antibody.

[0379] Figure 3 Humanized siglec-6 antibody hRND-3 with normal, effector-silent, or effector-enhanced Fc domains was shown to not induce mast cell degranulation as measured by β-hexosaminidase release. IFNγ treatment of mast cells to upregulate FcγRI did not result in degranulation by siglec-6 antibody. FcεRIα cross-linking antibody plus SCF and PMA / ionomycin treatment served as positive controls. DETAILED DESCRIPTION

[0380] definition

[0381] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0382] As used herein, when used in this article to refer to a value, the terms "about", "approximately" and "equivalent thereto" refer to a value similar to the reference value in the context of a reference value. Generally speaking, those skilled in the art who are familiar with the context will understand the relevant degree of variation covered by "about", "approximately" and "equivalent thereto" in this context. For example, in some embodiments, the terms "about", "approximately" and "equivalent thereto" can be covered in 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 value mentioned.

[0383] As used herein, the terms "antagonize," "neutralize," or "block" when used in reference to an antibody or antigen-binding fragment thereof, are intended to mean that binding of the antibody or fragment thereof to its target results in inhibition of at least some biological activity of the target.

[0384] As used herein, "antibody" refers to a polypeptide whose amino acid sequence includes an immunoglobulin and a fragment thereof that specifically binds to a specified antigen, or a fragment thereof. The antibody 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). It will be appreciated by those of ordinary skill in the art that the characteristic sequence or portion of an antibody may include amino acids found in one or more regions of the antibody (e.g., variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof). In addition, it will be appreciated by those of ordinary skill in the art that the characteristic sequence or portion of an antibody may include one or more polypeptide chains, and may include sequence elements found in the same polypeptide chain or in different polypeptide chains.

[0385] As used herein, "antibody mimics" refers to any molecule that can simulate the ability of an antibody to bind to an antigen, but is not limited to the antibody structure. Examples of antibody mimics include, but are not limited to, Affibodies, Affilins, Affimers, Afftins, Alphabodies, Anticalins, Avimers, Centyrins, DARPins, Fynomers, monobodies, and nanoCLAMPs.

[0386] An "antigen-binding fragment" of an antibody comprises a portion of an intact antibody that is still capable of binding antigen. In certain embodiments, papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. The Fab fragment consists of an entire light chain and the variable region domain (V H ) and the first constant domain of a heavy chain (C H 1) composition. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. In certain embodiments, treatment of an antibody with pepsin produces a single large F(ab')2 fragment that roughly corresponds to two disulfide-linked Fab fragments with different antigen binding activities and is still capable of cross-linking antigen. The Fab' fragment differs from the Fab fragment in that it has a single C HA few residues are added to the carboxyl terminus of the constant domain, including one or more cysteines from the antibody hinge region. Fab'-SH denotes Fab' in which the cysteine ​​residue(s) of the constant domain have a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0387] As used herein, the term "antigen binding site" refers to any molecule or any part of a molecule that can bind to an antigen. In human antibodies, the antigen binding site is formed by amino acid residues of the N-terminal variable region ("V") of the heavy chain ("H") and the light chain ("L"). The three highly divergent segments within the heavy and light chain V regions are called "hypervariable regions", which are located between more conservative flanking segments called "framework regions" or "FRs". Therefore, the term "FR" refers to the amino acid sequence naturally occurring between and adjacent to the hypervariable regions of immunoglobulins. In human antibodies, 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 the three hypervariable regions of the heavy chain and the light chain are each called "complementarity determining region" or "CDR". In some animals, such as camels and cartilaginous fish, the antigen binding site may be formed by a single antibody chain, thereby providing a "single domain antibody". The antigen binding site may also be an antibody mimetic or present in an antibody mimetic.

[0388] CDR 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 according to these definitions usually include overlaps or subsets 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, 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 CDR and light chain CDR of an antibody are defined using different conventions. For example, in certain embodiments, the heavy chain CDR is defined according to MacCallum (supra) and the light chain CDR is defined according to Kabat (supra). CDRH1, CDRH2 and CDRH3 represent heavy chain CDRs, CDRL1, CDRL2 and CDRL3 represent light chain CDRs.

[0389] As used herein, the "binding affinity" of a molecule (e.g., antigen binding site) of the present disclosure to a given target (e.g., FCER1 or c-Kit) can be determined by a variety of methods known to those skilled 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).

[0390] As used herein, "bispecific" refers to a molecule that is capable of specifically binding to at least two different targets. Typically, a bispecific molecule comprises two antigen binding sites, each site being specific for a different target. In some embodiments, a bispecific molecule is capable of binding to two targets simultaneously.

[0391] As used herein, unless otherwise specified, "c-Kit" refers to human c-Kit as defined by UniProt P10721, including variants, isoforms and species homologs of human c-Kit. c-Kit is also known as "KIT", "CD117", "mast cell / stem cell growth factor receptor Kit", "SCFR", "proto-oncogene c-Kit" or "tyrosine protein kinase Kit", which are used interchangeably.

[0392] As used herein, "detectable affinity" refers to the ability to bind a given target with an affinity constant, typically expressed as K. D or EC 50 The affinity constant is about 10 at most. -5 M or lower (lower K D or EC 50 Lower affinities cannot be measured by common methods such as ELISA (enzyme-linked immunosorbent assay) and are therefore of secondary significance.

[0393] As used herein, the term "epitope" is an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule, which is called a paratope and consists of the six complementary determining regions of an antibody. A single antigen can have more than one epitope. Epitopes may be conformational or linear. Conformational epitopes consist of spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes consist of adjacent amino acid residues in a polypeptide chain.

[0394] The "Fc" fragment contains the carboxyl-terminal portions of two heavy chains linked together by disulfide bonds. The effector functions of an antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcR) on certain cell types.

[0395] "Fragment" with respect to an antigen (e.g., siglec-6 or c-Kit) refers to an N-terminal and / or C-terminal truncation or protein domain of the antigen. In some embodiments, a fragment of an antigen retains the ability of the full-length antigen to be recognized and / or bound by an antibody or antigen-binding fragment thereof of the present disclosure.

[0396] As used herein, "fucosylation" or "fucosylated" refers to the presence of fucose residues within oligosaccharides attached to the antibody peptide backbone. Specifically, a fucosylated antibody comprises α(l,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or two N-linked oligosaccharides attached to the Fc region of the antibody. "Non-fucosylated," "afucosylated," or "fucose-deficient" antibodies refer to glycosylated antibody variants comprising an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose. In some embodiments, the non-fucosylated or fucose-deficient antibodies have reduced fucose relative to the amount of fucose on the same antibody produced in a cell line. In some embodiments, antibodies with reduced fucose or lack of fucose have improved ADCC function.

[0397] The "degree of fucosylation" is the percentage of fucosylated oligosaccharides relative to all oligosaccharides, which can be identified by methods known in the art, such as by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in an N-glycosidase F-treated antibody composition. In a composition of a "fully fucosylated antibody", substantially all oligosaccharides contain fucose residues, i.e., are fucosylated. In some embodiments, a composition of a fully fucosylated antibody has a degree of fucosylation of at least about 90%. In contrast, in a composition of a "completely non-fucosylated antibody", substantially no oligosaccharides are fucosylated. In some embodiments, a composition of a completely non-fucosylated antibody has a degree of fucosylation of less than about 10%. In a composition of a "partially fucosylated antibody", only a portion of the oligosaccharides contain fucose. The individual antibodies in such compositions may not contain fucose residues in the N-linked oligosaccharides in the Fc region, contain fucose residues in one N-linked oligosaccharide in the Fc region, or contain fucose residues in two N-linked oligosaccharides in the Fc region, provided that the composition does not contain substantially all single antibodies lacking fucose residues in the N-linked oligosaccharides in the Fc region, nor does it contain substantially all single antibodies containing fucose residues in both N-linked oligosaccharides in the Fc region. In one embodiment, the 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 and methods and cell lines for producing antibodies with altered, reduced or eliminated fucosylation are known in the art (see, e.g., WO 2023 / 044390A1).

[0398] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, each antibody constituting the population is identical, except for possible naturally occurring mutations that may be present in trace amounts. Monoclonal antibodies have a high degree of specificity (for a single antigenic site). In addition, compared with conventional (polyclonal) antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody is for a single determinant on the antigen. The modifier "monoclonal" indicates the characteristics of the antibody obtained from a substantially homogeneous antibody population and should not be interpreted as requiring the antibody to be produced by any ad hoc method. For example, monoclonal antibodies can be made by a hybridoma method, or can be made by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibody" can also be separated from a phage antibody library.

[0399] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use, either in vivo or ex vivo.

[0400] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound described herein, which, after administration to a subject, can provide a compound described herein or its active metabolite or residue. As known to those skilled in the art, the "salts" of the compounds described herein can be derived from inorganic or organic acids and bases.

[0401] 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, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Other acids, such as oxalic acid, while not pharmaceutically acceptable in themselves, may be used to prepare salts useful as intermediates to obtain the compounds described herein and their pharmaceutically acceptable acid addition salts.

[0402] Exemplary bases include, but are not limited to, alkali metal (eg, sodium) hydroxides, alkaline earth metal (eg, magnesium) hydroxides, ammonia, and compounds of formula NW4 + Compounds (wherein W is C 1-4 Alkyl) etc.

[0403] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and the like. Other examples of salts include anions of the compounds described herein with suitable cations such as Na + NH4 + and NW4 + (W is C 1-4 Alkyl) and other compounds.

[0404] For therapeutic use, salts of the compounds described herein are considered pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0405] As used herein, unless otherwise indicated, "siglec-6" refers to human siglec-6 defined by UniProt O43699, including variants, isoforms and species homologs of human siglec-6. Siglec-6 is also known 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", which are used interchangeably.

[0406] 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., rodents, apes, equines, bovines, porcines, canines, felines, etc.), and more preferably include humans.

[0407] As used herein, the phrases "therapeutically effective amount" and "effective amount" are used interchangeably and refer to an amount effective to achieve the desired therapeutic outcome at the necessary dosage and for the necessary period of time. The therapeutically effective amount may vary depending on a variety of factors, such as the type of disease (e.g., cancer), the disease state, 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. An effective amount may also be an amount in which any toxic or deleterious effects of the immunoconjugate or its pharmaceutical composition are offset by the therapeutically beneficial effects.

[0408] As used herein, "treating" a condition or "treatment" of the condition (e.g., a condition described herein, such as cancer) is a method of obtaining a beneficial or desired result (e.g., a clinical result). Beneficial or desired results may include, but are not limited to, alleviation or improvement of one or more symptoms or conditions; reduction in the extent of a disease, disorder, or condition; stabilization (i.e., not worsening) of the state of a disease, disorder, or condition; prevention of the spread of a disease, disorder, or condition (e.g., primary cancer and / or secondary metastasis); delay or slowing of the progression of a disease, disorder, or condition; improvement or palliation of a disease, disorder, or condition; and partial or complete and detectable or undetectable remission.

[0409] Antigen binding site

[0410] Capable of binding to the antigen binding site of c-Kit

[0411] In another aspect, the present application provides an antigen binding site capable of binding to c-Kit.

[0412] In some embodiments, an antigen binding site capable of binding to c-Kit is provided, present in the form of an antibody mimetic. In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to c-Kit and present as an antibody mimetic as described herein, wherein such antigen binding site 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 an antigen binding site capable of binding to c-Kit and present as an antibody mimetic as described herein.

[0413] In some embodiments, an antigen binding site capable of binding to c-Kit is provided, present in the form of an antibody (e.g., a bispecific antibody). In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to c-Kit and present as an antibody described herein, wherein such antigen binding site 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 comprised by the antigen binding site capable of binding to c-Kit and present as an antibody described herein.

[0414] In some embodiments, an antigen binding site capable of binding to c-Kit is provided that 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, an antigen binding site capable of binding to c-Kit is provided that comprises VH and VL, wherein VH comprises CDR-H1, CDR-H2 and CDR-H3, VL comprises CDR-L1, CDR-L2 and CDR-L3, and wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are each independently selected from those of VH or VL described in Table 10. The CDRs of VH and VL are determined according to 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 of which is independently selected from those described in Table 10. In some embodiments, an antigen binding site is provided that is a variant of an antigen binding site described herein that is capable of binding to c-Kit, wherein such antigen binding site has a CDR sequence that differs from each of the CDR sequences described in Table 10 by no more than two amino acid residues (e.g., two or one amino acid residue). In some embodiments, an antigen binding site is provided that is a variant of an antigen binding site described herein that is capable of binding to c-Kit, wherein such antigen binding site has a set of six CDRs whose sequences, as a whole, differ from a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 selected from Table 10 by no more than two amino acid residues (e.g., two or one amino acid residue). In some embodiments, an antigen binding site is provided that is a variant of an antigen binding site described herein that is capable of binding to c-Kit, wherein such antigen binding site has a set of six CDRs whose sequences generally differ by no more than two amino acid residues (e.g., two or one amino acid residue) from the sequence of an anti-c-Kit having a set of VH and VL selected from Table 10.

[0415] In some embodiments, an antigen binding site capable of binding to c-Kit is provided comprising a VH sequence as described in Table 10 and a VL sequence as described in Table 10. In some embodiments, an antigen binding site is provided, which is an antigen binding site described herein that is capable of binding to c-Kit, such as an anti-c-Kit variant having a set of VH and VL selected from Table 10, wherein such 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 described 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 described in Table 10.

[0416] In some embodiments, an antigen binding site capable of binding to c-Kit is provided comprising a heavy chain sequence as described in Table 10 and a light chain sequence as described in Table 10. In some embodiments, an antigen binding site is provided, which is an antigen binding site described herein that is capable of binding to c-Kit, such as an anti-c-Kit variant having a set of heavy chains and light chains selected from Table 10, wherein such 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 described 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 described in Table 10.

[0417] In certain embodiments, the antigen binding sites described herein are derived from murine SR1. For example, in certain embodiments, the antigen binding sites described herein comprise 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 having VH and VL sequences of SEQ ID NOs: 40 and 45, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2 and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively. In certain embodiments, VL comprises CDR-L1, 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 CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively.

[0418] In certain embodiments, the antigen binding site described herein is derived from humanized SR1-1. For example, in certain embodiments, the antigen binding site described herein 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: 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 having the VH and VL sequences of SEQ ID NO: 52 and 87, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, 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, VL comprises CDR-L1, 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) 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) VL comprising CDR-L1, CDR-L2 and CDR-L3 comprising the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively.

