Antibodies to CD24 and uses thereof

Abstract

The present specification describes CD24 binding antibodies and their fragments.The antibodies described herein have the useful property of not binding to either B cells or activated T cells.Such antibodies can have an enhanced safety profile with reduced immune side effects.In one aspect, the present specification describes an antibody or its antigen-binding fragment that binds to CD24, and the antibody does not bind to T lymphocytes.In certain embodiments, the antibody does not bind to activated T lymphocytes.

Description

[Technical Field]

[0001] cross reference This patent application claims the benefit of International Patent Application No. PCT / CN2022 / 128003, filed October 27, 2022; International Patent Application No. PCT / CN2022 / 128297, filed October 28, 2022; and International Patent Application No. PCT / CN2023 / 121689, filed September 26, 2023; which applications are incorporated herein by reference in their entirety. [Background technology]

[0002] background CD24 is a highly glycosylated protein with a small protein core that is linked to the plasma membrane via a glycosyl-phosphatidylinositol anchor. CD24 is primarily expressed by immune cells but is frequently overexpressed in human tumors. In cancer, CD24 is a regulator of cell migration, invasion, and proliferation. Therefore, CD24 is an attractive target for monoclonal antibodies as cancer therapy. Summary of the Invention [Means for solving the problem]

[0003] Abstract The present invention describes an antibody that specifically binds to CD24.The antibody described herein has the useful property of not binding to either B cell or activated T cell.This antibody can have an improved safety profile and reduce immune side effects.

[0004] In one aspect, described herein is an antibody or antigen-binding fragment thereof that binds to CD24, wherein the antibody does not bind to T lymphocytes. In certain embodiments, the antibody does not bind to activated T lymphocytes. In certain embodiments, the antibody does not bind to B lymphocytes. In certain embodiments, the antibody binds to CD24 at a concentration of 1x10 -8 Less than K DIn certain embodiments, the antibody binds to CD24 at 1×10 -9 Less than K D In certain embodiments, the antibody binds to an epitope consisting of the amino acid sequence of SEQ ID NO:501. In certain embodiments, the antibody is a humanized antibody or a chimeric antibody. In certain embodiments, the antibody is a Fab, F(ab)2, or single-chain variable fragment (scFv). In certain embodiments, the antibody is a heavy-chain antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody is an IgG antibody. In certain embodiments, the antibody is an IgG1 isotype. In certain embodiments, the antibody is an IgG4 isotype. In certain embodiments, the antibody binds to the serine at residue 18 of SEQ ID NO:500. In certain embodiments, the antibody requires the presence of the serine at residue 18 of SEQ ID NO:500 for binding. In certain embodiments, the antibody binds to an epitope consisting of the amino acid sequence of SEQ ID NO:501.

[0005] In another aspect, also described herein is an antibody or antigen-binding fragment thereof that binds to CD24, wherein the antibody or antigen-binding fragment thereof comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 401; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 405; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 407; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 411. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 401; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 405; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 407; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 411. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 402; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 406; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 408; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 412. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 402; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 403;(c) light chain complementarity determining region 3 (LCDR3) set forth in SEQ ID NO: 406; (d) heavy chain complementarity determining region 1 (HCDR1) set forth in SEQ ID NO: 408; (e) heavy chain complementarity determining region 2 (HCDR2) set forth in SEQ ID NO: 409; and / or (f) heavy chain complementarity determining region 3 (HCDR3) set forth in SEQ ID NO: 412. In certain embodiments, the antibody comprises (a) light chain complementarity determining region 1 (LCDR1) set forth in SEQ ID NO: 402; (b) light chain complementarity determining region 2 (LCDR2) set forth in SEQ ID NO: 403; (c) light chain complementarity determining region 3 (LCDR3) set forth in SEQ ID NO: 406; (d) heavy chain complementarity determining region 1 (HCDR1) set forth in SEQ ID NO: 408; (e) heavy chain complementarity determining region 2 (HCDR2) set forth in SEQ ID NO: 409; and / or (f) heavy chain complementarity determining region 3 (HCDR3) set forth in SEQ ID NO: 412; and the antibody does not bind or does not substantially bind to either B cells or activated T cells. In certain embodiments, the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 23, 33, 43, 53, 63, 73, 83, 703, 713, 723, 733, and 743; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 24, 34, 44, 54, 64, 74, 84, 704, 714, 724, 734, and 744; (c) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 25, 35, 45, 55, 65, 75, 85, 705, 715 (d) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76, 86, 706, 716, 726, 736, and 746; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77, 87, 707, 717, 727, 737, and 747;and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 28, 38, 48, 58, 68, 78, 88, 708, 718, 728, 738, and 748. In certain embodiments, the antibody comprises a combination of LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, and HCDR3, including: (i) LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 14, LCDR3 comprising SEQ ID NO: 15, HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, and HCDR3 comprising SEQ ID NO: 18; (ii) LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ ID NO: 24, LCDR3 comprising SEQ ID NO: 25, HCDR1 comprising SEQ ID NO: 26, HCDR2 comprising SEQ ID NO: 27, and HCDR3 comprising SEQ ID NO: 28; (iii) LCDR1 comprising SEQ ID NO: 33, LCDR2 comprising SEQ ID NO: 34, LCDR3 comprising SEQ ID NO: 35, HCDR1 comprising SEQ ID NO: 36, HCDR2 comprising SEQ ID NO: 37, and HCDR3 comprising SEQ ID NO: 38; (iv) LCDR1 comprising SEQ ID NO: 43, LCDR2 comprising SEQ ID NO: 44, LCDR3 comprising SEQ ID NO: 45, and HCDR3 comprising SEQ ID NO: 46 (v) LCDR1 comprising SEQ ID NO:53, LCDR2 comprising SEQ ID NO:54, LCDR3 comprising SEQ ID NO:55, HCDR1 comprising SEQ ID NO:56, HCDR2 comprising SEQ ID NO:57, HCDR3 comprising SEQ ID NO:58; (vi) LCDR1 comprising SEQ ID NO:63, LCDR2 comprising SEQ ID NO:64, LCDR3 comprising SEQ ID NO:65, HCDR1 comprising SEQ ID NO:66, HCDR2 comprising SEQ ID NO:67, HCDR3 comprising SEQ ID NO:68; (vii) LCDR1 comprising SEQ ID NO:73, LCDR2 comprising SEQ ID NO:74, LCDR3 comprising SEQ ID NO:75, HCDR1 comprising SEQ ID NO:76, HCDR2 comprising SEQ ID NO:77, HCDR3 comprising SEQ ID NO:78; (viii) LCDR1 comprising SEQ ID NO:83, LCDR2 comprising SEQ ID NO:84, LCDR3 comprising SEQ ID NO:85, HCDR1 comprising SEQ ID NO:86, HCDR2 comprising SEQ ID NO:87, HCDR3 comprising SEQ ID NO:88;(ix) LCDR1 comprising SEQ ID NO: 703, LCDR2 comprising SEQ ID NO: 704, LCDR3 comprising SEQ ID NO: 705, HCDR1 comprising SEQ ID NO: 706, HCDR2 comprising SEQ ID NO: 707, HCDR3 comprising SEQ ID NO: 708; (x) LCDR1 comprising SEQ ID NO: 713, LCDR2 comprising SEQ ID NO: 714, LCDR3 comprising SEQ ID NO: 715, HCDR1 comprising SEQ ID NO: 716, HCDR2 comprising SEQ ID NO: 717, HCDR3 comprising SEQ ID NO: 718; (xi) LCDR1 comprising SEQ ID NO: 723, LCDR2 comprising SEQ ID NO: 724, LCDR3 comprising SEQ ID NO: 725, SEQ ID NO: 726 (xii) an HCDR1 comprising SEQ ID NO: 733, an LCDR2 comprising SEQ ID NO: 734, an LCDR3 comprising SEQ ID NO: 735, an HCDR1 comprising SEQ ID NO: 736, an HCDR2 comprising SEQ ID NO: 737, an HCDR3 comprising SEQ ID NO: 738; and (xiii) an LCDR1 comprising SEQ ID NO: 743, an LCDR2 comprising SEQ ID NO: 744, an LCDR3 comprising SEQ ID NO: 745, an HCDR1 comprising SEQ ID NO: 746, an HCDR2 comprising SEQ ID NO: 747, an HCDR3 comprising SEQ ID NO: 748. In certain embodiments, the antibody comprises a combination of LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, and HCDR3, the combinations being: (i) LCDR1 as set forth in SEQ ID NO: 13, LCDR2 as set forth in SEQ ID NO: 14, LCDR3 as set forth in SEQ ID NO: 15, HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18; (ii) LCDR1 as set forth in SEQ ID NO: 23, LCDR2 as set forth in SEQ ID NO: 24, LCDR3 as set forth in SEQ ID NO: 25, HCDR1 as set forth in SEQ ID NO: 26, HCDR2 as set forth in SEQ ID NO: 27, HCDR3 as set forth in SEQ ID NO: 28; (iii) LCDR1 as set forth in SEQ ID NO: 33, LCDR2 as set forth in SEQ ID NO: 34, LCDR3 as set forth in SEQ ID NO: 35, HCDR1 as set forth in SEQ ID NO: 36, HCDR2 as set forth in SEQ ID NO: 37, HCDR3 as set forth in SEQ ID NO: 38;(iv) LCDR1 as set forth in SEQ ID NO:43, LCDR2 as set forth in SEQ ID NO:44, LCDR3 as set forth in SEQ ID NO:45, HCDR1 as set forth in SEQ ID NO:46, HCDR2 as set forth in SEQ ID NO:47, HCDR3 as set forth in SEQ ID NO:48; (v) LCDR1 as set forth in SEQ ID NO:53, LCDR2 as set forth in SEQ ID NO:54, LCDR3 as set forth in SEQ ID NO:55, HCDR1 as set forth in SEQ ID NO:56, HCDR2 as set forth in SEQ ID NO:57, HCDR3 as set forth in SEQ ID NO:58; (vi) LCDR1 as set forth in SEQ ID NO:63, LCDR2 as set forth in SEQ ID NO:64, LCDR3 as set forth in SEQ ID NO:65, HCDR1 as set forth in SEQ ID NO:66, HCDR2 as set forth in SEQ ID NO:67, HCDR3 as set forth in SEQ ID NO:68; (vii) LCDR1 as set forth in SEQ ID NO:73, LCDR2 as set forth in SEQ ID NO:74, LCDR3 as set forth in SEQ ID NO:75, SEQ ID NO: (viii) LCDR1 as set forth in SEQ ID NO: 83, LCDR2 as set forth in SEQ ID NO: 84, LCDR3 as set forth in SEQ ID NO: 85, HCDR1 as set forth in SEQ ID NO: 86, HCDR2 as set forth in SEQ ID NO: 87, HCDR3 as set forth in SEQ ID NO: 88; (ix) LCDR1 as set forth in SEQ ID NO: 703, HCDR2 as set forth in SEQ ID NO: 704 (x) LCDR1 as set forth in SEQ ID NO: 713, LCDR2 as set forth in SEQ ID NO: 714, LCDR3 as set forth in SEQ ID NO: 715, HCDR1 as set forth in SEQ ID NO: 716, HCDR2 as set forth in SEQ ID NO: 717, HCDR3 as set forth in SEQ ID NO: 718;(xi) LCDR1 as set forth in SEQ ID NO: 723, LCDR2 as set forth in SEQ ID NO: 724, LCDR3 as set forth in SEQ ID NO: 725, HCDR1 as set forth in SEQ ID NO: 726, HCDR2 as set forth in SEQ ID NO: 727, HCDR3 as set forth in SEQ ID NO: 728; (xii) LCDR1 as set forth in SEQ ID NO: 733, LCDR2 as set forth in SEQ ID NO: 734, LCDR3 as set forth in SEQ ID NO: 735, HCDR1 as set forth in SEQ ID NO: 736, HCDR2 as set forth in SEQ ID NO: 737, HCDR3 as set forth in SEQ ID NO: 738; and (xiii) LCDR1 as set forth in SEQ ID NO: 743, LCDR2 as set forth in SEQ ID NO: 744, LCDR3 as set forth in SEQ ID NO: 745, HCDR1 as set forth in SEQ ID NO: 746, HCDR2 as set forth in SEQ ID NO: 747, HCDR3 as set forth in SEQ ID NO: 748. In certain embodiments, the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) an amino acid sequence set forth in SEQ ID NO: 14; (c) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 15; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 24; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 25; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 26; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 27; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 33; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 34; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 35; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 36; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 37; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 38.In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 43; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 44; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 45; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 46; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 47; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 53; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 54; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 55; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 56; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 57; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 58. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 63; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 64; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 65; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 66; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 68.In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 73; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 74; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 75; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 76; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 77; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 78. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 83; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 84; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 85; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 86; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88. In certain embodiments, the antibody comprises an immunoglobulin light chain amino acid variable region sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.In certain embodiments, the antibody comprises an immunoglobulin light chain amino acid variable region sequence identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320. In certain embodiments, the antibody comprises (i) a light chain variable region (VL) having an LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having an HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to any one of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, and North definition. In certain embodiments, the antibody comprises: (i) a light chain variable region (VL) having an LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having an HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined using a hybrid scheme of any two or three of the following: Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, and North definition.In certain embodiments, the antibody comprises (i) a light chain variable region (VL) having LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Kabat definition. In certain embodiments, the antibody comprises (i) a light chain variable region (VL) having LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Chothia definition. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 275, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 276. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 277, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 278.In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 277; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 278. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 309, and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 310. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 309; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 310. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 155, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 156. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 157, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 158. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 157; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 158. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 169, and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 170. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 169; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 170. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 267, and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 268. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 267; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 268. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 271, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 272. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is identical to SEQ ID NO: 271; and an immunoglobulin heavy chain variable region amino acid sequence that is identical to SEQ ID NO: 272. In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the immunoglobulin light chain variable region sequences in Table 1; and an immunoglobulin heavy chain amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the immunoglobulin light chain variable region sequences in Table 1. In certain embodiments, the antibody comprises an immunoglobulin light chain amino acid sequence that is identical to any one of the immunoglobulin light chain variable region sequences in Table 1; and an immunoglobulin heavy chain variable region amino acid sequence that is identical to any one of the immunoglobulin heavy chain variable region sequences in Table 1. In certain embodiments, the antibody comprises an arginine substitution at position 17 of the immunoglobulin light chain according to Kabat numbering. In certain embodiments, the antibody comprises a tyrosine substitution at position 12 of the immunoglobulin light chain according to Kabat numbering.In certain embodiments, described herein is a pharmaceutical composition comprising an antibody described herein and a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the antibody or pharmaceutical composition is formulated for intravenous administration. Also described herein is a nucleic acid encoding an antibody of the present disclosure. In certain embodiments, described herein is an expression vector comprising a nucleic acid of the present disclosure. Also described herein is a eukaryotic cell comprising the expression vector. In certain embodiments, the eukaryotic cell is a CHO cell line. Also described herein is a method of treating cancer or tumor, the method comprising administering to an individual suffering from the cancer or tumor an antibody of the present disclosure or a pharmaceutical composition comprising the antibody. In certain embodiments, the cancer or tumor is a solid tissue cancer. In certain embodiments, the cancer or tumor comprises breast cancer, colon cancer, or ovarian cancer. In certain embodiments, the breast cancer is triple-negative breast cancer. In certain embodiments, the cancer or tumor is a hematological cancer. Also described herein is a method for inhibiting CD24 signal transduction in cancer cells, comprising contacting the cancer cells with the antibody of the present disclosure or the pharmaceutical composition of the present disclosure.In certain embodiments, CD24 signal transduction is not inhibited in non-neoplastic B lymphocytes or non-neoplastic T lymphocytes.In certain embodiments, the cancer cells are cancer cells that exist in vivo in an individual.

[0006] BRIEF DESCRIPTION OF THE DRAWINGS The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features described herein or the advantages thereof may be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and in which the accompanying drawings are included: [Brief explanation of the drawings]

[0007] [Figure 1]1A and 1B illustrate the dose-response curves of mature CD24 antibodies (single mutations) against CD24(27-56aa)-GST(NEOX-4) by ELISA.

[0008] [Figure 2A] 2A and 2B illustrate the dose response curves of mature CD24 antibodies (combined mutations) against CD24(27-56aa)-GST(NEOX-4) by ELISA. [Figure 2B] 2A and 2B illustrate the dose response curves of mature CD24 antibodies (combined mutations) against CD24(27-56aa)-GST(NEOX-4) by ELISA.

[0009] [Figure 3] 3A and 3B illustrate the dose-response curve of CD24 antibody on NALM6 cells.

[0010] [Figure 4] 4A and 4B illustrate the dose-response curve of CD24 antibody on MDA-MB-468 cells.

[0011] [Figure 5] 5A, 5B, and 5C illustrate the ADCC effect induced by anti-CD24 antibody on NALM6 and MDA-MB-468 cells.

[0012] [Figure 6] 6A, 6B, 6C, and 6D illustrate the ADCP effect induced by anti-CD24 antibody on NALM6 and MDA-MB-468 cells.

[0013] [Figure 7AB] 7A, 7B, 7C and 7D illustrate dose-response binding curves with primary B cells and activated T cells. [Figure 7CD] 7A, 7B, 7C and 7D illustrate dose-response binding curves with primary B cells and activated T cells.

[0014] [Figure 8] FIG. 8 illustrates antibody binding by ELISA to NEOX-4 treated with N-glycanase, neuraminidase, or N-glycanase plus neuraminidase.

[0015] [Figure 9] Figures 9A, 9B, and 9C illustrate CD24 antibody binding to N-glycanase, neuraminidase, or N-glycanase + neuraminidase treated NALM6 (9A), MDA-MB-468 (9B), and primary B cells (9C).

[0016] [Figure 10] FIG. 10 illustrates epitope binding by blocking peptides.

[0017] [Figure 11] FIG. 11 illustrates epitope determination by alanine spanning mutagenesis.

[0018] [Figure 12] FIG. 12 illustrates the epitope comparison from the binding studies.

[0019] [Figure 13] FIG. 13 illustrates CD24 antibody in vivo efficacy in the MC38-hCD24 syngeneic model.

[0020] [Figure 14] Figures 14A and 14B illustrate CD24 antibody in vivo efficacy in the NALM6 CDX model.

[0021] [Figure 15] Figures 15A and 15B illustrate CD24 antibody in vivo efficacy in the MDA-MB-468 CDX model. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description In one aspect, an antibody or antigen-binding fragment thereof that binds to CD24 is described herein, wherein the antibody does not bind to T lymphocytes. In certain embodiments, the antibody does not bind to activated T lymphocytes. In certain embodiments, the antibody does not bind to B lymphocytes.

[0023] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the provided embodiments may be practiced without these details. Unless the context otherwise requires, throughout this specification and the following claims, the words "comprise" and variations thereof, such as "comprises" and "comprising," should be construed in an open-ended, inclusive sense, i.e., "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. It should be noted that the term "or" generally is employed in its sense including "and / or" unless the context clearly dictates otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0024] As used herein, the term "about" refers to an amount near the stated amount, to the extent of 10% or less.