[0419] In certain embodiments, the humanized SR1-1 variant comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, each of which differs by no more than one amino acid from CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 52 and 87, respectively. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3, each of which differs by no more than one amino acid from SEQ ID NOs: 41, 53, and 43, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3, each of which differs by no more than one amino acid from SEQ ID NOs: 46, 47, and 48, respectively. In certain embodiments, the humanized SR1-1 variant comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, whose sequences generally differ by at least one amino acid residue (e.g., one, two, or three) from CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art, of the VH and VL sequences of SEQ ID NOs: 52 and 87, respectively. In certain embodiments, the VH comprises CDR-H1, CDR-H2, and CDR-H3, whose sequences generally differ by at least one amino acid residue from SEQ ID NOs: 41, 53, and 43, respectively. In certain embodiments, the VL comprises CDR-L1, CDR-L2, and CDR-L3, whose sequences generally differ by at least one amino acid residue from SEQ ID NOs: 46, 47, and 48, respectively. 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.

[0420] In certain embodiments, the antigen binding site described herein is derived from humanized SR1-2. For example, in certain embodiments, the antigen binding site described herein 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: 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: 87. In certain embodiments, the antigen binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having VH and VL sequences of SEQ ID NOs: 55 and 87, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0421] In certain embodiments, the antigen binding sites described herein are derived from humanized SR1-3. For example, in certain embodiments, the antigen binding sites described herein comprise 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 having VH and VL sequences of SEQ ID NOs: 57 and 87, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0422] In certain embodiments, the antigen binding sites described herein are derived from humanized SR1-4. For example, in certain embodiments, the antigen binding sites described herein comprise 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having VH and VL sequences of SEQ ID NOs: 59 and 87, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0423] In certain embodiments, the antigen binding sites described herein are derived from humanized SR1-5. For example, in certain embodiments, the antigen binding sites described herein comprise 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 site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having VH and VL sequences of SEQ ID NOs: 52 and 89, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0424] In certain embodiments, the antigen binding sites described herein are derived from humanized SR1-6. For example, in certain embodiments, the antigen binding sites described herein comprise 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 having VH and VL sequences of SEQ ID NOs: 55 and 89, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0425] In each of the above embodiments, it is contemplated herein that the VH and / or VL sequences that bind to c-Kit may contain 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 VH and / or VL without affecting their ability to bind to c-Kit. For example, it is contemplated herein that the VH and VL sequences that bind to c-Kit may contain cysteine ​​heterodimerization mutations, thereby facilitating the formation of disulfide bridges between VH and VL to form scFv.

[0426] In certain embodiments, the antigen binding sites disclosed herein have a K less than or equal to DValues ​​(affinity greater than or equal to) bind to human c-Kit: 900uM, 500uM, 250uM, 100uM, 50uM, 25uM, 10uM, 9uM, 8uM, 7uM, 6uM, 5uM, 4uM, 3uM, 2uM, 1uM, 900nM, 800nM, 700nM, 600nM, 500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 45nM, 40nM, 35nM, In some embodiments, the antigen binding site of the present application has a K of greater than or equal to 1 pM. D Values ​​(affinity less than or equal to) bind to human c-Kit: 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 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 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, In some embodiments, the antigen binding sites of the present application have a K in the range of picomolar to micromolar. D In certain embodiments, the antigen binding site of the present application has a K value within the following range: DValues ​​bind to human c-Kit: about 1pM to about 900uM, 1pM to about 500uM, 1pM to about 100uM, 1pM to about 50uM, 1pM to about 10uM, 1pM to about 5uM, 1pM to about 1uM, 1pM to about 500nM, 1pM to about 100nM, 1pM to about 50nM, 1pM to about 10nM, 1pM to about 5nM, 1pM to about 1nM, 1pM to about 500pM, 1pM to about 100pM, 1pM to about 50pM, 1pM to about 10pM, 1pM to about 5pM, 5pM to about 900uM, 5pM to about 500uM, 5pM to about 100uM, 5pM to about 50uM, 5pM to about 10uM, 5pM to about 10uM, 5pM to about 5 10pM to about 900uM, 10pM to about 500uM, 10pM to about 100uM, 10pM to about 50uM, 10pM to about 10uM, 10pM to about 500nM, 5pM to about 100nM, 5pM to about 50nM, 5pM to about 10nM, 5pM to about 5nM, 5pM to about 10nM, 5pM to about 500pM, 5pM to about 100pM, 5pM to about 50pM, 5pM to about 10pM, 10pM to about 900uM, 10pM to about 500uM, 10pM to about 100uM, 10pM to about 50uM, 10pM to about 10uM, 10pM to about 50uM, 10pM to about 10uM, 10pM to about 500nM, 10pM to about 100nM, 10pM to about 50nM, 10pM to about 10nM, 10pM to about 5nM, 10pM to about 1nM, 10pM to about 500pM, 10pM to about 100pM, 10pM to about 50pM, 50pM to about 900uM, 50pM to about 500uM, 50pM to about 100uM, 50pM to about 50uM, 50pM to about 10uM, 50pM to about 5uM, 50pM to about 1uM, 50pM to about 500nM, 50pM to about 100nM, 50pM to about 50nM, 50pM to about 10nM, 50pM to about 5nM, 50pM to about 1nM, 50pM to about 500pM, 50pM to about 100pM, 100pM to about 900uM, 100pM to about 500uM, 100pM From about 100uM to about 100pM, from about 100pM to about 50uM, from about 100pM to about 10uM, from about 100pM to about 5uM, from about 100pM to about 1uM, from about 100pM to about 500nM, from about 100pM to about 100nM, from about 100pM to about 50nM, from about 100pM to about 10nM, from about 100pM to about 5nM, from about 100pM to about 1nM, from about 100pM to about 500pM, from about 500pM to about 500uM, from about 500pM to about 100uM, from about 500pM to about 50uM, from about 500pM to about 10uM, from about 500pM to about 5uM, from about 500pM to about 1uM, from about 500pM to about 500nM, from about 500pM to about 100nM, from about 500pM to about 50nM,500pM to about 10nM, 500pM to about 5nM, 500pM to about 1nM, 1nM to about 500uM, 1nM to about 100uM, 1nM to about 50uM, 1nM to about 10uM, 1nM to about 5uM, 1nM to about 1uM, 1nM to about 500nM, 1nM to about 100nM, 1nM to about 50nM, 1nM to about 10nM, 1nM to about 5nM, 5nM to about 100uM, 5nM to about 50uM, 5nM to about 10uM, 5nM to about 5uM, 5nM to about 1uM, 5nM to about 500nM, 1nM to about 100nM, 1nM to about 50nM, 1nM to about 10nM, 1nM to about 5nM, 5nM to about 100uM, 5nM to about 50uM, 5nM to about 10uM, 5nM to about 5uM, 5nM to about 1uM, 5nM to about 500nM, 5nM to about 100nM, 5nM to about 50nM, 5nM to about 10 In some embodiments, K, K, 10nM to about 10uM, 10nM to about 5uM, 10nM to about 1uM, 10nM to about 500nM, 10nM to about 100nM, 10nM to about 50nM, 50nM to about 10uM, 50nM to about 5uM, 50nM to about 1uM, 50nM to about 500nM, 50nM to about 100nM, 100nM to about 10uM, 100nM to about 5uM, 100nM to about 1uM, 100nM to about 500nM, 500nM to about 10uM, 500nM to about 5uM, 500nM to about 1uM, 1uM to about 10uM, 1uM to about 5uM, or 5uM to about 10uM are measured using a standard binding assay (e.g., biolayer interferometry). D value.

[0427] In some embodiments, the antigen binding sites disclosed herein bind to human c-Kit with an affinity comparable to that of human c-Kit. D In some embodiments, the affinity of the antigen binding site for cynomolgus c-Kit relative to its affinity for human c-Kit measured in the same assay is within 2-fold, 3-fold, 5-fold, or 10-fold (greater affinity for human c-Kit or greater affinity for cynomolgus c-Kit).

[0428] In some embodiments, the antigen binding sites disclosed herein bind to c-Kit expressed on the surface of a cell.

[0429] 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.

[0430] Capable of binding to the antigen binding site of siglec-6

[0431] In another aspect, the present application provides an antigen binding site capable of binding to siglec-6.

[0432] In some embodiments, an antigen binding site capable of binding to siglec-6 is provided, present in the form of an antibody mimetic. In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to siglec-6 and present as an antibody mimetic as described herein, wherein such an antigen binding site 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 capable of binding to siglec-6 and present as an antibody mimetic as described herein.

[0433] In some embodiments, an antigen binding site capable of binding to siglec-6 is provided, present in the form of an antibody (e.g., a bispecific antibody). In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to siglec-6 and present as an antibody described herein, wherein such an antigen binding site 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 comprised by the antigen binding site capable of binding to siglec-6 and present as an antibody described herein.

[0434] In some embodiments, an antigen binding site capable of binding to siglec-6 is provided that 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.

[0435] In some embodiments, an antigen binding site capable of binding to siglec-6 is provided comprising VH and VL, wherein VH comprises CDR-H1, CDR-H2 and CDR-H3, and VL comprises CDR-L1, CDR-L2 and CDR-L3, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are each independently selected from those of VH or VL described in Table 9. The CDRs of VH and VL are determined according to 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 of which is independently selected from those described in Table 9. In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to siglec-6 as described herein, wherein such antigen binding site has a CDR sequence that differs from each of the CDR sequences described in Table 9 by no more than two amino acid residues (e.g., two or one amino acid residue). In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to siglec-6 as described herein, wherein such antigen binding site has a set of six CDRs, whose sequences generally differ from a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 9 by no more than two amino acid residues (e.g., two or one amino acid residue). In some embodiments, an antigen binding site is provided, which is a variant of an antigen binding site capable of binding to siglec-6 as described herein, wherein such antigen binding site has a set of six CDRs, whose sequences generally differ from an anti-siglec-6 sequence having a set of VH and VL selected from Table 9 by no more than two amino acid residues (e.g., two or one amino acid residue).

[0436] In some embodiments, an antigen binding site capable of binding to siglec-6 is provided comprising a VH sequence as described in Table 9 and a VL sequence as described in Table 9. In some embodiments, an antigen binding site is provided, which is an antigen binding site described herein that is capable of binding to siglec-6, such as an anti-siglec-6 variant having a set of VH and VL selected from Table 9, wherein such 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 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.

[0437] In some embodiments, an antigen binding site capable of binding to siglec-6 is provided comprising a heavy chain sequence as described in Table 9 and a light chain sequence as described in Table 9. In some embodiments, an antigen binding site is provided, which is an antigen binding site described herein that is capable of binding to siglec-6, such as an anti-siglec-6 variant having a set of heavy chains and light chains selected from Table 9, wherein such 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 described 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 described in Table 9.

[0438] In certain embodiments, the antigen binding sites described herein are derived from murine RND. For example, in certain embodiments, the antigen binding sites described herein comprise 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 having VH and VL sequences of SEQ ID NOs: 2 and 10, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2 and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively. In certain embodiments, VL comprises CDR-L1, CDR-L2 and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 11, 12 and 13, 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: 3, 4, and 5, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively.

[0439] In certain embodiments, the antigen binding site described herein is derived from humanized RND-2. For example, in certain embodiments, the antigen binding site described herein 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: 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 having VH and VL sequences of SEQ ID NOs: 23 and 38, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2 and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 3, 21 and 5, respectively. In certain embodiments, VL comprises CDR-L1, CDR-L2 and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 32, 12 and 13, 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: 3, 21, and 5, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 12, or 13, respectively.

[0440] In certain embodiments, the antigen binding site described herein is derived from humanized RND-3. For example, in certain embodiments, the antigen binding site described herein 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: 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having VH and VL sequences of SEQ ID NOs: 25 and 38, respectively, as determined by Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2, and CDR-H3 comprising amino acid sequences of SEQ ID NOs: 3, 21, and 5, respectively.

[0441] In certain embodiments, the antigen binding site described herein is derived from humanized RND-4. For example, in certain embodiments, the antigen binding site described herein 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: 27, 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 having VH and VL sequences of SEQ ID NOs: 27 and 38, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2 and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively. In certain embodiments, VL comprises CDR-L1, CDR-L2 and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 32, 12 and 13, 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: 3, 4, and 5, respectively; and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 32, 12, or 13, respectively.

[0442] In certain embodiments, the antigen binding site described herein is derived from humanized RND-5. For example, in certain embodiments, the antigen binding site described herein 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: 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 site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having VH and VL sequences of SEQ ID NOs: 29 and 38, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0443] In certain embodiments, the antigen binding sites described herein are derived from JML-1. For example, in certain embodiments, the antigen binding sites described herein comprise 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 having VH and VL sequences of SEQ ID NOs: 121 and 126, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0444] In certain embodiments, the antigen binding sites described herein are derived from murine M2. For example, in certain embodiments, the antigen binding sites described herein comprise 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having VH and VL sequences of SEQ ID NOs: 132 and 136, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2, and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 133, 134, and 135, respectively. In certain embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 137, 138, and 139, 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: 133, 134 and 135, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively.

[0445] In certain embodiments, the antigen binding sites described herein are derived from murine M7. For example, in certain embodiments, the antigen binding sites described herein comprise 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 having VH and VL sequences of SEQ ID NOs: 140 and 144, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2 and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 141, 142 and 143, respectively. In certain embodiments, VL comprises CDR-L1, CDR-L2 and CDR-L3, comprising the amino acid sequences of SEQ ID NOs: 145, 146 and 147, 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: 141, 142 and 143, respectively; and (b) a VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 145, 146 and 147, respectively.

[0446] In certain embodiments, the antigen binding sites described herein are derived from murine M11. For example, in certain embodiments, the antigen binding sites described herein comprise 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 having VH and VL sequences of SEQ ID NOs: 148 and 152, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, 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, 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.

[0447] In certain embodiments, the antigen binding site described herein is derived from humanized M11-1. For example, in certain embodiments, the antigen binding site described herein 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: 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 comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 having VH and VL sequences of SEQ ID NOs: 159 and 164, respectively, which are determined according to Kabat, AbM, Chothia or any other CDR determination method known in the art. In certain embodiments, VH comprises CDR-H1, CDR-H2 and CDR-H3, comprising the amino acid sequences of SEQ ID NOs: 149, 160 and 151, respectively. In certain embodiments, VL comprises 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 comprises (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.

[0448] In certain embodiments, the antigen binding site described herein is derived from humanized M11-2. For example, in certain embodiments, the antigen binding site described herein 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 having VH and VL sequences of SEQ ID NOs: 162 and 167, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0449] In certain embodiments, the antigen binding site described herein is derived from humanized M11-3. For example, in certain embodiments, the antigen binding site described herein 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: 169. In certain embodiments, the antigen binding site comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 having VH and VL sequences of SEQ ID NOs: 162 and 169, respectively, which are determined according to Kabat, AbM, Chothia, or any other CDR determination method known in the art.

[0450] In certain embodiments, the antigen binding sites disclosed herein have a K less than or equal to DValues ​​(affinity greater than or equal to) bind to human siglec-6: 900nM, 800nM, 700nM, 600nM, 500nM, 400nM, 300nM, 200nM, 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 15nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 90 In certain embodiments, the antigen binding sites disclosed herein have a K of less than or equal to 10 nM. D In certain embodiments, the antigen binding sites disclosed herein bind to human siglec-6 with a K value of less than or equal to 10 nM. D Values ​​(affinity greater than or equal to) bind to human siglec-6: 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 , 2.4nM, 2.5nM, 2.6nM, 2.7nM, 2.8nM, 2.9nM, 3.0nM, 3.1nM, 3.2nM, 3.3nM, 3.4nM, 3.5nM, 3.6nM, 3.7nM, 3.8nM, 3.9nM, 4.0nM, 4.1nM, 4.2nM, 4.3nM, 4.4nM, 4.5nM, 4.6nM, 4.7nM, 4.8nM, 4.9nM or 5.0nM. In certain embodiments, the antigen binding sites of the present application have a K in the femtomolar to nanomolar range. D In certain embodiments, the antigen binding site of the present application has a K value within the following range: DValues ​​bind to human siglec-6: 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, 5fM to about 5nM, 5fM to about 1nM, 5fM to about 500pM, 5fM to about 100pM, 5fM to about 50pM, 5fM to about 10pM, 5fM to about 5pM, 5fM to about 1pM, 5fM to about 500fM, 5fM to about 100fM, 5fM to about 50fM, 5fM to about 10fM, 10fM to about 900nM, 10fM to about 500nM, 10fM to about 100nM, 10fM to about 50nM, 10fM to about 10nM, 10fM to about 50nM, 10fM to about 10nM, 10fM to about 5nM, 10fM to about 1nM, 10fM to about 500pM, 10fM to about 100pM, 10fM to about 50pM, 10fM to about 10pM, 10fM to about about 5pM, 10fM to about 1pM, 10fM to about 500fM, 10fM to about 100fM, 10fM to about 50fM, 50fM to about 900nM, 50fM to about 500nM, 50fM to about 100nM, 50fM to about 50nM, 50fM to about 10nM, 50fM to about 50nM, 50fM to about 10nM, 50fM to about 5nM, 50fM to about 1nM, 50fM to about 500pM, 50fM to about 100pM, 50fM to about 50pM, 50fM to about 10pM, 50fM to about 5pM, 50fM to about 1pM, 50fM to about 500fM, 50fM to about 100fM, 100fM to about 900nM, 100fM to about 500nM, 100fM To about 100nM, 100fM to about 50nM, 100fM to about 10nM, 100fM to about 5nM, 100fM to about 1nM, 100fM to about 500pM, 100fM to about 100pM, 100fM to about 50pM, 100fM to about 10pM, 100fM to about 5pM, 100fM to about 1pM, 100fM to about 500fM, 500fM to about 900nM, 500fM to about 500nM, 500fM to about 100nM, 500fM to about 50nM, 500fM to about 10nM, 500fM to about 50nM, 500fM to about 10nM, 500fM to about 5nM, 500fM to about 1nM, 500fM to about 500pM, 500fM to about 100pM,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 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 1 In some embodiments, K, K, 10pM to about 10nM, 10pM to about 5nM, 10pM to about 1nM, 10pM to about 500pM, 10pM to about 100pM, 10pM to about 50pM, 50pM to about 10nM, 50pM to about 5nM, 50pM to about 1nM, 50pM to about 500pM, 50pM to about 100pM, 100pM to about 10nM, 100pM to about 5nM, 100pM to about 1nM, 100pM to about 500pM, 50pM to about 10nM, 100pM to about 5nM, 100pM to about 1nM, 100pM to about 500pM, 500pM to about 10nM, 500pM to about 5nM, 500pM to about 1nM, 1nM to about 10nM, 1nM to about 5nM, or 5nM to about 10nM. In some embodiments, K ... D value.

[0451] In some embodiments, the antigen binding sites disclosed herein bind to human siglec-6 with an affinity (K D In some embodiments, the affinity of the antigen binding site for cynomolgus monkey siglec-6 relative to its affinity for human siglec-6 measured in the same assay is within 2-fold, 3-fold, 5-fold, or 10-fold (greater affinity for human siglec-6 or greater affinity for cynomolgus monkey siglec-6).

[0452] In some embodiments, the antigen binding sites disclosed herein bind to siglec-6 expressed on the surface of a cell. In some embodiments, the antigen binding sites disclosed herein bind to mast cells. In some embodiments, the antigen binding sites disclosed herein preferentially bind to mast cells rather than other immune cells (e.g., T cells, dendritic cells, macrophages, neutrophils, or basophils).

[0453] 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 siglec 1-5, 7-12, 14 or 15.

[0454] In some embodiments, the antigen binding sites disclosed herein do not induce mast cell degranulation.

[0455] Molecules containing antigen binding sites

[0456] Also provided herein are molecules containing the disclosed antigen binding sites. Such molecules can be, but are not limited to, antibodies or antigen binding fragments thereof, antibody fragments, nanobodies, antibody mimetics, and the like.

[0457] In some embodiments, antibodies or antigen-binding fragments thereof containing disclosed antigen-binding sites are provided. In some embodiments, the antibody or antigen-binding fragment thereof is multispecific, such as 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 a non-fucosylated antibody (afucosylated antibody). In some embodiments, the antibody is at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1% fucosylated. In some embodiments, the antibody is a non-fucosylated antibody. 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% afucosylated.

[0458] In some embodiments, the antibody or antigen binding fragment thereof is capable of binding to c-Kit. In some embodiments, the antibody or antigen binding fragment thereof is capable of binding to siglec-6. In some embodiments, the antibody or antigen binding fragment thereof is capable of binding to c-Kit and siglec-6.

[0459] In some embodiments, antibody fragments thereof containing the disclosed antigen binding sites are provided. For example, the fragment can be, but is not limited to, Fab, scFab (single-chain Fab), F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, or diabody (dimer of scFv). In some embodiments, the antibody fragment is multispecific, such as bispecific. In some embodiments, the antibody fragment is capable of binding to c-Kit. In some embodiments, the antibody fragment is capable of binding to siglec-6. In some embodiments, the antibody fragment is capable of binding to c-Kit and siglec-6.

[0460] In some embodiments, antibody mimetics containing disclosed antigen binding sites are provided. For example, the fragment can be, but is not limited to, an affinity body, an Affilin, an Affimer, an afftin, an Alphabody, an Anticalin, an avimer, a Centyrin, a DARPin, a Fynomer, a monoclonal antibody, or a nanoCLAMP. In some embodiments, the antibody mimetics are multispecific, such as bispecific. In some embodiments, the antibody mimetics are capable of binding to c-Kit. In some embodiments, the antibody mimetics are capable of binding to siglec-6. In some embodiments, the antibody mimetics are capable of binding to c-Kit and siglec-6.

[0461] In some embodiments, bispecific molecules comprising two antigen binding sites disclosed herein are provided.

[0462] In some embodiments, the bispecific molecule comprises an antigen binding site described herein that is capable of binding to c-Kit. In some embodiments, the bispecific molecule comprises an antigen binding site described herein that is capable of binding to siglec-6. In some embodiments, the bispecific molecule comprises an antigen binding site described herein that is capable of binding to c-Kit and an antigen binding site described herein that is capable of binding to siglec-6.

[0463] In some embodiments, a bispecific molecule is provided, comprising: (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, the 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, the 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, the VH region and the VL region are or are derived from the VH / VL region of a mouse RND, a 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, the VH region and the VL region are or are derived from the VH / VL region of a mouse SR1 or a humanized SR1 as described herein.

[0464] In some embodiments, the bispecific molecule is capable of binding to cells expressing silect-6. In some embodiments, the bispecific molecule is capable of binding to cells expressing c-Kit. In some embodiments, the bispecific molecule is capable of binding to mast cells. In some embodiments, the bispecific molecule preferentially binds to mast cells rather than another immune cell (e.g., T cells, dendritic cells, macrophages, neutrophils or basophils). In some embodiments, the bispecific molecule has an internalization rate comparable to or lower than a reference antigen binding site capable of binding siglect-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 comparable to or lower than a reference antigen binding site capable of binding c-Kit (e.g., an antibody comprising VH and VL of SEQ ID NOs: 40 and 45, respectively).

[0465] Various formats and uses of bispecific molecules are known in the art.The bispecific molecules according to the invention are not limited to any particular bispecific format or method of generating it.

[0466] In some embodiments, the bispecific molecule can be a bispecific antibody or a fragment or derivative thereof, including but not limited to: (i) a single antibody with two arms, each arm comprising a different antigen binding site; (ii) a bispecific scFv, for example, two scFvs connected via a peptide linker; (iii) a dual variable domain antibody (DVD-Ig), in which each light chain and heavy chain contains two variable domains connected in series by a short peptide bond; (iv) a bispecific (Fab′)2 fragment; (v) a diabody; (vi) a tandem diabody (TandAb), a fusion of two diabodies; and (vii) a "dock and lock (DNL)-Fab3", a trivalent bispecific consisting of two identical Fab fragments connected to different Fab fragments. Heterotropic binding proteins; (viii) IgG-like molecules with engineered Fc to enforce heterodimerization; (ix) recombinant IgG-like dual-targeting molecules, in which each side of the molecule contains Fab fragments or partial Fab fragments of at least two different antibodies; (x) IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or partial Fab fragment; (xi) Fc fusion molecules; (v) Fab fusion molecules; and (vi) scFv- and diabody-based heavy chain antibodies (e.g., domain antibodies, nanobodies), in which different scFvs, diabodies or heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused with a heavy chain constant domain, Fc region or a portion thereof.

[0467] Examples of IgG-like molecules with engineered Fc include, but are not limited to, triomabs, knobs-into-holes (kih) molecules (e.g., kih IgG with a common light chain), CrossMAbs, orthoFab IgG molecules, electrostatic matching molecules, LUZ-Y molecules, DIG-body and PIG-body molecules, chain exchange engineered domain bodies (SEEDbody) molecules, Biclonics molecules, FcΔAdp molecules, bispecific IgG1 and IgG2 molecules, Azymetric scaffold molecules, and DuoBody molecules. Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, dual-targeting (DT)-Ig molecules, two-in-one antibodies, mAbs 2 The example of IgG fusion molecules includes but is not limited to DVD-Ig molecules and IgG-scFv. The example of Fc fusion molecules includes but is not limited to scFv / Fc fusions, SCORPION molecules and Fc-DART molecules. The example of Fab fusion bispecific antibodies includes but is not limited to F(ab)2 molecules, DNL molecules and Fab-Fv molecules. The examples of domain antibodies based on scFv and double antibodies include but are not limited to bispecific T cell engagers (BiTE) molecules, tandem double antibody molecules (TandAb), dual affinity retargeting technology (DART) molecules, single-chain double antibody molecules, TCR-like antibodies (AIT, ReceptorLogics), human serum albumin scFv fusions, COMBODY molecules, dual-targeted nano antibodies and dual-targeted heavy chain domain antibodies only.

[0468] In certain embodiments, the disclosed bispecific molecules can be fusion proteins containing one or more antibody mimics. In certain embodiments, the disclosed bispecific molecules can be fusion proteins containing one or more antibodies or antigen-binding fragments thereof. In some embodiments, the disclosed bispecific molecules can be fusion proteins containing one or more antibody mimics and one or more antibodies or antigen-binding fragments thereof.

[0469] In some embodiments, the molecule containing the disclosed antigen binding site also comprises an antibody constant region or a fragment or variant thereof. In some embodiments, the antibody constant region can be, for example, a heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; in particular, selected from, for example, a (for example, human) heavy chain constant region of IgG1, IgG2, IgG3 and IgG4. In certain embodiments, the antibody constant region or a fragment or variant thereof has an amino acid sequence consistent with, for example, a heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE, preferably, for example, a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 at least 90% (for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%). In certain embodiments, the antibody constant region can be a light chain constant region selected from, for example, a (e.g., human) light chain constant region of κ or λ. The constant region can be changed, for example, mutated, to modify the properties of the antibody (e.g., to increase or decrease one or more of the following: 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 will not recruit effector cells or fix complement. In another embodiment, the antibody has reduced or no ability to bind to Fc receptors. For example, it is an isotype or subtype, fragment or other mutant that does not support binding to Fc receptors, for example, it has a mutagenic or missing Fc receptor binding region.

[0470] 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, which comprises one or more of the following mutations, based on EU index numbering: (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 P33 1S; (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 comprising one or more of the following mutations, numbered based on 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 comprising one or more of the following mutations, numbered based on 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.

[0471] 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 comprising one or more of the following mutations, numbered based on 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.

[0472] In some embodiments, the disclosed antigen binding sites are linked to antibody constant regions or fragments or variants thereof.In certain embodiments, the antigen binding sites are linked to IgG constant regions, including hinge, CH2 and CH3 domains (with or without CH1 domains).

[0473] In some embodiments, the molecules containing the disclosed antigen binding sites further comprise an afucosylated Fc region. In some embodiments, the Fc region is an afucosylated IgG1 Fc region.

[0474] Amino acid sequence modification

[0475] Amino acid sequence modifications of the antigen binding sites disclosed herein and molecules containing the antigen binding sites (e.g., antibodies or antigen binding fragments thereof) are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or antigen binding fragment. Amino acid sequence variants can be prepared, for example, by introducing appropriate nucleotide changes in the nucleic acid sequence encoding the antigen binding site or a molecule containing the antigen binding site, or by peptide synthesis. Such modifications may include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions may be performed, as long as the antigen binding site or a 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 processes, such as changing the number or position of glycosylation sites.

[0476] A useful method for identifying certain residues or regions as preferred mutagenesis positions is called "alanine scanning mutagenesis". In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys and Glu) is identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. Then, those amino acid positions that show functional sensitivity to substitution are improved by introducing additional or other variants at or for the substitution site. Therefore, although the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis can be performed at the target codon or region, and the expressed variants can be screened to obtain the desired activity.

[0477] Examples of amino acid sequence insertions include, but are not limited to, amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include, but are not limited to, N-terminal methionyl residues.