[0025] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of suffering from, or is at risk of developing at least one disease that the described compositions and methods are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0026] Among the antibodies provided are monoclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Antibodies include antibody-conjugates and molecules (e.g., chimeric molecules) comprising antibodies. Thus, antibodies include, but are not limited to, full-length and native antibodies, as well as fragments and portions thereof that retain their binding specificity (e.g., any specific binding portion thereof, including those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM)); and biologically relevant (antigen-binding) fragments or specific binding portions thereof (including, but not limited to, Fab, F(ab')2, Fv, and scFv (single chain or related entities)). Monoclonal antibodies are generally of substantially homogeneous antibody composition; thus, any individual antibodies comprised within a monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibody may comprise a human IgG1 constant region. The monoclonal antibody may comprise a human IgG4 constant region. As used herein, the human constant region may comprise a natural human immunoglobulin constant region or a variant thereof that further comprises common modifications that confer altered effector function, prolonged half-life, or other properties. Such modifications include, according to EU numbering, D356E, L358M, L234A, L235A, P329G, M252Y, S254T, and / or T256E.

[0027] The term "antibody" is used herein in the broadest sense and includes monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (e.g., heavy-chain antibodies), including single-chain variable fragments (sFv or scFv), and single-chain domain antibodies (e.g., sdAb, sdFv, nanobodies, VHH) or fragments thereof. The term also includes genetically engineered and / or otherwise modified forms of immunoglobulins (e.g., intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies). The term "antibody" includes antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs. Unless otherwise stated, the term "antibody" should be understood to include functional antibody fragments thereof. The term also includes intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0028] Many techniques known in the art can be used to prepare suitable antibodies, e.g., recombinant, monoclonal, or polyclonal antibodies (see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd ed. 1986)). Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells; for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Random combination of heavy and light chain gene products generates a large pool of antibodies with different antigen specificities (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for producing single-chain or recombinant antibodies (U.S. Pat. Nos. 4,946,778 and 4,816,567) can be adapted to produce the antibodies of the present disclosure.Also, transgenic mice, or other organisms such as other mammals, can be used to express humanized or human antibodies (see, e.g., U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also be generated that are bispecific, i.e., capable of recognizing two different antigens (see, e.g., WO 93 / 08829; Traunecker et al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods in Enzymology 121:210 (1986)). The antibody can also be a heteroconjugate (e.g., two covalently linked antibodies) or an immunotoxin (see, e.g., U.S. Pat. No. 4,676,980; WO 91 / 00360; WO 92 / 200373; and EP 03089).

[0029] "Linker" as used herein also refers to a "linker sequence," "spacer," "tethering sequence," or grammatical equivalents thereof. A "linker," as referred to herein, connects two separate molecules that themselves have target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides, or comprise separate domains of the same polypeptide. For example, two separate binding moieties or a heavy / light chain pair. Many strategies can be used to covalently link molecules together. The linkers described herein can be utilized to connect the light chain variable region and the heavy chain variable region in an scFv molecule; or can be used to tether an scFv or other antigen-binding fragment to the N- or C-terminus of an antibody heavy chain; or to the N- or C-terminus of a light chain to create a bispecific or multispecific binding molecule. These include, but are not limited to, polypeptide linkages between the N- and C-termini of proteins or protein domains, linkages via disulfide bonds, and linkages via chemical cross-linking reagents. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant technology or peptide synthesis. The linker peptide may primarily contain the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of a length suitable for connecting two molecules in a manner that allows them to assume the correct conformation relative to each other so that they retain the desired activity. In one embodiment, the linker is about 1 to 50 amino acids in length or about 1 to 30 amino acids in length. In one embodiment, a linker of 1 to 20 amino acids in length may be used. Useful linkers include glycine-serine polymers (e.g., (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n (where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers).Exemplary linkers for linking antibody fragments or single-chain variable fragments may include AAEPKSS, AAEPKSS, GGGG, or GGGGDKTHTCPPCP. Alternatively, various non-proteinaceous polymers may find use as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0030] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. The terms "framework region" and "FR" are known in the art to refer to non-CDR regions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The precise amino acid sequence boundaries of a given CDR or FR can be determined using any of a number of well-known schemes (Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al. (1997) JMB 273,927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003). Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8;309(3):657-70 ("Aho" numbering scheme); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modeling antibodies on the WEB", Protein Eng. 2000 Dec;13(12):819-24 ("AbM" numbering scheme).In certain embodiments, the CDRs of the antibodies described herein may be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0031] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of both the Kabat and Chothia schemes is based on the most common antibody region sequence length, and insertions are controlled by insertion letters, such as "30a", and deletions occur in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.

[0032] The term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that are involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, e.g., Kindt et al., Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). H or V L A V domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may contain complementary V domains. L or V H To screen a library of domains, V domains derived from antibodies that bind the above antigens are selected. H or V Ldomains can be isolated using (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0033] Specific binding or binding of an antibody molecule described herein refers to binding mediated by one or more CDR portions of an antibody. Not all CDRs may be required for specific binding. Specific binding can be demonstrated, for example, by ELISA for a specific described target or antigen, which shows a significant increase in binding compared to an isotype control antibody.

[0034] As used herein, "epitope" refers to a binding determinant of an antibody or fragment described herein that is minimally required for specific binding of the antibody or fragment to a target antigen. When the target antigen is a polypeptide, the epitope may be continuous or discontinuous. A continuous epitope is determined by a single region of the target antigen, while a discontinuous epitope may be formed from two or more distinct regions. A discontinuous epitope may be formed, for example, when the target antigen adopts a tertiary structure in which two amino acid sequences are combined to form a three-dimensional structure bound by an antibody. When the target antigen is a polypeptide, the epitope is generally multiple amino acids linked together in a polypeptide chain. A continuous epitope may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids. An epitope may comprise a contiguous polymer of amino acids, but not all of the amino acids in the polymer may be contacted by amino acid residues of an antibody. Such non-contacting amino acids may be important for the structure and linkage of the contacting amino acids and therefore still constitute part of the epitope. Those skilled in the art can determine whether any given antibody binds the epitope of a reference antibody, for example, by cross-blocking experiments with the reference antibody. In certain embodiments, antibodies that bind the same epitope as the described antibodies are described herein. In certain embodiments, antibodies that are competitively blocked by the described antibodies are described herein. In certain embodiments, antibodies that compete for binding with the described antibodies are described herein.

[0035] Among the antibodies provided are antibody fragments. The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment (e.g., scFv) comprising the heavy chain variable region and / or the light chain variable region.

[0036] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolysis of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment (e.g., a fragment containing a non-naturally occurring arrangement (e.g., one having two or more antibody regions or chains joined by a synthetic linker (e.g., a polypeptide linker) and / or one not produced by enzymatic digestion of a naturally occurring intact antibody). In some aspects, the antibody fragment is an scFv.

[0037] A "humanized" antibody is one in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has undergone humanization to reduce immunogenicity to humans while retaining the specificity and affinity of its parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve antibody specificity or affinity.

[0038] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or a non-human source utilizing a human antibody repertoire or other human antibody coding sequence (including a human antibody library). The term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions (e.g., in which all or substantially all CDRs are non-human).

[0039] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci or which are extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries (including phage display and cell-free libraries) containing antibody coding sequences derived from the human repertoire.

[0040] "CD24" refers to a sialoglycoprotein expressed on the surface of most B lymphocytes and activated T lymphocytes. The amino acid sequence of human CD24 can be found at www.uniprot.org / uniprotkb / P25063 / entry under accession number P2506. In certain embodiments, the anti-CD24 binding antibodies described herein bind to mammalian CD24. In certain embodiments, the anti-CD24 binding antibodies described herein bind to human CD24.

[0041] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains and other peptides, e.g., linkers and connecting peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The terms also include post-expression modifications of the polypeptide (e.g., glycosylation, sialylation, acetylation, phosphorylation, etc.). In some aspects, the polypeptide may contain modifications with respect to the native or naturally occurring sequence, so long as the protein maintains the desired activity. These modifications may be intentional, such as through site-directed mutagenesis, or accidental, such as through mutations of the host producing the protein or errors due to PCR amplification. In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences of the present invention, evaluating one or more biological activities of the polypeptide as described herein, and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at a final construct, provided that the final construct has the desired properties (e.g., antigen binding).

[0042] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of known ways using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code has been submitted, along with user documentation, to the U.S. Copyright Office (Washington, DC, 20559), where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.) or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All comparison parameters are set by the ALIGN-2 program and do not vary.

[0043] In situations where ALIGN-2 is employed for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to a given amino acid sequence B, with a given amino acid sequence B, or to a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y (where X is the number of amino acid residues scored by the sequence alignment program ALIGN-2 as identical matches in that program's alignment of A and B, and Y is the total number of amino acid residues in B). It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0044] In some embodiments, antibodies are altered to increase or decrease their glycosylation (e.g., by altering the amino acid sequence so that one or more glycosylation sites are created or removed). The carbohydrate attached to the Fc region of the antibody can be altered. Native antibodies from mammalian cells typically have an Asn-binding domain in the CH2 domain of the Fc region. 297(See, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharide can be a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, or fucose, attached to the GlcNAc in the stem of the biantennary oligosaccharide structure. Modification of the oligosaccharides in the antibody can be performed, for example, to generate antibody variants with certain improved properties. Antibody glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose can be determined by the ratio of Asn to ATP. 297 Asn compared to the total of all sugar structures bound to 297 The average amount of fucose in the glycan of Asn is determined by calculating the average amount of fucose in the glycan of Asn (see, for example, WO 08 / 077546). 297 refers to an asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues; see, e.g., Edelman et al. Proc Natl Acad Sci U S A. 1969 May; 63(1):78-85). However, Asn 297may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations in the antibody. Such fucosylation variants may have improved ADCC function (see, e.g., Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)). Cell lines, e.g., knockout cell lines and methods for their use, can be used to produce defucosylated antibodies (e.g., Lec13 CHO cells, which are deficient in protein fucosylation, and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, e.g., Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006))). Other antibody glycosylation variants are also included (see, e.g., U.S. Patent No. 6,602,684).

[0045] In some embodiments, the antibodies provided herein have an activity against the antibody target of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D In some embodiments, the antibodies provided herein have an affinity for the antibody target of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or greater (e.g., 10 -8M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 Dissociation constant (K D The target of the antibody may be CD24. D may be measured by any suitable assay. In certain embodiments, K D can be measured using a surface plasmon resonance assay (eg, using a BIACORE®-2000, BIACORE®-3000, or Octet).