[0478] In some embodiments, the antigen binding site or a molecule containing the antigen binding site is fused at one terminus to another polypeptide, such as a cytotoxic polypeptide, an enzyme, or a polypeptide that increases the serum half-life of the antibody or antigen binding fragment.

[0479] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the amino acid sequence of the molecule. The most interesting sites for substitution mutagenesis are usually hypervariable regions, but framework region changes are also contemplated. Examples of conservative substitutions are shown under the heading "preferred substitutions" in Table 1. More substantial changes under the heading "exemplary substitutions" in Table 1, or changes further described below with reference to amino acid classes, can be introduced and the resulting antibodies or antigen-binding fragments screened.

[0480] Table 1. Exemplary amino acid substitutions

[0481] Original residue Exemplary substitutions Preferred substitution Ala(A) Val, Leu, Ile Val Arg(R) Lys, Gln, Asn Lys Asn(N) Gln, His, Asp, Lys, Arg Gln Asp(D) Glu.Asn Glu Cys(C) Ser, Ala Ser Gln(Q) Asn、Glu Asn Glu(E) Asp、Gln Asp Gly(G) Ala Ala His(H) Asn, Gln, Lys, Arg Arg Ile(I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu(L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys(K) Arg, Gln, Asn Arg Met(M) Leu, Phe, Ile Leu Phe(F) Leu, Val, Ile, Ala, Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr、Phe Tyr Tyr(Y) Trp, Phe, Thr, Ser Phe Val(V) Ile, Leu, Met, Phe, Ala, norleucine Leu

[0482] Substantial modification of the biological properties of antibodies can be achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a 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 generally divided into several categories based on common side chain properties:

[0483] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0484] (2) Neutral hydrophilic: Cys, Ser, Thr;

[0485] (3) Acidic: Asp, Glu;

[0486] (4) Basic: Asn, Gln, His, Lys, Arg;

[0487] (5) Residues that affect chain orientation: Gly, Pro; and

[0488] (6) Aromatic: Trp, Tyr, Phe.

[0489] Non-conservative substitutions may entail exchanging a member of one of these classes for another class.

[0490] Additionally or alternatively, cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody or antigen-binding fragment may be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0491] In some embodiments, substitution variants comprise substitutions within one or more hypervariable region residues of a parent antibody (e.g., a human antibody). Typically, variants with improved biological properties relative to the parent antibody from which they were generated are selected for further development.

[0492] The method for producing such substitution variants relates to affinity maturation using phage display. In one example of such a method, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The resulting antibody variants are displayed in a monovalent manner, such as from filamentous phage particles fused to the M13 gene III product packaged within each particle. The phage display variants are then screened for biological activity (e.g., binding affinity).

[0493] In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly 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 technology described in detail herein. Once such variants are generated, the variant group is screened, and antibodies with excellent properties in one or more related assays can be selected for further development.

[0494] In some embodiments, the original glycosylation pattern of the parent antibody is altered. Such alterations may include deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites not present in the antibody.

[0495] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate 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 except proline) are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in 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 may also be used.

[0496] Addition of glycosylation sites to the antibody can be achieved by altering the amino acid sequence of the antibody or antigen-binding fragment so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0497] 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 variants or non-variants of earlier preparations of antibodies or their antigen-binding fragments.

[0498] In some embodiments, modifications that increase serum half-life are used. For example, a salvage receptor binding epitope can be incorporated into a molecule containing a disclosed antigen binding site, as described, for example, in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0499] Methods for preparing antigen binding sites and molecules containing antigen binding sites

[0500] The above proteins can be prepared using recombinant DNA techniques well known to those skilled in the art.

[0501] For example, one or more nucleic acid sequences encoding a protein containing a disclosed antigen binding site can be cloned into one or more expression vectors; the expression vector can be stably transfected into a host cell capable of expressing the gene. After transfection, individual clones can be isolated for cell bank generation using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or ClonePix. Clones can be cultured under conditions suitable for bioreactor amplification and maintenance of expression of proteins containing antigen binding sites disclosed herein. Proteins 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.

[0502] Thus, also provided herein are isolated nucleic acids encoding antigen binding sites and molecules (eg, proteins) containing the antigen binding sites, vectors and host cells comprising the nucleic acids, and recombinant techniques for production.

[0503] In certain embodiments, one or more isolated nucleic acids are provided, which comprise sequences encoding the immunoglobulin heavy chain and / or immunoglobulin light chain variable regions of any antibody disclosed herein, and one or more expression vectors, which express the immunoglobulin heavy chain and / or immunoglobulin light chain variable regions of any antibody disclosed herein. A host cell comprising one or more of the aforementioned expression vectors and / or isolated nucleic acids is also provided.

[0504] In some embodiments, the antigen binding sites and molecules (eg, proteins) containing the antigen binding sites of the present disclosure can be fused to another agent, eg, another therapeutic agent. The construction of fusion proteins is within the ordinary skill in the art.

[0505] Monoclonal antibodies

[0506] Monoclonal antibodies may be made using the hybridoma method first described, or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0507] In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively or in addition, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells.

[0508] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

[0509] The example of suitable fusion partner includes but is not limited to myeloma cell, and the myeloma cell effectively fuses, supports the stable high-level production of antibody by the selected antibody-producing cell, and is sensitive to the selective culture medium of the unfused parental cell. The example of suitable myeloma cell line includes but is not limited to mouse myeloma cell line, such as those derived from MOPC-21 and MPC-11 mouse tumors, which can be obtained from Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives, such as X63-Ag8-653 cells that can be obtained from American Type Culture Collection (American Type Culture Collection, Rockville, Md. USA). Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies are also described.

[0510] According to the hybridoma method, the culture medium in which the hybridoma cells are grown is then assayed for production of monoclonal antibodies directed against the antigen. For example, the binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0511] The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis.

[0512] Once hybridoma cells producing antibodies with desired specificity, affinity and / or activity have been identified, 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 culture media. In addition, hybridoma cells can be grown in vivo in animals as ascites tumors, for example, by injecting cells intraperitoneally into mice.

[0513] The antibody can be purified by conventional antibody purification procedures, such as affinity chromatography (e.g. using protein A or protein ) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, etc., to appropriately separate the monoclonal antibodies secreted by the subclones from the culture medium, ascites or serum.

[0514] DNA encoding monoclonal antibodies can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding mouse antibody heavy and light chains). Hybridoma cells can be used as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into a host cell that does not originally produce antibody protein, such as an Escherichia coli cell, ape COS cell, Chinese hamster ovary (CHO) cell, or myeloma cell, to obtain the synthesis of monoclonal antibodies in recombinant host cells.

[0515] In certain embodiments, monoclonal antibodies or antibody fragments are isolated from antibody phage libraries. Human antibodies with high affinity (nM range) can be produced, for example, by chain shuffling. Combinatorial infection and in vivo recombination can provide strategies for building very large phage libraries. These techniques are feasible alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0516] The DNA encoding the antibody can be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy and light chain constant domain (CH and CL) sequences for the homologous murine sequences (see, e.g., U.S. Pat. No. 4,816,567), or by fusing the immunoglobulin coding sequence to all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide).

[0517] Human antibodies and phage display methods

[0518] Human antibodies can be produced by methods known in the art, including methods described herein. For example, transgenic animals (e.g., mice) can be produced that are capable of producing a complete human antibody library in the absence of endogenous immunoglobulin production after immunization. For example, homozygous deletions of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice have been described, resulting in complete inhibition of endogenous antibody production. Transferring human germline immunoglobulin gene arrays to such germline mutant mice will result in the production of human antibodies after antigen attack. See, e.g., U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852.

[0519] Alternatively, phage display technology can be used to produce human antibodies and antibody fragments from immunoglobulin variable (V) domain gene libraries of unimmunized donors in vitro. According to this technology, the antibody V domain gene frame is cloned into the major or minor coat protein gene of filamentous phage (such as M13 or fd), and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of phage genomes, the selection based on the functional properties of antibodies also results in the selection of genes encoding antibodies that show those properties. Therefore, phage simulates some properties of B cells. Phage display can be carried out in various forms. Several sources of V gene segments can be used for phage display, such as random combinatorial libraries from V genes, such as libraries derived from immune mouse spleens. V gene libraries from unimmunized human donors can be constructed, and antibodies for antigens (including self-antigens) of different arrays can be separated substantially according to the method described in the art. See, for example, U.S. Patent No. 5,565,332 and No. 5,573,905.

[0520] Human antibodies can also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0521] Competition assays for determining whether an antibody binds to the same epitope as a disclosed antibody or competes for binding with a disclosed antibody are known in the art. Exemplary competition assays include immunoassays (e.g., ELISA assays, RIA assays), surface plasmon resonance (e.g., Biacore analysis), biolayer interferometry, and flow cytometry.

[0522] Typically, competitive assays involve the use of antigens, test antibodies, and reference antibodies that are bound to a solid surface or expressed on a cell surface. The reference antibody is labeled, while the test antibody is unlabeled. Competitive inhibition is measured by determining the amount of the labeled reference antibody bound to a solid surface or cell in the presence of the test antibody. Typically, the test antibody is present in excess (e.g., 1x, 5x, 10x, 20x, or 100x). Antibodies identified by competitive assays (e.g., competitive antibodies) include antibodies that bind to the same epitope or similar (e.g., overlapping) epitopes as the reference antibody, and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antibody to occur sterically hindered.

[0523] Competition assays can be performed in two orientations to ensure that the presence of the label does not interfere or otherwise inhibit binding. For example, in the first orientation, the reference antibody is labeled and the test antibody is not labeled, and in the second orientation, the test antibody is labeled and the reference antibody is not labeled.

[0524] A test antibody competes with a reference antibody for specific binding to an antigen if an excess of one antibody (e.g., 1x, 5x, 10x, 20x, or 100x) inhibits binding of the other antibody, e.g., by at least 50%, 75%, 90%, 95%, or 99%, as measured in a competitive binding assay.

[0525] If substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody, then it can be determined that the two antibodies bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody, then it can be determined that the two antibodies bind to overlapping epitopes.

[0526] The antibodies disclosed herein can be further optimized (e.g., affinity maturation) to improve biochemical characteristics (including affinity and / or specificity), improve biophysical properties (including aggregation, stability, precipitation and / or non-specific interactions), and / or reduce immunogenicity. Affinity maturation procedures are within the ordinary skill in the art. For example, diversity can be introduced into immunoglobulin heavy chains and / or immunoglobulin light chains by DNA shuffling, chain shuffling, CDR shuffling, random mutagenesis and / or site-specific mutagenesis.

[0527] In certain embodiments, the isolated human antibody contains one or more somatic mutations. In these cases, the antibody can be modified to a human germline sequence to optimize the antibody (e.g., by a process known as germlining).

[0528] Typically, an optimized antibody has at least the same or substantially the same affinity for an antigen as the non-optimized (or parent) antibody from which it was derived. For example, in certain embodiments, an optimized antibody has a higher affinity for an antigen when compared to a parent antibody.

[0529] Pharmaceutical composition

[0530] In certain embodiments, provided molecules containing disclosed antigen binding sites are incorporated into pharmaceutical compositions suitable for administration to a subject together with one or more pharmaceutically acceptable carriers. As used herein, "pharmaceutically acceptable carrier" refers to any of a variety of physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, saline, phosphate buffered saline, glucose, glycerol, ethanol, etc., and combinations thereof.

[0531] In some embodiments, the pharmaceutical composition comprises one or more tonicity agents or stabilizers. Non-limiting examples of such tonicity agents or stabilizers include sugars (e.g., sucrose), polyols (e.g., mannitol or sorbitol), and sodium chloride.

[0532] In some embodiments, the pharmaceutical composition comprises one or more bulking agents and / or lyoprotectants (e.g., mannitol or glycine), buffers (e.g., phosphate, acetate, or histidine buffers), surfactants (e.g., polysorbates), antioxidants (e.g., methionine), and / or metal ions or chelators (e.g., ethylenediaminetetraacetic acid (EDTA)).

[0533] In some embodiments, the pharmaceutical composition comprises one or more auxiliary substances, such as wetting or emulsifying agents, preservatives (eg, benzyl alcohol), or buffers, which can enhance the shelf life and / or effectiveness of the immunoconjugates disclosed herein.

[0534] Pharmaceutical compositions can be provided in any of a variety of forms. These include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The suitability of some forms may depend on the intended mode of administration and therapeutic application.

[0535] In some embodiments, the pharmaceutical composition is in the form of an injectable or infusible solution.

[0536] Pharmaceutical compositions are usually sterile and stable under manufacturing, transportation and storage conditions. Pharmaceutical compositions can be formulated into, for example, solutions, microemulsions, dispersions, liposomes or other ordered structures. In some embodiments, pharmaceutical compositions are formulated into structures that are particularly suitable for high drug concentrations. For example, sterile injectable solutions can be prepared by incorporating therapeutic agents (e.g., immunoconjugates) in the desired amount into a suitable solvent having one or a combination of the components listed herein, optionally followed by sterilization (e.g., filtration sterilization). Typically, dispersions can be prepared by incorporating immunoconjugates into a sterile vehicle containing a basic dispersion medium and other components (e.g., those other components mentioned herein). In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods include vacuum drying and freeze drying, to produce, for example, powders of immunoconjugates and any other desired components from their previously sterile filtered solutions.

[0537] The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining a certain particle size (for example, in the case of a dispersion) and / or by the use of a surfactant. Prolonged absorption of an injectable composition can be achieved, for example, by including in the composition an agent that delays absorption (e.g., monostearate and / or gelatin).

[0538] Treatment

[0539] The present application provides methods for treating diseases or conditions using molecules (e.g., proteins, such as antibodies or antigen-binding fragments thereof) or pharmaceutical compositions described herein. The methods of treatment disclosed herein generally include administering to a subject in need thereof (e.g., a human subject) a therapeutically effective amount of a disclosed molecule containing an antigen binding site or a pharmaceutical composition thereof.

[0540] In some embodiments, the disease or condition is a mast cell-related disease or condition. In some embodiments, a mast cell-related disease or condition is associated with undesirable mast cell activity, such as excessive proliferation of mast cells. Non-limiting examples of mast cell-related diseases or conditions include upper airway diseases such as allergic rhinitis and sinusitis, foreign body aspiration, glottic stenosis, tracheal stenosis, laryngotracheomalacia, vascular rings, chronic obstructive pulmonary disease (COPD) and congestive heart failure, eosinophilic bronchitis, polychondritis, sarcoidosis, papillomatosis, arthritis (e.g., rheumatoid arthritis), Wegener's granulomatosis, primary biliary cirrhosis, allergies that respond to oral immunotherapy, bile duct lesions caused by mast cells, and pulmonary edema. secondary sclerosing cholangitis due to leukemia, pruritus, pruritus caused by or associated with chronic kidney disease or primary biliary cirrhosis, bronchiolitis obliterans syndrome after lung transplantation or hematopoietic stem cell transplantation, inflammatory bowel syndrome, functional dyspepsia, infantile bronchopulmonary dysplasia, cerebral ischemia, seizures, pneumonia due to influenza, sepsis, scleroderma, cluster headaches, cardiac fibrosis, primary sclerosing cholangitis (PSC), Morbihan disease, aspirin-intolerant asthma, vulvodynia, chronic pelvic pain syndrome (prostatitis), and cancer (e.g., lung cancer).

[0541] In some embodiments, the disease or condition is an autoimmune disease. In some embodiments, the disease or condition is allergy. In some embodiments, the disease or condition is inflammation. In some embodiments, the disease or condition is pruritus. In some embodiments, the disease or condition is cancer.

[0542] The therapeutically effective amount can be administered via a single dose or via multiple doses (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses). When administered via multiple doses, any of a variety of suitable treatment regimens can be used, including regular administration (e.g., once every other day, once every three days, once every four days, once every five days, three times a week, twice a week, once a week, once every two weeks, once every three weeks, etc.).

[0543] The dosage regimen (e.g., the amount of each therapeutic agent, the relative duration of therapy, etc.) that is effective in the treatment method may depend on the severity of the disease or disorder and the weight and general condition of the subject. For example, the therapeutically effective amount of a particular composition comprising a therapeutic agent applied to a mammal (e.g., a human) may be determined by a person of ordinary skill in the art taking into account individual differences in age, weight, and condition of the mammal. The therapeutically effective amount and / or the optimal amount may also be determined empirically by a person skilled in the art.

[0544] The molecules containing the antigen binding site or pharmaceutical compositions thereof can be administered by any of a variety of suitable routes, including but not limited to systemic routes, such as parenteral (eg, intravenous or subcutaneous) or enteral routes.

[0545] List of Exemplary Embodiments

[0546] The present invention is further described by the following non-limiting exemplary embodiments:

[0547] Embodiment 1. An antigen binding site capable of binding to FCER1A, FCER1B and / or FCER1G.

[0548] Embodiment 2. The antigen binding site of embodiment 1, wherein the antigen binding site is capable of binding to FCER1A.

[0549] Embodiment 3. The antigen binding site of embodiment 1, wherein said antigen binding site is capable of binding to FCER1B.

[0550] Embodiment 4. The antigen binding site of embodiment 1, wherein the antigen binding site is capable of binding to FCER1G.

[0551] Embodiment 5. An antigen binding site capable of binding to c-Kit.

[0552] Embodiment 6. An antigen binding site capable of binding to siglec-6.

[0553] Embodiment 7. An antigen binding site capable of binding to siglec-8.

[0554] Embodiment 8. The antigen binding site of any one of embodiments 1-7, wherein said antigen binding site is present as a single chain variable fragment (scFv).

[0555] Embodiment 9. The antigen binding site of any one of embodiments 1-7, wherein the antigen binding site exists as an antibody mimetic (e.g., an affinity body, an affilin, an affimer, an afftin, an alphabody, an anticalin, an avimer, a centyrin, a DARPin, a fynomer, a monoclonal antibody, or a nanoCLAMP).

[0556] Embodiment 10. The antigen binding site of any one of embodiments 5 and 8-9, wherein the antigen binding site comprises

[0557] (a) the complementarity determining region 1 (CDR1), CDR2 and / or CDR3 of the heavy chain (HC) shown in any one of SEQ ID NO: 39, 49, 51, 54, 56 or 58; and / or

[0558] (b) CDR1, CDR2 and / or CDR3 of the light chain (LC) shown in any one of SEQ ID NO:44, 50, 86 or 88.

[0559] Embodiment 11. The antigen binding site of any one of embodiments 5 and 8-10, wherein the antigen binding site comprises

[0560] (a) a heavy chain variable domain (VH) comprising the following complementarity determining regions:

[0561] CDR-H1, comprising the amino acid sequence: SYNMH (SEQ ID NO: 41),

[0562] CDR-H2, comprising the amino acid sequence: VIYSGNGDTSYNQKFKG (SEQ ID NO:42) or IYSGNGDTSYNQKFKGK (SEQ ID NO:33), and

[0563] CDR-H3, comprising the amino acid sequence: ERDTRFGN (SEQ ID NO: 43); and / or

[0564] (b) a light chain variable domain (VL) comprising the following complementarity determining regions:

[0565] CDR-L1, comprising the amino acid sequence: RASESVDIYGNSFMH (SEQ ID NO: 46);

[0566] CDR-L2, comprising the amino acid sequence: LASNLES (SEQ ID NO:47); and

[0567] CDR-L3, comprising the amino acid sequence: QQNNEDPYT (SEQ ID NO: 48).

[0568] Embodiment 12. The antigen binding site of any one of embodiments 5 and 8-11, wherein the antigen binding site comprises

[0569] (a) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown in SEQ ID NO: 39; and / or

[0570] A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown in SEQ ID NO:44; or

[0571] (b) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown in SEQ ID NO: 49; and / or

[0572] A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown in SEQ ID NO: 50; or

[0573] (c) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown in any one of SEQ ID NOs: 51, 54, 56 and 58; and / or

[0574] A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown in SEQ ID NO:86 or 88.

[0575] Embodiment 13. The antigen binding site of any one of embodiments 5 and 8-12, wherein the antigen binding site comprises

[0576] (a) a HC comprising CDR-H1, CDR-H2 and CDR-H3 of the HC shown in SEQ ID NO: 39 and having at least 85% amino acid sequence identity with SEQ ID NO: 39; and / or

[0577] An LC comprising CDR-L1, CDR-L2 and CDR-L3 of the LC shown in SEQ ID NO:44 and having at least 85% amino acid sequence identity to SEQ ID NO:44; or

[0578] (b) a HC comprising CDR-H1, CDR-H2 and CDR-H3 of the HC shown in SEQ ID NO: 49 and having at least 85% amino acid sequence identity with SEQ ID NO: 49; and / or

[0579] An LC comprising CDR-L1, CDR-L2 and CDR-L3 of the LC shown in SEQ ID NO:50 and having at least 85% amino acid sequence identity to SEQ ID NO:50; or

[0580] (c) a HC comprising CDR-H1, CDR-H2 and CDR-H3 of the HC shown in any one of SEQ ID NOs: 51, 54, 56 and 58 and having at least 85% amino acid sequence identity thereto; and / or

[0581] An LC comprising CDR-L1, CDR-L2 and CDR-L3 of the LC shown in SEQ ID NO: 86 or 88 and having at least 85% amino acid sequence identity thereto.

[0582] Embodiment 14. The antigen binding site of embodiment 12 or 13, wherein the sequence identity is at least 90%, 95%, 96%, 97%, 98% or 99%.

[0583] Embodiment 15. The antigen binding site of any one of embodiments 5 and 8-14, wherein the antigen binding site comprises

[0584] (a) the VH of the HC shown in SEQ ID NO: 39; and / or the VL of the LC shown in SEQ ID NO: 44; or

[0585] (b) the VH of the HC shown in SEQ ID NO:49; and / or the VL of the LC shown in SEQ ID NO:50; or

[0586] (c) the VH of the HC shown in any one of SEQ ID NOs: 51, 54, 56 and 58; and / or the VL of the LC shown in SEQ ID NOs: 86 or 88.

[0587] Embodiment 16. The antigen binding site of any one of embodiments 6 and 8-9, wherein the antigen binding site comprises

[0588] (a) CDR1, CDR2 and / or CDR3 of the HC set forth in any one of SEQ ID NO: 1, 17, 19, 22, 24, 26 or 28; and / or

[0589] (b) CDR1, CDR2 and / or CDR3 of LC shown in any one of SEQ ID NO: 9, 18, 30, 35 or 37.

[0590] Embodiment 17. The antigen binding site of any one of embodiments 6, 8-9 and 16, wherein the antigen binding site comprises

[0591] (a) VH comprising the following complementarity determining regions:

[0592] CDR-H1, comprising the amino acid sequence: SYWMH (SEQ ID NO: 3),

[0593] CDR-H2, comprising the amino acid sequence: EIYPTNGGTTYNEKFKR (SEQ ID NO: 4), and

[0594] CDR-H3 comprising the amino acid sequence: EDFYAMDY (SEQ ID NO: 5); and / or

[0595] (b) VL comprising the following complementarity determining regions:

[0596] CDR-L1, comprising the amino acid sequence: KSSQSLLDSDGKTCLN (SEQ ID NO: 11);

[0597] CDR-L2, comprising the amino acid sequence: LVSKLDS (SEQ ID NO: 12); and

[0598] CDR-L3, comprising the amino acid sequence: WQGTHFPYT (SEQ ID NO: 13).

[0599] Embodiment 18. The antigen binding site of any one of embodiments 6, 8-9 and 16, wherein the antigen binding site comprises

[0600] (a) VH comprising the following complementarity determining regions:

[0601] CDR-H1, comprising the amino acid sequence: GYAFTSYWMH (SEQ ID NO: 6), CDR-H2, comprising the amino acid sequence: EIYPTNGGTT (SEQ ID NO: 7), and

[0602] CDR-H3 comprising the amino acid sequence: EDFYAMDY (SEQ ID NO: 8); and / or

[0603] (b) VL comprising the following complementarity determining regions:

[0604] CDR-L1, comprising the amino acid sequence: KSSQSLLDSDGKTCLN (SEQ ID NO: 14);

[0605] CDR-L2, comprising the amino acid sequence: LVSKLDS (SEQ ID NO: 15); and

[0606] CDR-L3, comprising the amino acid sequence: WQGTHFPYT (SEQ ID NO: 16).

[0607] Embodiment 19. The antigen binding site of any one of embodiments 6, 8-9 and 16-18, wherein the antigen binding site comprises

[0608] (a) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown in SEQ ID NO: 1; and / or

[0609] A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown in SEQ ID NO:9; or

[0610] (b) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown in SEQ ID NO: 17; and / or

[0611] A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown in SEQ ID NO: 18; or

[0612] (c) a VH comprising an amino acid sequence having at least 85% identity to the VH of the HC shown in any one of SEQ ID NOs: 19, 22, 24, 26 and 28; and / or

[0613] A VL comprising an amino acid sequence having at least 85% identity to the VL of the LC shown in any one of SEQ ID NOs: 30, 35 and 37.

[0614] Embodiment 20. The antigen binding site of any one of embodiments 6, 8-9 and 16-19, wherein the antigen binding site comprises

[0615] (a) a 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 with SEQ ID NO: 1; and / or

[0616] 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

[0617] (b) a 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 with SEQ ID NO: 17; and / or

[0618] 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

[0619] (c) a 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

[0620] 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.

[0621] Embodiment 21. The antigen binding site of embodiment 19 or 20, wherein the sequence identity is at least 90%, 95%, 96%, 97%, 98% or 99%.

[0622] Embodiment 22. The antigen binding site of any one of embodiments 6, 8-9 and 16-21, wherein the antigen binding site comprises

[0623] (a) the VH of the HC shown in SEQ ID NO: 1; and / or the VL of the LC shown in SEQ ID NO: 9; or

[0624] (b) the VH of the HC shown in SEQ ID NO: 17; and / or the VL of the LC shown in SEQ ID NO: 18; or

[0625] (c) the VH of the HC shown in any one of SEQ ID NOs: 19, 22, 24, 26 and 28; and / or the VL of the LC shown in any one of SEQ ID NOs: 30, 35 and 37.

[0626] Embodiment 23. The antigen binding site of any one of embodiments 6-9, wherein the antigen binding site comprises

[0627] (a) CDR1, CDR2 and / or CDR3 of the VH shown in SEQ ID NO: 121; and / or

[0628] (b) CDR1, CDR2 and / or CDR3 of LC shown in SEQ ID NO:126.

[0629] Embodiment 24. The antigen binding site of any one of embodiments 6-9 and 23, wherein the antigen binding site comprises

[0630] (a) VH comprising the following complementarity determining regions:

[0631] CDR-H1, comprising the amino acid sequence: DYGMH (SEQ ID NO: 122),

[0632] CDR-H2, comprising the amino acid sequence: GISWNSGSIGYADSVKG (SEQ ID NO: 123), and

[0633] CDR-H3 comprising the amino acid sequence: GGQTIDI (SEQ ID NO: 124); and / or

[0634] (b) VL comprising the following complementarity determining regions:

[0635] CDR-L1, comprising the amino acid sequence: RASQSISSYLN (SEQ ID NO: 127);

[0636] CDR-L2, comprising the amino acid sequence: AASSLQS (SEQ ID NO: 128); and

[0637] CDR-L3, comprising the amino acid sequence: QQSYSTPFT (SEQ ID NO: 129).

[0638] Embodiment 25. The antigen binding site of any one of embodiments 6-9 and 23-24, wherein the antigen binding site comprises

[0639] A VH comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 121; and / or

[0640] A VL comprising an amino acid sequence at least 85% identical to SEQ ID NO:126.

[0641] Embodiment 26. The antigen binding site of any one of embodiments 6-9 and 23-25, wherein the antigen binding site comprises

[0642] A HC comprising 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 with SEQ ID NO: 120; and / or

[0643] An LC comprising 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.

[0644] Embodiment 27. The antigen binding site of embodiment 25 or 26, wherein the sequence identity is at least 90%, 95%, 96%, 97%, 98% or 99%.

[0645] Embodiment 28. The antigen binding site of any one of embodiments 6-9 and 23-27, wherein the antigen binding site comprises 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.

[0646] Embodiment 29. An antibody or antigen-binding fragment thereof comprising the antigen-binding site of any one of embodiments 1-4.

[0647] Embodiment 30. An antibody or antigen-binding fragment thereof comprising the antigen-binding site of any one of embodiments 5 and 10-15.

[0648] Embodiment 31. The antibody or antigen-binding fragment thereof of embodiment 30, which is capable of blocking c-kit signaling.

[0649] Embodiment 32. An antibody or antigen-binding fragment thereof comprising the antigen-binding site of any one of embodiments 6 and 16-22.

[0650] Embodiment 33. The antibody or antigen-binding fragment thereof of embodiment 32, which is capable of agonizing siglec-6.

[0651] Embodiment 34. An antibody or antigen-binding fragment thereof comprising the antigen-binding site of embodiment 7.