[0046] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region herein refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. Fc regions include native sequence Fc regions and variant Fc regions. The Fc region variant can comprise a human Fc region sequence containing amino acid modifications (e.g., substitutions) at one or more amino acid positions (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region).

[0047] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region herein refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. Fc regions include native sequence Fc regions and variant Fc regions. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0048] In some cases, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), which result in target cell killing, albeit by different mechanisms. Thus, in some embodiments, the antibodies described herein comprise a variable domain of the invention combined with a constant domain comprising a different Fc region selected based on the biological activity of the antibody for the intended use. In certain instances, for example, human IgG is classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, each of which contains an Fc region with a unique profile with respect to binding to one or more of the Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16) and the inhibitory receptor FcγRIIB) and with respect to the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA. H131 and with weaker affinity to FcγRIIA R131 FcγRIIIA V158 IgG4 binds to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 the inhibitory receptor, FcγRIIB, has low affinity for IgG1, IgG2, and IgG3 but not for all other Fcγ receptors. Studies have shown that FcγRI does not bind IgG2, and FcγRIIIB does not bind IgG2 or IgG4 (Ibid.). In general, with regard to ADCC and activity, human IgG1≧IgG3>>IgG4≧IgG2.

[0049] In some embodiments, antibodies of the present disclosure are variants with reduced effector function, making them desirable candidates for applications where certain effector functions (e.g., complement fixation and ADCC) are unnecessary or deleterious. Such antibodies may have reduced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, antibodies of the present disclosure are variants with increased effector function for applications where increased immunogenicity is beneficial. Such antibodies may have increased CDC, ADCC, or ADCP, or a combination thereof. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods may be employed (e.g., ACTI TM and CytoTox 96® non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0050] The antibody may have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such antibodies may comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn, including those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 according to the EU numbering system (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260 and 5,624,821; and WO94 / 29351).

[0051] In some embodiments, it may be desirable to generate cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In some embodiments, the substituted residues are present at accessible sites of the antibody. A reactive thiol group may be positioned at the site for conjugation to another moiety, such as a drug moiety or a linker-drug moiety, to generate an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0052] In some embodiments, the antibodies provided herein can be further modified to contain additional nonproteinaceous moieties that are known and available. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when two or more polymers are attached, they can be the same molecule or different molecules.

[0053] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are a type of polynucleotide containing two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to introduce the polynucleotide encoding the polypeptide into cells. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid linked to it. One type of vector is a genome-integrating vector, or "integrated vector," which can become integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. Vectors that can direct the expression of genes to which they are operably linked are referred to herein as "expression vectors." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements (e.g., promoters, enhancers, polyadenylation signals used to control transcription) can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene for facilitating recognition of transformants may also be incorporated. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc. may be employed. Plasmid vectors may be linearized for integration into the genome region. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a lentivirus, adenovirus, or adeno-associated virus. In certain embodiments, the expression vector is an adenovirus. In certain embodiments, the expression vector is an adeno-associated virus. In certain embodiments, the expression vector is a lentivirus.

[0054] As used herein, the terms "homologous," "homology," or "percent homology," when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, may be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990 (as revised in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula has been incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent sequence homology may be determined using the most recent version of BLAST available as of the filing date of this application.

[0055] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise render appropriate cells transgenic for the nucleic acid, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct; 2(5): 466-477. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are cell lines useful for producing antibodies, such as Chinese hamster ovary (CHO) cells, NS0 mouse myeloma cells, or PER.C6® cells. In certain embodiments, the nucleic acids encoding the antibodies are integrated into the genomic loci of cells useful for producing antibodies. In certain embodiments, described herein are methods of producing an antibody, the method comprising culturing cells containing nucleic acid encoding the antibody under in vitro conditions sufficient to allow for the production and secretion of the antibody.

[0056] In certain embodiments, the present invention provides a master cell bank comprising (a) a mammalian cell line comprising the nucleic acid encoding the antibody described herein, which is integrated at a genomic location; and (b) a cryoprotectant.In certain embodiments, the cryoprotectant comprises glycerol or DMSO.In certain embodiments, the master cell bank is contained in a suitable vial or container that can withstand freezing by liquid nitrogen.

[0057] Also provided herein are methods for producing the antibodies described herein. Such methods include incubating cells or cell lines containing nucleic acids encoding the antibodies in a cell culture medium under conditions sufficient to allow the expression and secretion of the antibodies, and further recovering the antibodies from the cell culture medium. The recovering step may further include one or more purification steps to remove living cells, cell debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the additional purification steps include centrifugation, ultracentrifugation, Protein A, Protein G, Protein A / G, or Protein L purification, and / or ion exchange chromatography.

[0058] "Treat," "treatment," or "Treating," as used herein, refers to the deliberate intervention in a physiological disease state that results in, for example, a reduction in the disease or severity; a reduction in the duration of a condition; an improvement in or elimination of one or more symptoms associated with a disease or condition; or a beneficial effect on a subject with a disease or condition. Treatment does not require a cure of the underlying disease or condition.

[0059] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject against disease onset or promotes disease regression, as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those of skill in the art, for example, by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0060] As used herein, "pharmaceutically acceptable" with reference to a "carrier," "excipient," or "diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some aspects, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0061] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids (e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like), and non-toxic organic acids (e.g., aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like). Base addition salts include those derived from alkaline earth metals (e.g., sodium, potassium, magnesium, calcium, and the like), and non-toxic organic amines (e.g., N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like).

[0062] CD24 binding antibody The present invention describes an antibody that specifically binds to CD24.The antibody described herein has the useful property of not binding to either B cell or activated T cell.This antibody can have an improved safety profile and reduced immune side effects.

[0063] In certain embodiments, the antibody does not bind to T lymphocytes. In certain embodiments, the antibody has weak binding to T lymphocytes. In certain embodiments, the antibody does not bind to activated T lymphocytes. In certain embodiments, the antibody has weak binding to activated T lymphocytes. In certain embodiments, the activated T lymphocytes are activated human T lymphocytes. In certain embodiments, the antibody does not bind to B lymphocytes. In certain embodiments, the antibody has weak binding to B lymphocytes. In certain embodiments, the B lymphocytes are human B lymphocytes. In certain embodiments, the antibody binds to CD24 at 1×10 -8 Less than K D In certain embodiments, the antibody binds to CD24 at 1×10 -9 Less than K DIn certain embodiments, the antibody binds to the serine at residue 18 of SEQ ID NO:500. In certain embodiments, the antibody requires the presence of serine at residue 18 of SEQ ID NO:500 for binding. In certain embodiments, the antibody binds to the serine at residue 18 of SEQ ID NO:500 but does not bind to the asparagine at residue 17 of SEQ ID NO:500. In certain embodiments, the antibody binds to an epitope consisting of the amino acid sequence of SEQ ID NO:501. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 401; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 405; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 407; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 411.

[0064] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 402; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 406; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 408; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 412.

[0065] In certain embodiments, the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 23, 33, 43, 53, 63, 73, 83, 703, 713, 723, 733, and 743; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 24, 34, 44, 54, 64, 74, 84, 704, 714, 724, 734, and 744; and (c) a light chain complementarity determining region 3 (LCDR4) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 25, 35, 45, 55, 65, 75, 85, 705, 715, 725, 735, and 745. (d) heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76, 86, 706, 716, 726, 736, and 746; (e) heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77, 87, 707, 717, 727, 737, and 747; and / or (f) heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 28, 38, 48, 58, 68, 78, 88, 708, 718, 728, 738, and 748.

[0066] In certain embodiments, the antibody comprises a combination of LCDR1-3 and HCDR1-3, the combinations being: (i) LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 14, LCDR3 comprising SEQ ID NO: 15, HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, and HCDR3 comprising SEQ ID NO: 18; (ii) LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ ID NO: 24, LCDR3 comprising SEQ ID NO: 25, HCDR1 comprising SEQ ID NO: 26, HCDR2 comprising SEQ ID NO: 27, and HCDR3 comprising SEQ ID NO: 28; (iii) LCDR1 comprising SEQ ID NO: 33, LCDR2 comprising SEQ ID NO: 34, LCDR3 comprising SEQ ID NO: 35, HCDR1 comprising SEQ ID NO: 36, HCDR2 comprising SEQ ID NO: 37, and HCDR3 comprising SEQ ID NO: 38; (iv) LCDR1 comprising SEQ ID NO: 43, LCDR2 comprising SEQ ID NO: 44, LCDR3 comprising SEQ ID NO: 45, HCDR1 comprising SEQ ID NO: 46, HCDR2 comprising SEQ ID NO: 47, and HCDR3 comprising SEQ ID NO: 48; (v) LCDR1 comprising SEQ ID NO: 53, and LCDR2 comprising SEQ ID NO: 54 (vi) LCDR1 comprising SEQ ID NO: 63, LCDR2 comprising SEQ ID NO: 64, LCDR3 comprising SEQ ID NO: 65, HCDR1 comprising SEQ ID NO: 66, HCDR2 comprising SEQ ID NO: 67, HCDR3 comprising SEQ ID NO: 68; (vii) LCDR1 comprising SEQ ID NO: 73, LCDR2 comprising SEQ ID NO: 74, LCDR3 comprising SEQ ID NO: 75, HCDR1 comprising SEQ ID NO: 76 , HCDR2 comprising SEQ ID NO:77, HCDR3 comprising SEQ ID NO:78; (viii) LCDR1 comprising SEQ ID NO:83, LCDR2 comprising SEQ ID NO:84, LCDR3 comprising SEQ ID NO:85, HCDR1 comprising SEQ ID NO:86, HCDR2 comprising SEQ ID NO:87, HCDR3 comprising SEQ ID NO:88; (ix) LCDR1 comprising SEQ ID NO:703, LCDR2 comprising SEQ ID NO:704, LCDR3 comprising SEQ ID NO:705, HCDR1 comprising SEQ ID NO:706, HCDR2 comprising SEQ ID NO:707, HCDR3 comprising SEQ ID NO:708;(x) LCDR1 comprising SEQ ID NO: 713, LCDR2 comprising SEQ ID NO: 714, LCDR3 comprising SEQ ID NO: 715, HCDR1 comprising SEQ ID NO: 716, HCDR2 comprising SEQ ID NO: 717, HCDR3 comprising SEQ ID NO: 718; (xi) LCDR1 comprising SEQ ID NO: 723, LCDR2 comprising SEQ ID NO: 724, LCDR3 comprising SEQ ID NO: 725, HCDR1 comprising SEQ ID NO: 726, HCDR2 comprising SEQ ID NO: 727, HCDR3 comprising SEQ ID NO: 728; (xii) LCDR1 comprising SEQ ID NO: 733, LCDR2 comprising SEQ ID NO: 734, LCDR3 comprising SEQ ID NO: 735, HCDR1 comprising SEQ ID NO: 736, HCDR2 comprising SEQ ID NO: 737, HCDR3 comprising SEQ ID NO: 738; and (xiii) LCDR1 comprising SEQ ID NO: 743, LCDR2 comprising SEQ ID NO: 744, LCDR3 comprising SEQ ID NO: 745, HCDR1 comprising SEQ ID NO: 746, HCDR2 comprising SEQ ID NO: 747, HCDR3 comprising SEQ ID NO: 748.