[0652] Embodiment 35. An antibody or antigen-binding fragment thereof comprising the antigen-binding site of any one of embodiments 23-28.

[0653] Embodiment 36. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-35, wherein the antibody is a humanized antibody.

[0654] Embodiment 37. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-35, wherein the antibody is a human antibody.

[0655] Embodiment 38. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-35, wherein the antibody is a multispecific antibody.

[0656] Embodiment 39. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-35, wherein the antibody is a bispecific antibody.

[0657] Embodiment 40. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-35, wherein the antigen-binding fragment is Fab, F(ab')2, Fab', scFv, Fv fragment, Fd fragment or diabody.

[0658] Embodiment 41. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-35, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region.

[0659] Embodiment 42. The antibody or antigen-binding fragment thereof of Embodiment 41, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region.

[0660] Embodiment 43. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-42, wherein the antibody or antigen-binding fragment thereof has reduced or ineffective effector function.

[0661] Embodiment 44. The antibody or antigen-binding fragment thereof of any one of Embodiments 29-43, wherein the antibody or antigen-binding fragment thereof is afucosylated.

[0662] Embodiment 45. The antibody or antigen-binding fragment thereof of Embodiment 44, wherein said antibody or antigen-binding fragment thereof is at least about 75% afucosylated.

[0663] Embodiment 46. The antibody or antigen-binding fragment thereof of embodiment 44 or 45, wherein the antibody or antigen-binding fragment thereof is capable of depleting mast cells.

[0664] Embodiment 47. A protein comprising the antigen binding site of any one of embodiments 1-4.

[0665] Embodiment 48. A protein comprising the antigen binding site of any one of embodiments 5 and 10-15.

[0666] Embodiment 49. A protein comprising the antigen binding site of any one of embodiments 6 and 16-22.

[0667] Embodiment 50. A protein comprising the antigen binding site of embodiment 7.

[0668] Embodiment 51. A protein comprising the antigen binding site of any one of embodiments 23-28.

[0669] Embodiment 52. A protein comprising two or more antigen binding sites selected from: (i) an antigen binding site of any one of embodiments 1-4; (ii) an antigen binding site of any one of embodiments 5 and 10-15; (iii) an antigen binding site of any one of embodiments 6 and 16-22; (iv) an antigen binding site of embodiment 7; and (v) an antigen binding site of any one of embodiments 23-28.

[0670] Embodiment 53. A protein comprising two antigen binding sites selected from the group consisting of: (i) an antigen binding site of any one of embodiments 1-4; (ii) an antigen binding site of any one of embodiments 5 and 10-15; (iii) an antigen binding site of any one of embodiments 6 and 16-22; (iv) an antigen binding site of embodiment 7; and (v) an antigen binding site of any one of embodiments 23-28.

[0671] Embodiment 54. A protein comprising

[0672] (i) the antigen binding site of any one of embodiments 5 and 10-15; and

[0673] (ii) an antigen binding site selected from the group consisting of: (a) the antigen binding site of embodiment 2, (b) the antigen binding site of embodiment 3, or (c) the antigen binding site of one of embodiments 6 and 16-22.

[0674] Embodiment 55. The protein of any one of Embodiments 47-54, further comprising an antibody heavy chain constant region.

[0675] Embodiment 56. The protein of Embodiment 55, wherein the antibody heavy chain constant region is a human IgG heavy chain constant region.

[0676] Embodiment 57. The protein of any one of embodiments 47-56, wherein said protein has reduced or ineffective effector function.

[0677] Embodiment 58. The protein of any one of embodiments 47-57, wherein the protein is afucosylated.

[0678] Embodiment 59. The protein of embodiment 58, wherein said protein is at least about 75% afucosylated.

[0679] Embodiment 60. The protein of embodiment 58 or 59, wherein said protein is capable of depleting mast cells.

[0680] Embodiment 61. An isolated nucleic acid encoding the antigen binding site of any one of embodiments 1-28, the antibody or antigen binding fragment thereof of any one of embodiments 29-46, or the protein of any one of embodiments 47-60.

[0681] Embodiment 62. An expression vector comprising the isolated nucleic acid of embodiment 61.

[0682] Embodiment 63. A host cell comprising the isolated nucleic acid molecule of embodiment 61 or the expression vector of embodiment 62.

[0683] Embodiment 64. A pharmaceutical composition comprising an antigen binding site of any one of embodiments 1-28, an antibody or antigen binding fragment thereof of any one of embodiments 29-46, or a protein of any one of embodiments 47-60, and a pharmaceutically acceptable carrier.

[0684] Embodiment 65. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of an antigen binding site of any one of embodiments 1-28, an antibody or antigen binding fragment thereof of any one of embodiments 29-46, a protein of any one of embodiments 47-60, or a pharmaceutical composition of embodiment 64.

[0685] Embodiment 66. The method of Embodiment 65, wherein the disease or disorder is a mast cell-related disease or disorder.

[0686] Embodiment 67. The method of Embodiment 65, wherein the disease or disorder is an autoimmune disease.

[0687] Embodiment 68. The method of Embodiment 65, wherein the disease or condition is allergy.

[0688] Embodiment 69. The method of Embodiment 65, wherein the disease or condition is inflammation.

[0689] Embodiment 70. The method of embodiment 65, wherein the disease or condition is pruritus.

[0690] Embodiment 71. The method of Embodiment 65, wherein the disease or condition is cancer.

[0691] Embodiment 72. The antibody or antigen-binding fragment thereof of embodiment 12 or 13, comprising an amino acid sequence selected from any one of SEQ ID NOs: 1, 9, 17-19, 22, 24, 26, 28, 30, 35, 37, 120, 121, 125 and 126, or a fragment thereof.

[0692] Embodiment 73. The antibody or antigen-binding fragment thereof of embodiment 12 or 13, comprising at least one amino acid sequence or a fragment thereof selected from any one of SEQ ID NOs: 1, 9, 17-19, 22, 24, 26, 28, 30, 35, 37, 120, 121, 125 and 126.

[0693] Embodiment 74. The antibody or antigen-binding fragment thereof of embodiment 12 or 13, comprising at least one CDR sequence found in a sequence selected from any one of SEQ ID NOs: 1, 9, 17-19, 22, 24, 26, 28, 30, 35, 37, 120, 121, 125 and 126.

[0694] Embodiment 75. The antibody or antigen-binding fragment thereof of embodiment 12 or 13, comprising at least two CDR sequences found in a sequence selected from any one of SEQ ID NOs: 1, 9, 17-19, 22, 24, 26, 28, 30, 35, 37, 120, 121, 125 and 126.

[0695] Embodiment 76. The antibody or antigen-binding fragment thereof of embodiment 12 or 13, comprising at least three CDR sequences found in a sequence selected from any one of SEQ ID NOs: 1, 9, 17-19, 22, 24, 26, 28, 30, 35, 37, 120, 121, 125 and 126.

[0696] Embodiment 77. The antibody or antigen-binding fragment thereof of Embodiment 11, comprising an amino acid sequence selected from any one of SEQ ID NOs: 39, 44, 49-51, 54, 56, 58, 86 and 88, or a fragment thereof.

[0697] Embodiment 78. The antibody or antigen-binding fragment thereof of embodiment 11, comprising at least one amino acid sequence selected from any one of SEQ ID NOs: 39, 44, 49-51, 54, 56, 58, 86 and 88, or a fragment thereof.

[0698] Embodiment 79. The antibody or antigen-binding fragment thereof of embodiment 11, comprising at least two amino acid sequences selected from any one of SEQ ID NOs: 39, 44, 49-51, 54, 56, 58, 86 and 88, or a fragment thereof.

[0699] Embodiment 80. The antibody or antigen-binding fragment thereof of 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.

[0700] Embodiment 81. The antibody or antigen-binding fragment thereof of 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.

[0701] Embodiment 82. The antibody or antigen-binding fragment thereof of 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.

[0702] 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.

[0703] Example

[0704] Example 1. Antibody Generation

[0705] Human antibodies targeting FCER1, c-Kit, and siglec (eg, siglec-6 and siglec8) are generated by methods known in the art.

[0706] In some embodiments, transgenic mice that cannot express functional endogenous immunoglobulins but express human immunoglobulin genes are used. Specifically, human heavy chain and light chain immunoglobulin gene complexes are introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions and diversity regions are also introduced into mouse embryonic stem cells. The introduction of human immunoglobulin loci by homologous recombination occurs alone or simultaneously with the non-functionalization of mouse heavy chain and light chain immunoglobulin genes. Specifically, the homozygous deletion in the JH region prevents endogenous antibody production. The modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then cultivated to produce homozygous offspring expressing human antibodies. With selected antigens, such as all or part of an antigen (e.g., FCER1, c-Kit or siglec), transgenic mice are immunized in a normal manner. Monoclonal antibodies against antigens can be obtained from immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgene carried by transgenic mice is rearranged during B cell differentiation, and then undergoes class switching and somatic mutation.

[0707] In some other embodiments, antibodies targeting FCER1, c-Kit, and siglec (eg, siglec-6 and siglec 8) are generated by screening yeast display libraries.

[0708] In addition, the c-Kit / siglec-6 bispecific antibody is produced by methods known in the art. In some embodiments, the c-Kit / siglec-6 bispecific antibody is in the form of a CrossMab.

[0709] Example 2. Antibody Binding to c-Kit

[0710] The targeted binding of c-Kit antibodies to cells expressing c-Kit 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 c-Kit antibodies in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide) and incubated at 4°C for 30 minutes, washed and incubated with PE-labeled anti-human secondary antibodies. The cells were incubated in the dark at 4°C for 30 minutes. After washing, cell fluorescence analysis was performed using BD FACS Symphony A3 (BD ​​Biosciences). Cells were sorted by scattering properties, and cells were separated for single cell analysis using side scatter width versus side scatter area. The binding of antibodies to cells was quantified by measuring the fluorescence emission at an excitation wavelength of 561nm using a 586 / 15nm 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.

[0711] Table 2. Binding of c-Kit antibodies to c-Kit expressed on the cell surface of M-07e cells

[0712]

[0713]

[0714] In addition, the binding affinity of c-Kit antibodies to human c-Kit and cynomolgus monkey c-Kit was measured by biolayer interferometry (BLA) using an Octect instrument. Briefly, the capture sensor was loaded with a 3-fold dilution of the test antibody. The association and dissociation rates were determined and used to calculate the equilibrium dissociation constant K D (Table 3). For the humanized c-Kit antibodies tested (hSR-1-1 to hSR1-6), it was observed that D The affinities measured for c-Kit were in the subnanomolar range, and for the chimeric c-Kit antibody tested (chSR1), the affinities were in the single-digit nanomolar range.

[0715] Table 3. Binding affinity to human and cynomolgus monkey c-Kit as determined by BLI

[0716]

[0717] Example 3. Analysis of cross-reactivity and non-specific binding of c-Kit antibodies by ELISA

[0718] An ELISA-based assay was used to evaluate cross-reactivity and nonspecific binding of c-Kit antibodies to human c-Kit, cynomolgus c-Kit, and c-Kit family members.

[0719] His-tagged human c-Kit, His-tagged cynomolgus c-Kit, and His-tagged PDGFRa, PDGFRb, CSF1R, and FLT3 extracellular domains were fixed on 96-well ELISA plates. The plates were washed with PBST (phosphate buffered saline + 0.05% Tween 20) and blocked with 1% BSA in PBS for 1 hour. After washing with PBST, mAbs were added to the coated plates and incubated at room temperature for 2 hours. The plates were washed and incubated with HRP-conjugated goat anti-human IgG secondary antibodies at room temperature for 1 hour. After washing, antibody binding was detected by adding 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric detection and incubated at room temperature for 10-20 minutes, followed by the addition of 2.5N sulfuric acid stop solution. The absorbance at 450nm was measured on a plate reader (PerkinElmer). Data were analyzed using GraphPadPrism.

[0720] The results for exemplary c-Kit antibodies are depicted in Table 4. The c-Kit antibodies were able to bind to human and cynomolgus c-Kit, but did not cross-react with c-Kit family members PDGFRa, PDGFRb, CSF1R, or FLT3.

[0721] Table 4. Cross-reactivity of humanized and chimeric c-Kit antibodies

[0722]

[0723] Example 4. Binding of antibodies to siglec-6

[0724] The targeted binding of siglec-6 antibodies to cells expressing siglec-6 was evaluated by flow cytometry. The 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 siglec-6 antibodies in FACS buffer (PBS + 0.5% bovine serum albumin + 0.09% sodium azide) and incubated at 4 ° C for 30 minutes, washed and incubated with PE-labeled anti-human or anti-mouse secondary antibodies. The cells were incubated in the dark at 4 ° C for 30 minutes. After washing, cell fluorescence analysis was performed using BD FACS Symphony A3 (BD ​​Biosciences). Cells were sorted by scattering properties, and cells were separated for single cell analysis using side scatter width to side scatter area. The binding of antibodies to cells was quantified by measuring the fluorescence emission at an excitation wavelength of 561nm using a 586 / 15nm filter. Data analysis was performed using FlowJo software and summarized in Table 5. All tested siglec-6 antibodies were able to bind to cells expressing siglec-6.

[0725] Table 5. Binding of humanized and chimeric siglec-6 antibodies to siglec-6 expressed on the cell surface of HMC1.2 cells combine

[0726]

[0727] In addition, the binding affinity of siglec-6 antibodies to human siglec-6 was measured by BLA using the Octect instrument. Briefly, the capture sensor was loaded with a 3-fold dilution of the test antibody. The association rate and dissociation rate were determined and used to calculate the equilibrium dissociation constant K D (Table 6). For the humanized and chimeric siglec-6 antibodies tested, the K D The values ​​measured affinities for siglec-6 were in the subnanomolar range.

[0728] Table 6. Binding affinity to human siglect-6 as determined by BLI

[0729]

[0730] Example 5. Analysis of cross-reactivity and nonspecific binding of siglec-6 antibodies by ELISA

[0731] An ELISA-based assay was used to assess cross-reactivity and nonspecific binding of siglec-6 antibodies to human siglec-6, cynomolgus siglec-6, and siglec-6 family members siglec 1-5, 7-12, 14, and 15.

[0732] Cultured HEK293 cells expressing human siglec-6, cynomolgus monkey siglec-6 or siglec-6 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) and incubated at 4 ° C for 30 minutes, washed and incubated with PE-labeled anti-human secondary antibodies. The cells were incubated in the dark at 4 ° C for 30 minutes. After washing, cell fluorescence analysis was performed using BD FACS Symphony A3 (BD ​​Biosciences). Cells were sorted by scattering properties, and cells were separated for single cell analysis using side scatter width versus side scatter area. The binding of antibodies to cells was quantified by measuring the fluorescence emission at an excitation wavelength of 561nm using a 586 / 15nm filter. Data analysis was performed using FlowJo software.

[0733] The results for exemplary siglec-6 antibodies are depicted in Table 7. The siglec-6 antibodies were able to bind to human and cynomolgus monkey siglec-6, but did not cross-react with siglec-6 family members siglec 1-5, 7-12, 14, or 15.

[0734] Table 7. Cross-reactivity of humanized and chimeric siglec-6 antibodies

[0735]

[0736] Example 6. Inhibition of cell proliferation by c-Kit antibody

[0737] The ability of c-Kit antibodies to inhibit stem cell factor (SCF)-dependent cell proliferation was evaluated.

[0738] M-07e cells were cultured as recommended by the distributor. Cells were starved of SCF overnight (16 hours). SCF-starved cells were seeded in a 96-well plate at a density of 15,000 cells / well in a medium without SCF. c-Kit antibodies (SR1 antibodies or CDX-0159) or controls were titrated onto the cells and the plates were incubated at 37°C, 5% CO2 for 2 hours. After incubation for 2 hours, 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 TiterGlo (Promega). Cell Titer Glo was added to each well and the plates were incubated on a benchtop shaker in the dark for 2 minutes. Luminescence was measured on a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism.

[0739] Figure 1An exemplary result is depicted in Figure 2, where antibody concentration was plotted as a function of relative luminescence and curve fitting was performed using a four-parameter nonlinear regression algorithm. The IC 50 The values ​​are summarized in Table 8. The tested SR1 antibodies inhibited SCF-dependent proliferation in M-07e cells.

[0740] Table 8. Inhibition of SCF-dependent proliferation of M-07e cells

[0741] Antibody <![CDATA[IC 50 (nM)]]> muSR1 0.2828 hSR1-1 0.1415 hSR1-2 0.1417 hSR1-3 0.1129 hSR1-4 0.1385 hSR1-5 0.1415 hSR1-6 0.1517 chSR1 0.2769 CDX-0159 0.4082

[0742] Example 7. Inhibition of cell proliferation by c-Kit / siglec 6 bispecific antibody

[0743] The ability of c-Kit / Siglec 6 antibodies to inhibit SCF-dependent cell proliferation was evaluated.

[0744] M-07e cells were starved of SCF overnight (16 hours). SCF-starved cells were seeded in a 96-well plate at a density of 15,000 cells / well in a medium without SCF. 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 incubation for 2 hours, SCF was added to a final concentration of 50 ng / mL, and the plate was incubated at 37°C, 5% CO2 for 72 hours. Cell viability was measured using Cell Titer Glo (Promega). Cell TiterGlo was added to each well, and the plate was incubated on a benchtop shaker in the dark for 2 minutes. Luminescence was measured on a plate reader (PerkinElmer). Data were analyzed using GraphPad Prism and curve fitting was performed using a four-parameter nonlinear 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.

[0745] The results are Figure 2 The c-Kit / siglec-6 bispecific antibodies tested inhibited SCF-dependent proliferation in M-07e cells; the siglec-6 monospecific antibody did not inhibit proliferation.

[0746] Example 8. Mast cell degranulation assay

[0747] Experiments were performed to evaluate whether the disclosed siglect-6 antibodies induce mast cell degranulation.

[0748] CD34+ progenitor cells from whole blood are cultured in mast cell medium containing cytokines and growth factors. The cultures mature into mast cells within 6-8 weeks after the differentiation process is initiated.

[0749] To induce FcγRI expression in mast cells, cells were treated with 100UI / mL IFN-γ for 72 hours. Mast cells treated with and without IFN-γ were starved for cytokines for 4 hours. The cells were washed in Tyrode buffer and seeded in 96-well plates at 35,000 cells per well. The plates were incubated at 37°C and 5% CO2 for 30 minutes. Siglec 6 antibodies with normal human IgG1Fc, effector-silenced IgG1 Fc (containing L234A / L235A / P329G mutations) or effector-enhanced IgG1 Fc (without fucosylation) and PMA / ionomycin were added to the appropriate wells together with SCF, and the plates were incubated at 37°C (in air) for 30 minutes. The culture medium from the treated mast cell culture was added to a 96-well plate containing a PNAG solution (0.35% p-nitrophenyl N-acetyl-bD-glucamide) and incubated at 37°C (in air) for 90 minutes. Similarly, the remaining mast cells were lysed in 0.1% triton X-100 and added to the PNAG solution for 90 minutes. Glycine buffer (400mM, pH 10.7) was added to all wells and the absorbance at 405nm was read in a plate reader. β-hexosaminidase was measured by calculating the percentage of β-hexosaminidase released from the β-hexosaminidase contained in the mast cells.

[0750] The results are Figure 3 All three Siglec-6 hRND-3 antibodies with modifications in the Fc domain did not induce degranulation of CD34-derived mast cells.

[0751] Sequence Appendix

[0752] In some sequences, the variable regions are shown in bold and the CDRs are underlined.

[0753] Table 9. Sequences of exemplary antigen binding sites capable of binding to silect-6

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763]

[0764]

[0765] Table 10. Sequences of exemplary antigen binding sites capable of binding to c-Kit

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775] Table 11. Other sequences

[0776]

[0777] Incorporated by Reference

[0778] Unless otherwise indicated to the contrary, the entire disclosure of each patent document and scientific article cited herein is incorporated by reference for all purposes.

[0779] Equivalent solutions / other implementation plans

[0780] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. An antibody or an antigen-binding fragment thereof, comprising a first antigen-binding domain capable of binding to siglec-6 and a second antigen-binding domain capable of binding to c-Kit.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen-binding domain is capable of binding to siglec-6 with a higher affinity than the second antigen-binding domain is capable of binding to c-Kit.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the first antigen-binding domain is capable of binding to D The antibody binds siglec-6 with an affinity of 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.

4. The antibody or antigen-binding fragment thereof of claim 3, wherein the first antigen-binding domain is capable of binding to D The affinity of the peptides or peptides may be between about 1 nM and about 900 uM, between about 5 nM and about 500 uM, between about 10 nM and about 100 uM, between about 20 nM and about 10 10 uM, or between about 25 and about 500 nM to bind c-Kit.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment is capable of binding to cells expressing siglect-6.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is capable of binding to mast cells.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the antibody or antigen-binding fragment preferentially binds to mast cells rather than another immune cell.

8. The antibody or antigen-binding fragment thereof of claim 6, wherein the immune cell is a T cell, a dendritic cell, a macrophage, a neutrophil or a basophil.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof does not induce mast cell degranulation.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof has a comparable or lower internalization rate than a reference antibody capable of binding to silect-6.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the reference antibody capable of binding to siglect-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.

12. The antibody or antigen-binding fragment thereof of any one of claims 1-11, wherein the antibody or antigen-binding fragment thereof has comparable or lower toxicity than a reference antibody capable of binding to c-Kit.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the reference antibody capable of binding 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.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the first antigen-binding domain comprises: (a) a VH region comprising: A heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 3, or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 4 or 21, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 5, or a sequence having 1 or 2 amino acid differences therefrom; and (b) a VL region comprising: A heavy chain complementarity determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 11 or 32, or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 12 or a sequence having 1 or 2 amino acid differences therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 13, or a sequence having 1 or 2 amino acid differences therefrom.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the first antigen-binding domain comprises: (a) a VH region comprising: HCDR1 having the amino acid sequence of SEQ ID NO: 3, a HCDR2 having the amino acid sequence of SEQ ID NO: 4 or 21, and a HCDR3 having the amino acid sequence of SEQ ID NO: 5; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 11 or 32, LCDR2 having the amino acid sequence of SEQ ID NO: 12, and LCDR3 having the amino acid sequence of SEQ ID NO:

13.

16. The antibody or antigen-binding fragment thereof of claim 14, wherein the first antigen-binding domain comprises: (a) a VH region comprising: (i) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 4 and 5, respectively; or (ii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 21 and 5, respectively; and (b) a VL region comprising: (i) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 11, 12 and 13, respectively; or (ii) LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 32, 12 and 13, respectively.

17. The antibody or antigen-binding fragment thereof of any one of claims 14 to 16, wherein 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.

18. The antibody or antigen-binding fragment thereof of claim 17, wherein the first antigen-binding domain comprises: (a) a VH region having an 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.

19. The antibody or antigen-binding fragment thereof of claim 17 or 18, wherein 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.

20. The antibody or antigen-binding fragment thereof of claim 19, wherein 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.

21. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the first antigen-binding domain comprises: (a) a VH region comprising: A HCDR1 having the amino acid sequence of SEQ ID NO: 122 or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 123, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 124, or a sequence having 1 or 2 amino acid differences therefrom; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 127 or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 128 or a sequence having 1 or 2 amino acid differences therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 129, or a sequence having 1 or 2 amino acid differences therefrom.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the first antigen-binding domain comprises: (a) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 122, a HCDR2 having the amino acid sequence of SEQ ID NO: 123, and a HCDR3 having the amino acid sequence of SEQ ID NO: 124; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 127, LCDR2 having the amino acid sequence of SEQ ID NO: 128, and LCDR3 having the amino acid sequence of SEQ ID NO:

139.

23. The antibody or antigen-binding fragment thereof of claim 21 or 22, wherein 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.

24. The antibody or antigen-binding fragment thereof of claim 23, wherein 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.

25. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the first antigen-binding domain comprises: (a) a VH region comprising: A HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141 or 149, or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151, or a sequence having 1 or 2 amino acid differences therefrom; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165, or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, or a sequence having 1 or 2 amino acid differences therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155, or a sequence having 1 or 2 amino acid differences therefrom.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the first antigen-binding domain comprises: (a) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141 or 149, a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, and A HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165, LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, and A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155.

27. The antibody or antigen-binding fragment thereof of claim 25 or 26, wherein the first antigen-binding domain comprises: (a) a VH region comprising: (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; and (b) a VL region comprising: (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.

28. The antibody or antigen-binding fragment thereof of any one of claims 25-27, wherein 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.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein the first antigen-binding domain comprises: (a) a VH region having an 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.

30. The antibody or antigen-binding fragment thereof of claim 28 or 29, wherein 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.

31. The antibody or antigen-binding fragment thereof of claim 28 or 29, wherein 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.

32. The antibody or antigen-binding fragment thereof of any one of claims 1 to 31, wherein the second antigen-binding domain comprises: (a) a VH region comprising: A HCDR1 having the amino acid sequence of SEQ ID NO: 41 or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 42 or 53, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence having 1 or 2 amino acid differences therefrom; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 46 or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 47 or a sequence having 1 or 2 amino acid differences therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence having 1 or 2 amino acid differences therefrom.

33. The antibody or antigen-binding fragment thereof of claim 32, wherein the second antigen-binding domain comprises: (a) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO:41, a HCDR2 having the amino acid sequence of SEQ ID NO: 42 or 53, and a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO:46, LCDR2 having the amino acid sequence of SEQ ID NO:47, and LCDR3 having the amino acid sequence of SEQ ID NO:

48.

34. The antibody or antigen-binding fragment thereof of claim 32, wherein the second antigen-binding domain comprises a VH region comprising: (a) a HCDR1 having the amino acid sequence of SEQ ID NO: 60, 61 or 62, (b) a HCDR2 having the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67 or 68, and (c) a HCDR3 having the amino acid sequence of SEQ ID NO: 69, 70, 71 or 72.

35. The antibody or antigen-binding fragment thereof of claim 32, wherein the second antigen-binding domain comprises a VH region comprising: (a) LCDR1 having the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94 or 95, (b) LCDR2 having the amino acid sequence of SEQ ID NO: 96 or 97, and (c) LCDR3 having the amino acid sequence of SEQ ID NO: 98, 99, 100, 101, 102, 103 or 104.

36. The antibody or antigen-binding fragment thereof of any one of claims 32-35, wherein the second antigen-binding domain comprises: (a) a VH region comprising: (i) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 42 and 43, respectively; (ii) HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 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; (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; and (b) a VL region comprising: (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.

37. The antibody or antigen-binding fragment thereof of any one of claims 32-36, wherein 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.

38. The antibody or antigen-binding fragment thereof of claim 37, wherein the second antigen-binding domain comprises: (a) a VH region having an 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.

39. The antibody or antigen-binding fragment thereof of claim 37 or 38, wherein 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.

40. The antibody or antigen-binding fragment thereof of claim 39, wherein 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.

41. The antibody or antigen-binding fragment thereof of any one of claims 1 to 40, wherein the antibody or antigen-binding fragment thereof comprises: (a) a first heavy chain comprising the 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 the 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 the VH region and a second CH region or a fragment thereof of the second antigen-binding domain; and (d) a second light chain comprising the VL region of the second antigen-binding domain and a second CL region or a fragment thereof.

42. An antibody or antigen-binding fragment thereof as described in claim 41, wherein the first CH region and the second CH region each comprise 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:

130.

43. An antibody or antigen-binding fragment thereof as described in claim 34 or 35, wherein the first heavy chain and the second heavy chain form a heterodimer, optionally wherein 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, wherein the constant regions are numbered according to the EU index.

44. An antibody or antigen-binding fragment thereof as described in any one of claims 41-43, wherein 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 antigen-binding fragment.

45. The antibody or antigen-binding fragment thereof of claim 44, wherein the first CH region and / or the second CH region is a human IgG1 CH comprising the following mutations: M252Y, S254T and T256E, wherein the numbering of the constant regions is according to the EU index.

46. ​​An antibody or antigen-binding fragment thereof as described in any one of claims 43-45, wherein the first CH region and / or the second CH region comprises one or more mutations that reduce or eliminate effector function.

47. The antibody or antigen-binding fragment thereof of claim 46, wherein the first CH region and / or the second CH region is a human IgG1 CH comprising one of the following mutations, wherein the numbering of the constant regions is 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.

48. The antibody or antigen-binding fragment thereof of claim 46, wherein the first CH region and / or the second CH region is a human IgG2 CH comprising one of the following mutations, wherein the numbering of the constant region is 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.

49. The antibody or antigen-binding fragment thereof of claim 46, wherein the first CH region and / or the second CH region is a human IgG4 CH comprising one of the following mutations, wherein the numbering of the constant region is 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.

50. The antibody or antigen-binding fragment thereof of any one of claims 41-45, wherein the first CH region and / or the second CH region comprises one or more mutations that enhance effector function.