[0067] In certain embodiments, the antibody comprises a combination of LCDR1-3 and HCDR1-3, the combination being (i) LCDR1 as set forth in SEQ ID NO: 13, LCDR2 as set forth in SEQ ID NO: 14, LCDR3 as set forth in SEQ ID NO: 15, HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, and HCDR3 as set forth in SEQ ID NO: 18; (ii) LCDR1 as set forth in SEQ ID NO: 23, LCDR2 as set forth in SEQ ID NO: 24, and HCDR3 as set forth in SEQ ID NO: 25. (iii) LCDR1 as set forth in SEQ ID NO: 33, LCDR2 as set forth in SEQ ID NO: 34, LCDR3 as set forth in SEQ ID NO: 35, HCDR1 as set forth in SEQ ID NO: 36, HCDR2 as set forth in SEQ ID NO: 37, HCDR3 as set forth in SEQ ID NO: 38; (iv) LCDR1 as set forth in SEQ ID NO: 43, HCDR2 as set forth in SEQ ID NO: 44 (v) LCDR1 as set forth in SEQ ID NO:53, LCDR2 as set forth in SEQ ID NO:54, LCDR3 as set forth in SEQ ID NO:55, HCDR1 as set forth in SEQ ID NO:56, HCDR2 as set forth in SEQ ID NO:57, HCDR3 as set forth in SEQ ID NO:58; (vi) LCDR1 as set forth in SEQ ID NO:63, LCDR2 as set forth in SEQ ID NO:54, LCDR3 as set forth in SEQ ID NO:55, HCDR1 as set forth in SEQ ID NO:56, HCDR2 as set forth in SEQ ID NO:57, HCDR3 as set forth in SEQ ID NO:58; CDR1, LCDR2 as set forth in SEQ ID NO:64, LCDR3 as set forth in SEQ ID NO:65, HCDR1 as set forth in SEQ ID NO:66, HCDR2 as set forth in SEQ ID NO:67, HCDR3 as set forth in SEQ ID NO:68; (vii) LCDR1 as set forth in SEQ ID NO:73, LCDR2 as set forth in SEQ ID NO:74, LCDR3 as set forth in SEQ ID NO:75, HCDR1 as set forth in SEQ ID NO:76, HCDR2 as set forth in SEQ ID NO:77, HCDR3 as set forth in SEQ ID NO:78;(viii) LCDR1 as set forth in SEQ ID NO: 83, LCDR2 as set forth in SEQ ID NO: 84, LCDR3 as set forth in SEQ ID NO: 85, HCDR1 as set forth in SEQ ID NO: 86, HCDR2 as set forth in SEQ ID NO: 87, HCDR3 as set forth in SEQ ID NO: 88; (ix) LCDR1 as set forth in SEQ ID NO: 703, LCDR2 as set forth in SEQ ID NO: 704, LCDR3 as set forth in SEQ ID NO: 705, HCDR1 as set forth in SEQ ID NO: 706, HCDR2 as set forth in SEQ ID NO: 707, HCDR3 as set forth in SEQ ID NO: 708; (x) LCDR1 as set forth in SEQ ID NO: 713, LCDR2 as set forth in SEQ ID NO: 714, LCDR3 as set forth in SEQ ID NO: 715, HCDR1 as set forth in SEQ ID NO: 716, HCDR2 as set forth in SEQ ID NO: 717, HCDR3 as set forth in SEQ ID NO: 718; (xi) SEQ ID NO: 723 (xii) LCDR1 as set forth in SEQ ID NO: 733, LCDR2 as set forth in SEQ ID NO: 734, LCDR3 as set forth in SEQ ID NO: 735, HCDR1 as set forth in SEQ ID NO: 736, HCDR2 as set forth in SEQ ID NO: 737, HCDR3 as set forth in SEQ ID NO: 738; and (xiii) LCDR1 as set forth in SEQ ID NO: 743, LCDR2 as set forth in SEQ ID NO: 744, LCDR3 as set forth in SEQ ID NO: 745, HCDR1 as set forth in SEQ ID NO: 746, HCDR2 as set forth in SEQ ID NO: 747, HCDR3 as set forth in SEQ ID NO: 748.

[0068] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0069] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 24; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 25; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 26; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 27; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 33; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 34; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 35; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 36; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 37; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 38.

[0070] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 43; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 44; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 45; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 46; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 47; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 48.

[0071] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 53; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 54; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 55; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 56; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 57; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 58.

[0072] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 63; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 64; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 65; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 66; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 68.

[0073] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 73; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 74; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 75; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 76; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 77; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 78.

[0074] In certain embodiments, the antibody comprises (a) a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 83; (b) a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 84; (c) a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 85; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 86; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87; and / or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88.

[0075] In certain embodiments, the antibody comprises an immunoglobulin light chain amino acid variable region sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.

[0076] In certain embodiments, the antibody comprises an immunoglobulin light chain amino acid variable region sequence identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.

[0077] In certain embodiments, the antibody comprises (i) a light chain variable region (VL) having an LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having an HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to any one of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, and North definition.

[0078] In certain embodiments, the antibody comprises: (i) a light chain variable region (VL) having an LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having an HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined using a hybrid scheme of any two or three of the following: Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, and North definition.

[0079] In certain embodiments, the antibody comprises (i) a light chain variable region (VL) having LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Kabat definition.

[0080] In certain embodiments, the antibody comprises (i) a light chain variable region (VL) having LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Chothia definition.

[0081] In certain embodiments, the antibody comprises (i) a light chain variable region (VL) having LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; the CDRs are defined according to Kabat in combination with the Chothia definition.

[0082] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:275, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:276.

[0083] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO:275; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:276.

[0084] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:277, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:278.

[0085] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO:277; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:278.

[0086] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:309, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:310.

[0087] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO:309; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:310.

[0088] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 155, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 156.

[0089] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO:155; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:156.

[0090] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 157, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 158.

[0091] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO:157; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:158.

[0092] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 169, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 170.

[0093] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 169; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 170.

[0094] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:267, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:268.

[0095] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO:267; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:268.

[0096] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:271, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:272.

[0097] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO:271; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:272.

[0098] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to an immunoglobulin light chain variable region sequence of Table 1; and an immunoglobulin heavy chain amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to a heavy chain immunoglobulin sequence of Table 1.

[0099] In certain embodiments, the antibody comprises an arginine substitution at position 17 of the immunoglobulin light chain, according to Kabat numbering.

[0100] In certain embodiments, the antibody comprises a tyrosine substitution at position 12 of the immunoglobulin light chain according to Kabat numbering.

[0101] In certain embodiments, the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% or 100% identical to any one of the immunoglobulin light chain variable region sequences in Table 1; and an immunoglobulin heavy chain amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% or 100% identical to any one of the immunoglobulin heavy chain variable region sequences in Table 1.

[0102] In certain embodiments, the antibody comprises an immunoglobulin light chain amino acid sequence identical to any one of the immunoglobulin light chain variable region sequences in Table 1; and an immunoglobulin heavy chain variable region amino acid sequence identical to any one of the immunoglobulin heavy chain variable region sequences in Table 1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] [Table 1-18] [Table 1-19] *CDRs are defined according to the Kabat definition in Table 1 and in the Examples hereinafter.

[0103] Treatment method Described herein are methods for inhibiting CD24 signaling in cancer cells, the method comprising contacting the cancer cells with an antibody of the present disclosure. In certain embodiments, CD24 signaling is not inhibited in non-neoplastic B lymphocytes or non-neoplastic T lymphocytes. In certain embodiments, the cancer cells are cancer cells present in vivo in an individual. In certain embodiments, CD24-mediated signaling is reduced by about 10%, 20%, 35%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In certain embodiments, CD24-mediated signaling is reduced to an undetectable level above background levels. In certain embodiments, SHP2-mediated signaling is reduced. In certain embodiments, SHP2-mediated signaling is reduced by about 10%, 20%, 35%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. Signaling through SHP2 can be evidenced by the level of phosphorylation of SHP2.

[0104] In certain aspects, described herein are methods for treating cancer, the methods comprising administering an antibody of the present disclosure to an individual suffering from cancer or a tumor. The antibodies of the present disclosure can be used in methods for treating cancer. The antibodies of the present disclosure can be used in the manufacture of a medicament for treating cancer. In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a blood cancer or tumor. In certain embodiments, the cancer or tumor includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, and liver. In certain embodiments, tumors that may be treated with the antibodies of the invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma.In certain embodiments, the tumor / cancer is selected from the following group: acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, focal hemangioendothelioma, nodular hyperplasia, gastronoma, germ cell tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioma, hepatic adenoma, hepatic adenoma, hepatic adenomatosis adenomatosis, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, squamous intraepithelial neoplasia, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, nerve sheath tumor, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar cancer, VIPoma (VIPpoma), and Wilms' tumor.In certain embodiments, the tumors / cancers to be treated with one or more antibodies of the present disclosure include: brain cancer, head and neck cancer, colorectal cancer, acute myeloid leukemia, precursor B-cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III, or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell carcinoma, and non-small cell carcinoma, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and breast cancer, preferably ductal carcinoma and / or breast cancer. In certain embodiments, the cancer treated with an antibody of the present disclosure includes glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes lung cancer. In certain embodiments, the cancer to be treated with one or more antibodies of the present disclosure comprises prostate cancer. In certain embodiments, the cancer to be treated with one or more antibodies of the present disclosure comprises colon cancer. In certain embodiments, the cancer to be treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is refractory to other treatments. In certain embodiments, the cancer to be treated is recurrent.

[0105] In certain embodiments, the antibody may be administered to a subject in need thereof by any route suitable for administering a pharmaceutical composition comprising the antibody (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, intratumorally, or intracerebrally). In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered according to an appropriate dosing schedule, such as once weekly, twice weekly, once monthly, twice monthly, once every two weeks, once every three weeks, or once monthly. In certain embodiments, the antibody is administered once every three weeks. The antibody may be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 20 mg / kg. A therapeutically effective amount includes an amount sufficient to ameliorate one or more symptoms associated with the disease or affliction being treated.

[0106] Pharmaceutically Acceptable Excipients, Carriers, and Diluents In certain embodiments, the anti-CD24 antibody of the present disclosure is contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents may be included to increase the shelf life, stability, or administerability of the antibody. Such compounds include salts, pH buffers, detergents, anticoagulants, and preservatives. In certain embodiments, the antibody of the present disclosure is administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5% dextrose. In certain embodiments, the solution further comprises one or more of the following: a buffer (e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris)); a surfactant (e.g., polysorbate 80 (Tween® 80), polysorbate 20 (Tween® 20), and poloxamer 188); a polyol / disaccharide / polysaccharide (e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40); an amino acid (e.g., glycine or arginine); an antioxidant (e.g., ascorbic acid, methionine); or a chelating agent (e.g., EDTA or EGTA).

[0107] In certain embodiments, antibodies of the present disclosure can be transported and / or stored, lyophilized, and reconstituted prior to administration. In certain embodiments, lyophilized antibody formulations include a bulking agent (e.g., mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof). The lyophilized formulation can be contained in a vial composed of glass or other suitable non-reactive material. When formulated, the antibody, whether reconstituted or not, can be buffered to a specific pH, generally below 7.0. In certain embodiments, the pH can be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

[0108] Also described herein are kits comprising one or more of the antibodies described herein in a suitable container and one or more additional components selected from instructions for use; diluents, excipients, carriers, and devices for administration (e.g., needles and / or syringes).

[0109] In certain embodiments, described herein are methods of preparing a cancer treatment, the methods comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents and an antibody of the present disclosure. In certain embodiments, described herein are methods of preparing a cancer treatment for storage or transport, the methods comprising lyophilizing one or more antibodies of the present disclosure. [Example]

[0110] Example The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0111] Example 1 – CD24 antigen design, expression, and purification The amino acid sequences of antigens and proteins used to raise antibodies of the present disclosure were obtained from UniProt (human CD24, Uniprot number: P25063). SUMO-his tag and GST tag were fused to the CD24 protein. Genes encoding SUMO-his tag and GST tag fusion proteins were cloned into the pET21b vector (Novagen, catalog number 69741), expressed in an E. coli expression system, and purified. Genes encoding GST-tag fusion proteins were cloned into the pTT5 vector, transiently expressed in Expi293F cells (Thermo, catalog number A14527), and purified. The CD24 antigen referred to in this section refers to the human CD24 protein unless otherwise specified.

[0112] Fusion protein of CD24(27-56aa) extracellular domain and GST for mouse immunization and detection, i.e., CD24(27-56aa)-GST (NEOX-2, SEQ ID NO: 503): [ka] NOTE: The underlined part indicates CD24 (27-56 aa), and the italicized part indicates the GST tag.

[0113] A fusion protein of CD24(27-56aa) extracellular domain and SUMO-his for detection, i.e., CD24(27-56aa)-SUMO(NEOX-27, SEQ ID NO: 504) [ka] NOTE: The underlined part indicates CD24 (27-56 aa), and the italicized part indicates the SUMO-his tag.

[0114] A fusion protein of CD24 (27-56aa) extracellular domain and GST for detection, i.e., CD24 (27-56aa)-GST (NEOX-4, SEQ ID NO: 505) [ka] NOTE: The bold part indicates the signal peptide, the underlined part indicates CD24 (27-56 aa), and the italic part indicates the GST tag.

[0115] Purification of CD24(27-56aa)-GST(NEOX-2) The cultured sample was centrifuged (13,000 rpm, 4°C, 20 min) to collect bacterial cells. The bacterial cells were resuspended in an appropriate volume of Buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5) and disrupted with a sonicator (Fisher Scientific, FB505 / 705) (3 s / 3 s, 30 min). Then, the supernatant sample was collected by high-speed centrifugation (13,000 rpm, 4°C, 30 min). A 5 ml GST resin affinity column (Cytiva, Cat No. 17075605) was pre-equilibrated with Buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5), and the supernatant sample was loaded onto the column. The column was rinsed with buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5) until no protein was detectable with Coomassie Brilliant Blue G-250. The column was then eluted with buffer F (buffer A containing 10 mM glutathione, RSH) to collect the target CD24(27-56aa)-GST protein. Finally, the CD24(27-56aa)-GST protein was exchanged from buffer F to PBS buffer by dialysis. The presence and purity of the CD24(27-56aa)-GST protein were confirmed by SDS-PAGE and HPLC.

[0116] Purification of CD24(27-56aa)-SUMO his(NEOX-27) The cultured sample was centrifuged (13,000 rpm, 4°C, 20 min) to collect bacterial cells. The bacterial cells were resuspended in an appropriate volume of Buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5) and disrupted with a sonicator (Fisher Scientific, FB505 / 705) (3 s / 3 s, 30 min). The supernatant sample was then collected by high-speed centrifugation (13,000 rpm, 4°C, 30 min). A 5 ml Ni Sepharose excel affinity column (Cytiva, Cat No. 17371201) was pre-equilibrated with Buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5), and the supernatant sample was loaded onto the column. The column was rinsed with Buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5) until no protein was detectable with Coomassie Brilliant Blue G-250. The column was then rinsed sequentially with Buffer B (Buffer A containing 20 mM imidazole) and Buffer C (Buffer A containing 50 mM imidazole). The target CD24(27-56aa)-SUMO his protein was then eluted with Buffer E (Buffer A containing 250 mM imidazole). Finally, the CD24(27-56aa)-SUMO his protein was exchanged from Buffer E into PBS buffer by dialysis. The presence and purity of the CD24(27-56aa)-SUMO his protein were confirmed by SDS-PAGE and HPLC.

[0117] Purification of CD24(27-56aa)-GST(NEOX-4) The culture samples were centrifuged at high speed (13,000 rpm, 4°C, 30 min) to collect the culture supernatant. A 1 ml GST resin affinity column (Cytiva, Cat. No. 17075605) was pre-equilibrated with Buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5), and the supernatant sample was loaded onto the column. The column was rinsed with Buffer A (50 mM Tris, 500 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.5) until no protein was detectable with Coomassie Brilliant Blue G-250. The column was then eluted with Buffer F (Buffer A containing 10 mM glutathione, RSH), and the target CD24(27-56aa)-GST protein was collected. Finally, the CD24(27-56aa)-GST protein was exchanged by dialysis from buffer F into PBS buffer. The presence and purity of the CD24(27-56aa)-GST protein was confirmed by SDS-PAGE and HPLC.

[0118] Recombinant antibody purification The supernatant sample expressed by the cells was centrifuged at high speed (13,000 rpm, 4°C, 30 minutes) to remove impurities, and the supernatant of the expressed recombinant antibody was purified on a Protein A column. The target protein was eluted with 100 mM acetic acid, pH 3.0, and neutralized with 1 M Tris-HCl, pH 8.0. The eluted sample was concentrated and further purified on a PBS-equilibrated gel chromatography column, Superdex 200 (GE), and the depolymerized peak was collected and divided into aliquots for further use.

[0119] Example 2 – Generation of anti-CD24 antibodies immunity Anti-human CD24 monoclonal antibodies were generated by immunizing mice. SJL white mice (female, 6-8 weeks old) (Shanghai Slac Laboratory Animal Co. Ltd., Animal Production License Number: SCXK(Shanghai) 2017-0005) were used in the experiments. The breeding environment was SPF grade. After purchasing, the mice were raised in a laboratory environment for one week under a 12 / 12 h light / dark cycle at a temperature of 20-25°C and humidity of 40-60%. Mice adapted to the above environment were immunized according to the following protocol. The immunizing antigen was a GST-tagged human CD24 extracellular domain fusion protein (CD24(27-56aa)-GST(NEOX-2)). The mice were immunized with CFA / IFA / Alum / CpG. The antigen-to-adjuvant ratio was 1:1, and the antigen and adjuvant were rapidly and thoroughly mixed before inoculation. Primary immunization: 50 μg antigen / mouse / injection; booster immunization: 25 μg antigen / mouse / injection. The injection interval was 7 days, and a total of five immunizations were performed. Starting from the third immunization, blood was collected 7 days after each immunization, and the antibody titers in the mouse serum were determined by ELISA. Mice with high serum antibody titers that tended to plateau were subjected to splenocyte fusion. Three days before splenocyte fusion, booster inoculations were performed by intraperitoneal (IP) injection of an antigen solution prepared in saline at a dose of 50 μg / mouse.