51. The antibody or antigen-binding fragment thereof of claim 50, wherein the first CH region and / or the second CH region is a human IgG1 CH comprising one of the following mutations, wherein the numbering of the constant regions is 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.

52. The antibody or antigen-binding fragment thereof of any one of claims 43-51, wherein at least one of the first heavy chain and the second heavy chain is non-fucosylated.

53. The antibody or antigen-binding fragment thereof of claim 50, wherein the antibody or antigen-binding fragment thereof is produced in a cell line, the cell line (a) having a knockout of α-1,6-fucosyltransferase (Fut8); or (b) Overexpression of β1,4-N-acetylglucosaminyltransferase III (GnT-III) and optionally overexpression of Golgi μ-mannosidase II (ManII).

54. The antibody or antigen-binding fragment thereof of any one of claims 43-53, wherein the first CL region and / or the second CL region is a human kappa constant region.

55. The antibody or antigen-binding fragment thereof of any one of claims 43-54, wherein: (a)(i) the first antigen binding domain comprises: A VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 3, 21 and 5, respectively; and A VL region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 32, 12 and 13, respectively; and (ii) the second antigen-binding domain comprises: a VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 43, respectively; and A VL region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively; or (b)(i) the first antigen binding domain comprises: A VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 122, 123 and 124, respectively; and A VL region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 127, 128 and 129, respectively; and (ii) the second antigen-binding domain comprises: a VH region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 41, 53 and 43, respectively; and The VL regions comprise LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 46, 47 and 48, respectively.

56. The antibody or antigen-binding fragment thereof of claim 55, wherein: (a) the first antigen-binding domain comprises a VH region having an amino acid sequence of SEQ ID NO: 25 and a VL region having an amino acid sequence of SEQ ID NO: 38; and The second antigen binding domain comprises a VH region having an amino acid sequence of SEQ ID NO: 52 and a VL region having an amino acid sequence of SEQ ID NO: 87; or (b) the first antigen-binding domain comprises a VH region having an amino acid sequence of SEQ ID NO: 121 and a VL region having an 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.

57. The antibody or antigen-binding fragment thereof of any one of claims 1-56, wherein the antigen-binding fragment is Fab, Fab', Fab2, F(ab')2, Fv, single-chain Fv (scFv) or a diabody.

58. The antibody or antigen-binding fragment thereof of any one of claims 1-57, wherein the antibody or antigen-binding fragment thereof is conjugated to an agent.

59. The antibody or antigen-binding fragment thereof of claim 58, wherein the agent is a cytotoxic agent or a label.

60. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-59.

61. The composition of claim 60, wherein the antibody or antigen-binding fragment comprises an Fc region and N-glycoside-linked carbohydrate chains attached to the Fc region, optionally wherein less than 50% of the N-glycoside-linked carbohydrate chains contain fucose residues.

62. The composition of claim 61, wherein substantially none of the N-glycosidically linked carbohydrate chains contain fucose residues.

63. An antibody or antigen-binding fragment thereof capable of binding to siglec-6, comprising: (a) a heavy chain variable (VH) region comprising: A heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 133, 141 or 149, or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151, or a sequence having 1 or 2 amino acid differences therefrom; and (b) a light chain variable (VL) region comprising: A light chain complementary determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165, or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, or a sequence having 1 or 2 amino acid differences therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155, or a sequence having 1 or 2 amino acid differences therefrom.

64. The antibody or antigen-binding fragment thereof of claim 63, comprising: (a) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 133, 141 or 149, a HCDR2 having the amino acid sequence of SEQ ID NO: 134, 142, 150 or 160, and A HCDR3 having the amino acid sequence of SEQ ID NO: 135, 143 or 151; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 137, 145, 153 or 165, LCDR2 having the amino acid sequence of SEQ ID NO: 138, 146 or 154, and A LCDR3 having the amino acid sequence of SEQ ID NO: 139, 147 or 155.

65. The antibody or antigen-binding fragment thereof of claim 63, comprising: (a)(i) a VH region comprising: A HCDR1 having the amino acid sequence of SEQ ID NO: 133 or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 134, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 135, or a sequence having 1 or 2 amino acid differences therefrom; and (ii) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 137 or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 138 or a sequence having 1 or 2 amino acid differences therefrom, and LCDR3 having the amino acid sequence of SEQ ID NO: 139, or a sequence having 1 or 2 amino acid differences therefrom; or (b)(i) a VH region comprising: A HCDR1 having the amino acid sequence of SEQ ID NO: 141 or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 142, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 143, or a sequence having 1 or 2 amino acid differences therefrom; and (ii) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 145 or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 146 or a sequence having 1 or 2 amino acid differences therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 147, or a sequence having 1 or 2 amino acid differences therefrom; or (c)(i) a VH region comprising: A HCDR1 having the amino acid sequence of SEQ ID NO: 149 or a sequence having 1 or 2 amino acid differences therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 150 or 160, or a sequence having 1 or 2 amino acid differences therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 151, or a sequence having 1 or 2 amino acid differences therefrom; and (ii) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 153 or 165, or a sequence having 1 or 2 amino acid differences therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 154, or a sequence having 1 or 2 amino acid differences therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 155, or a sequence having 1 or 2 amino acid differences therefrom.

66. The antibody or antigen-binding fragment thereof of claim 65, comprising: (a)(i) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 133, a HCDR2 having the amino acid sequence of SEQ ID NO: 134, and a HCDR3 having the amino acid sequence of SEQ ID NO: 135; and (ii) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 137, LCDR2 having the amino acid sequence of SEQ ID NO: 138, and LCDR3 having the amino acid sequence of SEQ ID NO: 139; or (b)(i) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 141, a HCDR2 having the amino acid sequence of SEQ ID NO: 142, and a HCDR3 having the amino acid sequence of SEQ ID NO: 143; and (ii) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 145, LCDR2 having the amino acid sequence of SEQ ID NO: 146, and LCDR3 having the amino acid sequence of SEQ ID NO: 147; or (c)(i) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO: 149, a HCDR2 having the amino acid sequence of SEQ ID NO: 150 or 160, and a HCDR3 having the amino acid sequence of SEQ ID NO: 151; and (ii) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO: 153 or 165, LCDR2 having the amino acid sequence of SEQ ID NO: 154, and LCDR3 having the amino acid sequence of SEQ ID NO:

155.

67. The antibody or antigen-binding fragment thereof of any one of claims 63-66, comprising: (a) a VH region comprising: (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; and (b) a VL region comprising: (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.

68. The antibody or antigen-binding fragment thereof of any one of claims 63-67, wherein 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.

69. The antibody or antigen-binding fragment thereof of any one of claims 63-68, wherein 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.

70. The antibody or antigen-binding fragment thereof of any one of claims 63-69, wherein (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.

71. The antibody or antigen-binding fragment thereof of any one of claims 63-69, wherein (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.

72. The antibody or antigen-binding fragment thereof of any one of claims 63-71, wherein the antibody is a humanized antibody.

73. A humanized antibody or antigen-binding fragment thereof capable of binding to siglec-6, comprising: (a) a heavy chain variable domain (VH) comprising: a heavy chain complementarity determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 3, a HCDR2 having the amino acid sequence of SEQ ID NO: 21, and a HCDR3 having the amino acid sequence of SEQ ID NO: 5; and (a) a light chain variable domain (VL) comprising: a light chain complementary 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.

74. The antibody or antigen-binding fragment thereof of claim 73, wherein 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.

75. The antibody or antigen-binding fragment thereof of claim 73 or 74, wherein The VH region comprises the amino acid sequence of SEQ ID NO: 20, 23, 25, 27 or 29; and The VL region comprises the amino acid sequence of SEQ ID NO:31, 36 or 38.

76. The antibody or antigen-binding fragment thereof of any one of claims 73-75, wherein (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.

77. The antibody or antigen-binding fragment thereof of claim 76, wherein 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.

78. The antibody or antigen-binding fragment thereof of any one of claims 63-77, wherein the antibody or antigen-binding fragment thereof is capable of activating the antigen-binding activity with a K of at most about 10 nM, at most about 5 nM, or at most about 1 nM. D Value binding siglec-6.

79. The antibody or antigen-binding fragment thereof of any one of claims 63-78, wherein the antibody or antigen-binding fragment thereof (a) Ability to bind to human siglec-6; (b) able to bind to cynomolgus monkey siglec-6; (c) capable of binding to mast cells, optionally preferentially over another immune cell (e.g., a T cell, a dendritic cell, a macrophage, a neutrophil, or a basophil); (d) does not induce mast cell degranulation; and / or (e) having an internalization rate that is comparable or lower than a reference antibody capable of binding to silect-6, optionally wherein 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.

80. A humanized antibody or antigen-binding fragment thereof capable of binding to c-Kit, comprising: (a) a heavy chain variable domain (VH) comprising: A heavy chain complementary determining region 1 (HCDR1) having the amino acid sequence of SEQ ID NO: 41 or a sequence having 1 amino acid difference therefrom, a HCDR2 having the amino acid sequence of SEQ ID NO: 53 or a sequence having 1 amino acid difference therefrom, and A HCDR3 having the amino acid sequence of SEQ ID NO: 43, or a sequence having 1 amino acid difference therefrom; and (a) a light chain variable domain (VL) comprising: A light chain complementary determining region 1 (LCDR1) having the amino acid sequence of SEQ ID NO: 46 or a sequence having 1 amino acid difference therefrom, LCDR2 having the amino acid sequence of SEQ ID NO: 47 or a sequence having 1 amino acid difference therefrom, and A LCDR3 having the amino acid sequence of SEQ ID NO: 48, or a sequence having 1 amino acid difference therefrom.

81. The antibody or antigen-binding fragment thereof of claim 80, comprising (a) a VH region comprising: a HCDR1 having the amino acid sequence of SEQ ID NO:41, a HCDR2 having the amino acid sequence of SEQ ID NO:53, and a HCDR3 having the amino acid sequence of SEQ ID NO: 43; and (b) a VL region comprising: LCDR1 having the amino acid sequence of SEQ ID NO:46, LCDR2 having the amino acid sequence of SEQ ID NO:47, and LCDR3 having the amino acid sequence of SEQ ID NO:

48.

82. The antibody or antigen-binding fragment thereof of claim 80, wherein the VH comprises: (a) a HCDR1 having the amino acid sequence of SEQ ID NO: 60, 61 or 62, (b) a HCDR2 having the amino acid sequence of SEQ ID NO: 63, 64, 65, 66, 67 or 68, and (c) a HCDR3 having the amino acid sequence of SEQ ID NO: 69, 70, 71 or 72.

83. The antibody or antigen-binding fragment thereof of claim 80 or 82, wherein the VL comprises: (a) LCDR1 having the amino acid sequence of SEQ ID NO: 90, 91, 92, 93, 94 or 95, (b) LCDR2 having the amino acid sequence of SEQ ID NO: 96 or 97, and (c) LCDR3 having the amino acid sequence of SEQ ID NO: 98, 99, 100, 101, 102, 103 or 104.

84. The antibody or antigen-binding fragment thereof of any one of claims 80 to 83, wherein: (a) the VH region comprises: (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; and (b) the VL region comprises: (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.

85. The antibody or antigen-binding fragment thereof of any one of claims 80-84, wherein: 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: 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 NO:87, 89, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 or 119.

86. The antibody or antigen-binding fragment thereof of any one of claims 80-85, wherein: The VH region 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; and The VL region 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.

87. The antibody or antigen-binding fragment thereof of any one of claims 80-86, wherein (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 comprises the amino acid sequence of SEQ ID NO: 52, and the VL region comprises the amino acid sequence of SEQ ID NO: 89; or (g) 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:

89.

88. The antibody or antigen-binding fragment thereof of claim 87, wherein 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.

89. The antibody or antigen-binding fragment thereof of any one of claims 80-88, wherein the antibody or antigen-binding fragment thereof is capable of quantifying the antigen-binding activity of the antibody or antigen-binding fragment thereof with a K between about 10 and about 500 nM, between about 20 and about 100 nM; or between about 25 and about 80 nM. D Value binding siglec-6.

90. The antibody or antigen-binding fragment thereof of any one of claims 80-89, wherein the antibody or antigen-binding fragment thereof (a) Capable of binding to human c-Kit; (b) capable of binding to cynomolgus monkey c-Kit; and / or (c) has comparable or lower toxicity than a reference antibody capable of binding to c-Kit, optionally wherein the reference antibody 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.

91. The antibody or antigen-binding fragment thereof of any one of claims 63-90, wherein the antigen-binding fragment is Fab, F(ab')2, Fab', single-chain Fv (scFv), Fv fragment, Fd fragment or diabody.

92. The antibody or antigen-binding fragment thereof of any one of claims 63-91, wherein the antibody or antigen-binding fragment thereof comprises an antibody heavy chain constant region, optionally wherein the constant region is a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region is an IgG1 heavy chain constant region.

93. The antibody or antigen-binding fragment thereof of claim 92, wherein the constant region comprises one or more mutations that increase the serum half-life of the antibody or antigen-binding fragment thereof.

94. The antibody or antigen-binding fragment thereof of claim 92 or 93, wherein the constant region comprises one or more mutations that reduce or eliminate effector function.

95. The antibody or antigen-binding fragment thereof of claim 92 or 93, wherein the constant region comprises one or more mutations that enhance effector function.

96. The antibody or antigen-binding fragment thereof of any one of claims 63-95, wherein at least one of the first heavy chain and the second heavy chain is afucosylated (eg, at least 75% afucosylated).

97. The antibody or antigen-binding fragment thereof of any one of claims 63-96, conjugated to a therapeutic agent or a label.

98. A polynucleotide encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-59 and 63-97.

99. An expression vector comprising the polynucleotide of claim 96.

100. A host cell comprising the polynucleotide of claim 98 or the expression vector of claim 99.

101. The host cell of claim 100, wherein the host cell is a mammalian or insect cell.

102. The host cell of claim 100 or 101, wherein the host cell (a) comprises a Fut8 knockout; and / or (b) overexpresses GnT-III and optionally overexpresses ManII.

103. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-59 and 63-97 and a pharmaceutically acceptable carrier.

104. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1-59 and 63-97, the composition of any one of claims 58-60, or the pharmaceutical composition of claim 103.

105. The method of claim 104, wherein the disease or disorder is a mast cell-related disease or disorder.

106. The method of claim 105, wherein the disease or condition is an autoimmune disease, allergy, inflammation, pruritus, or cancer.

107. The method of any one of claims 104-106, wherein the disease or disorder is associated with expression of siglec-6 and / or c-Kit.

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