[0120] Hybridoma formation Mouse splenic lymphocytes were mixed with Sp2 / 0 myeloma cells (CRL-8287™) at a ratio of 1:1 to 10:1 using a TBX electroporator. Hybridoma cells were obtained by one round of electric shock (60 V, 30 s; 40 ms pulse, 1700 V). The fused hybridoma cells were resuspended in complete medium (containing 20% ​​FBS, 1x HAT, and 1x DMEM medium) at a density of 4-5E5 / ml and seeded in 96-well plates at 200 μl / well. The cells were cultured at 37°C and 5% CO2 for 7 days. After 8 days of culture, the culture medium was switched to HT complete medium (containing 20% ​​FBS, 1x HT, and 1x DMEM medium) at 200 μl / well. The cells were cultured at 37°C and 5% CO2 for 2 days and then subjected to ELISA detection.

[0121] Screening of hybridoma cells Positive hybridomas were screened using ELISA and FACS (antigen detected: CD24(27-56aa)-SUMO his(NEOX-27)). Positive clones were expanded by ELISA and FACS and subjected to 2-3 rounds of subclone screening until single-cell clones were obtained. Hybridoma monoclonal G14 was obtained through the above experimental screening.

[0122] Cloning and sequencing of positive hybridoma clones Hybridoma cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen, Cat. No. 15596-018) according to the kit instructions. TM The cDNA was reverse transcribed using a Reverse Transcriptase Kit (Takara, Cat No. 2680A). The cDNA obtained by reverse transcription was PCR amplified using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) to amplify the antibody heavy and light chain variable region sequences, which were then cloned into a vector and subjected to sequencing. The amino acid sequences corresponding to the DNA sequences of the heavy and light chain variable regions of mouse monoclonal antibody G14 were obtained as follows:

[0123] G14 heavy chain variable region (G14VH, SEQ ID NO: 2) [ka]

[0124] G14 light chain variable region (G14VL, SEQ ID NO: 1) [ka]

[0125] Example 3 - Humanization of anti-human CD24 mouse monoclonal antibody By aligning the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy and light chain variable region germline genes with high homology to the mouse monoclonal antibody G14 antibody were selected as templates, respectively, to form humanized variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 by grafting the CDRs of the mouse antibody into the corresponding humanized templates.

[0126] 1.1 G14 Humanized Framework Grafting - Design 1 While keeping the light chain variable region sequence of the murine antibody G14 unchanged (murine), the heavy chain variable region was humanized using the germline IGHV3-15*01 IGHJ6*019 as a template. The humanized variable region sequence combination huG14-1_graft was as follows:

[0127] G14VL [ka]

[0128] G14VH_Graft [ka] The underlined portions indicate the CDR sequences.

[0129] 1.2 G14 humanized fragment grafting – Design 1 mutation Three mutations (G49A, N79S, L81V) were introduced into the FR region of the G14VH_Graft sequence to obtain the heavy chain variable region sequence huG14VH, which was paired with the light chain variable region sequence G14VL to obtain antibody huG14-1. The VH / VL combination is shown below:

[0130] G14VL (SEQ ID NO: 101) [ka]

[0131] huG14VH (SEQ ID NO: 102) [ka] The underlined portions indicate the CDR sequences.

[0132] 2.1 G14 Humanized Framework Grafting - Design 2 For G14 humanized framework grafting - Design 2, the light chain variable region was humanized using germline IGKV2-28*01 IGKJ2*01 as template and the heavy chain variable region was humanized using germline IGHV3-15*01 IGHJ6*019 as template. The grafted variable region sequence combination huG14-2_graft was obtained as follows:

[0133] G14VL_Graft1: [ka]

[0134] G14VH_Graft: [ka] The underlined portions indicate the CDR sequences.

[0135] 2.2 G14 humanized framework grafting – Design 2 mutations A number of residues in the FR region of the G14VL_Graft1 sequence were mutated (sites selected from the following: I2, Y41, L51, E17, P12, Q105), resulting in the following light chain variable region sequence: huG14VL1(I2V, Y41L, L51R), huG14VL1-1(I2V, Y41L, L51R, E17R), huG14VL1-2(I2V, Y41L, L51R, P12Y), huG14VL1-3(I2V, Y41L, L51R, Q105I), huG14VL1-15(I2V, Y41L, L51R, P12Y, E17R), huG14VL1-16(I2V, Y41L, L51R, S10R, P12Y, E17R, Q105I). Furthermore, three mutations (G49A, N79S, L81V) were introduced into the FR region of the G14VH_Graft sequence to obtain the heavy chain variable region sequence huG14VH. The resulting pair of humanized VH / VL combinations was as follows:

[0136] Humanized variable region sequence combination huG14-2: huG14VL1 (SEQ ID NO: 201) [ka]

[0137] huG14VH (SEQ ID NO: 202) [ka]

[0138] Humanized variable region sequence combination huG14-201: huG14VL1-1 (SEQ ID NO: 209): [ka]

[0139] huG14VH (SEQ ID NO: 210) [ka]

[0140] Humanized variable region sequence combination huG14-202: huG14VL1-2 (SEQ ID NO: 211): [ka]

[0141] huG14VH (SEQ ID NO: 212): [ka]

[0142] Humanized variable region sequence combination huG14-203: huG14VL1-3 (SEQ ID NO: 213): [ka]

[0143] huG14VH (SEQ ID NO: 214): [ka]

[0144] Humanized variable region sequence combination huG14-2011: huG14VL1-15 (SEQ ID NO: 279): [ka]

[0145] huG14VH (SEQ ID NO: 280): [ka]

[0146] Humanized variable region sequence combination huG14-2012: huG14VL1-16 (SEQ ID NO: 281): [ka]

[0147] huG14VH (SEQ ID NO: 282): [ka]

[0148] 3.1 G14 humanized framework grafting – Design 3 For G14 humanized framework grafting - Design 3, the light chain variable region was humanized using germline GKV2-30*01 IGKJ2*01 as template, and the heavy chain variable region was humanized using germline IGHV3-15*01 IGHJ6*019 as template. The humanized variable region sequence combination huG14-3_graft was obtained as follows:

[0149] G14VL_Graft2: [ka]

[0150] G14VH_Graft: [ka]

[0151] 3.2 G14 humanized framework grafting – Design 3 mutations Two mutations (F41L, V88L) were introduced into the FR region of the G14VL_Graft2 sequence to obtain the humanized light chain variable region sequence huG14VL2. Three mutations (G49A, N79S, L81V) were introduced into the FR region of the G14VH_Graft sequence to obtain the humanized heavy chain variable region sequence huG14VH. The humanized antibody huG14-3 was obtained. Its VH / VL is expressed as follows:

[0152] huG14VL2 (SEQ ID NO: 301): [ka]

[0153] huG14VH (SEQ ID NO: 302): [ka]

[0154] For illustrative purposes, all references to mutation sites in the FR regions in Example 3 employ a sequential numbering system, and a comparison table between the above numbering system and Kabat numbering is shown in the table below:

[0155] Regarding Humanized Framework - Design 2: [Table 10]

[0156] Humanized Framework - Regarding Design 3: [Table 11]

[0157] Example 4 - Affinity maturation of anti-human CD24 humanized antibodies Single and multiple point mutations were made in the CDRs of the VH and VL in the variable regions, and these mutant antibody proteins were expressed in CHO cells. The EC50 values ​​and / or affinities of the mutants and CD24 protein were determined by ELISA and / or SPR. Affinity-matured antibodies were thus obtained.

[0158] 1. Affinity maturation based on humanized design 1 (huG14-1): single mutation site design and multiple mutation combination design for CDRs Based on the humanized sequence of antibody huG14-1, single and multiple point mutations were introduced into the CDR regions of the heavy chain variable region VH and the light chain variable region VL. The final VH and VL sequences obtained are shown in the table below: [Table 12]

[0159] The above VH and VL sequences were paired to obtain a number of heavy / light chain variable region sequence combinations (i.e., affinity matured antibodies) that were expressed as follows: [Table 13]

[0160] 2. Affinity maturation based on humanized design 2 (e.g., huG14-2): Multiple mutation combination design of CDRs Based on the humanized sequences of Design 2, single and multiple point mutations were introduced into the CDR regions of the heavy chain variable region VH and the light chain variable region VL. The final VH and VL sequences obtained are shown in the table below: [Table 14]

[0161] The above VH and VL sequences were paired to obtain a number of heavy / light chain variable region sequence combinations (i.e., affinity matured antibodies) that were expressed as follows: [Table 15]

[0162] 3. Affinity maturation based on human design 3 (huG14-3): CDR multiple mutation combinatorial design Based on the humanized sequence of antibody huG14-3, single and multiple point mutations were made to the CDR regions of the heavy chain variable region VH and the light chain variable region VL. [Table 16]

[0163] The above VH and VL sequences were combined to obtain a number of heavy / light chain variable region sequence combinations (i.e., affinity matured antibodies) that were expressed as follows: [Table 17]

[0164] Example 5 - Binding and affinity of antibodies with mutations introduced to remove deamidation sites were maintained or higher Deamidation is a common chemical modification of antibodies that can potentially affect their stability in solution. We reduced deamidation, oxidation, or isomerization of several amino acids in the CDR regions of the described antibodies by introducing mutations that prevent such undesired reactions. The amino acid sequence "NG" is prone to deamidation, and the above antibody has NG at positions 33-34. Therefore, we constructed a CDR1 mutation of the CD24 antibody light chain KSSQSLLYSX1X2ETYLN (where X1 and X2 are amino acid residues at positions 33-34 in the variable region of the CD24 antibody light chain, respectively; X1 can be selected from Ser, Tyr, or Gln, and X2 can be selected from His, Lys, Arg, Trp, or Ala).

[0165] Using the light chain G14VL14 sequence of the huG14-124 variable region as an example sequence, sequences containing the CDR1 (KSSQSLLYSX1X2ETYLN) X1 or X2 mutation sites are shown in the table below (G14VL16 (G34H), G14VL17 (G34K), G14VL18 (G34R), G14VL19 (G34W), G14VL20 (G34Y), G14VL21 (G34A), G14VL22 (N33Y), G14VL23 (N33Q), and G14VL24 (N33S)). These single point mutation sequences were paired with the heavy chain variable region huG14VH1 sequence to generate nine CD24 antibodies (using the same constant region as in Example 6), as shown in the table below. [Table 18]

[0166] The affinity values ​​of these mutant antibodies to biotinylated CD24 protein (ACRO, Cat. No. CD4-H82E9) were determined by the Biacore affinity determination experimental method in Example 9, and the results were as follows: Table: Affinity of mutant antibodies to CD24 protein [Table 19]

[0167] The results showed that the affinity (KD) of the above mutant antibodies was higher than that of the huG14-124 antibody. Mutation of the X1 and X2 sites in the light chain CDR1 (KSSQSLLYSX1X2ETYLN) to X1 (Ser, Tyr, or Gln) and X2 (His, Lys, Arg, Trp, or Ala) did not decrease affinity.

[0168] Example 6 - Preparation of chimeric, humanized and affinity matured antibodies The above heavy chain variable regions can be recombinantly expressed with the human heavy chain IgG1 constant region shown in SEQ ID NO: 607 to obtain a full-length heavy chain sequence. The above light chain variable regions can be recombinantly expressed with the kappa light chain constant region sequence shown in SEQ ID NO: 608 to obtain a full-length light chain sequence. The above heavy and light chain variable regions can also be recombined with other heavy and light chain constant regions of the IgG family or constant regions of variant IgG families commonly known in the art to form complete heavy and light chain sequences of an antibody. Exemplary heavy and light chain constant regions employed in the examples of the present invention are as follows:

[0169] Sequence of the heavy chain constant region (CH1-Fc, SEQ ID NO: 607) [ka]

[0170] Sequence of the light chain constant region (CL, SEQ ID NO: 608) [ka]

[0171] Molecular cloning of recombinant chimeric antibody G14 The positive antibody molecules obtained by hybridoma screening were sequenced to obtain the gene sequences encoding the variable regions. Head and tail primers were designed based on the sequences obtained by the above sequencing, and the sequenced genes were used as templates to construct the VH / VK gene fragments of the G14 antibody by PCR. The VH / VK gene fragments were then subjected to homologous recombination with the expression vector pcDNA3.4 (Thermo Fisher, catalog number V001453) (containing the signal peptide and hIgG1 / κ constant region gene (CH1-Fc / CL) fragment) to construct the recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pcDNA3.4 / VL-CL-pcDNA3.4, forming the G14 chimeric antibody expression construct.

[0172] The full-length amino acid sequence of the chimeric antibody (G14) is as follows: G14 full-length light chain (SEQ ID NO: 611): [ka]

[0173] G14 full length heavy chain (SEQ ID NO: 612): [ka] Note: The italicized and underlined parts in the sequences indicate the variable region sequences (SEQ ID NOs: 1 and 2, respectively), and the rest are the constant region sequences.

[0174] 2: Molecular cloning of humanized and affinity matured antibodies The antibody sequences of the humanized and affinity-matured antibodies were codon-optimized to generate coding gene sequences that favor human codons. Primers were designed to construct the VH / VK gene fragments of each antibody (e.g., huG14-1 to huG14-3, and huG14-101 to huG14-139, huG14-235, huG14-306, etc.) via PCR. These fragments were then subjected to homologous recombination with the expression vector pcDNA3.4 (Thermo Fisher Scientific, catalog number V001453) (containing the signal peptide and hIgG1 / κ constant region gene (CH1-Fc / CL) fragment) to construct the recombinant humanized antibody full-length expression plasmid VH-CH1-Fc-pcDNA3.4 / VL-CL-pcDNA3.4.

[0175] The full-length amino acid sequence of an exemplary humanized antibody (huG14-1) is as follows (similar for other humanized / affinity matured antibodies):

[0176] huG14-1 full length light chain (SEQ ID NO: 609): [ka]

[0177] huG14-1 full length heavy chain (SEQ ID NO: 610): [ka] Note: The italicized and underlined parts in the sequences indicate the variable region sequences (SEQ ID NOs: 101 and 102, respectively), and the remainder is the constant region sequence.

[0178] Expression and purification of chimeric, humanized and affinity matured antibodies Plasmids expressing antibody light chains and heavy chains were transfected into CHO cells at a 1.5:1 ratio. After 4–6 days, the expression supernatant was collected and centrifuged at high speed (13,000 rpm, 4°C, 30 min) to remove impurities and purified on a Protein A column. The target antibody protein was eluted with a glycine eluent at pH 3.0–3.5 and neutralized with 1 M Tris-HCl at pH 8.0–9.0. The target antibody protein was exchanged by dialysis against PBS buffer, aliquoted, and frozen for later use. If the antibody purity was less than 85%, the antibody was further purified by gel chromatography on a Superdex 200 (GE) column equilibrated with PBS to remove aggregates. The monomer peak was collected, aliquoted, and frozen for later use.

[0179] Example 7. Determining the binding of chimeric, humanized, and affinity-matured antibodies to CD24 protein ELISA was used to determine the binding affinity between CD24 protein and a chimeric antibody (G14), a humanized antibody (huG14-1, whose VH / VL sequences are shown in SEQ ID NO: 102 and SEQ ID NO: 101, respectively), and an affinity-matured antibody (designated as huG14-101-139 and huG14-204-233, respectively, whose VH / VL combinations are detailed in the table in Example 4, and whose constant regions are the same as above). ELISA was performed as follows: 1) Coating of CD24 protein: CD24(27-56aa)-GST(NEOX-2) or CD24(27-56aa)-GST(NEOX-4) was diluted to 1 μg / ml with PBS, added to an ELISA plate at 100 μl / well, and coated overnight at 4°C. 2) Blocking: The plate was washed three times with 0.05% PBST solution, and 200 μl of a pre-adjusted 5% non-fat dry milk PBS solution was added to each well of the ELISA detection plate and incubated at room temperature for 2 hours. 3) Primary antibody dilution and sample loading: The plate was washed three times with PBS. The CD24 antibody was diluted to 200 μg / ml, then subjected to a 4-fold gradient dilution, and added to the ELISA detection plate at 100 μl / well and incubated at 37°C for 1 hour. 4) Secondary antibody addition: The plate was washed three times with PBS. HRP-labeled anti-human IgG Fc antibody (Jackson, catalog number 109-035-098) was diluted 5000-fold and added to each well at 100 μl and incubated at 37°C for 1 hour. 5) Color development: The plate was washed six times with PBS. 50 μl of TMB (Beijing Zhizhou Biotechnology Co., Ltd., Catalog No. ZZ180001) was added to each well and incubated at room temperature for 10 minutes in the dark. 50 μl of 2M H2SO4 was then added to terminate the color reaction. 5) Data collection and analysis: OD450 absorbance values ​​were read using a microplate reader (Thermo, Multiskan FC) and EC50 values ​​were calculated.

[0180] The results, as shown in Figures 1A and 1B and Table 2, indicate that the binding EC50 values ​​of the affinity-matured single point mutant antibodies to CD24(27-56aa)-GST(NEOX-4) were lower than that of the corresponding humanized antibody huG14-1, indicating that the experimental single point mutant affinity-matured CD24 antibodies have strong affinity to CD24(27-56aa)-GST(NEOX-4).

[0181] The results, as shown in Figures 2A and 2B and Tables 3-1 and 3-2, indicate that the binding EC50 values ​​of the affinity-matured single point mutation antibodies to CD24(27-56aa)-GST(NEOX-4) were lower than those of the humanized antibody huG14-1 and the chimeric antibody G14, indicating that the affinity-matured CD24 antibodies with the combined mutations have strong affinity to CD24(27-56aa)-GST(NEOX-4).

[0182] [Table 2] [Table 3-1] [Table 3-2]

[0183] Example 8 - Determination of binding of chimeric antibody G14 and affinity matured CD24 antibodies to tumor cells highly expressing CD24 NALM6 and MDA-MB-486 are human tumor cell lines that express high levels of CD24. The CD24 antibody was co-incubated with NALM6 and MDA-MB-486 cells to determine whether the CD24 antibody can specifically bind to NALM6 and MDA-MB-486 cells. The control positive antibody was P6373. Its sequence is shown in SEQ ID NOs: 603 and 604 (see WO 2019 / 222082 A1). The process of cell binding experiments was as follows: adding 5x10E4 cells to each well of a 96-well plate and removing the supernatant after centrifugation; diluting the CD24 antibody with PBS: starting from an initial concentration of 30µg / mL, diluting 4-fold gradient dilutions for a total of 7 dilutions; adding 100µl of the diluted CD24 antibody to each well of the 96-well plate, resuspending the cells, and then incubating at 4°C for 1 hour; removing the supernatant by centrifugation, washing the cells three times with 200µl staining buffer (1xPBS + 1% BSA), and then adding 50µl of a 1:200 diluted fluorescent secondary antibody goat anti-human IgG(Fc)-PE (Jackson, Cat. No. 109-116-098) to each well; resuspending and incubating at 4°C for 30 minutes in the dark; Wash three times with 50 μl staining buffer (1×PBS+1% BSA), resuspend the cells in 50 μl staining buffer (1×PBS+1% BSA) and place the cells on a Beckman cyto FLFX for detection and data analysis.

[0184] As shown in Figures 3A and 3B and Table 4, both the chimeric antibody G14 and the affinity-matured CD24 antibody were able to specifically bind to CD24 protein on the surface of NALM6 cells, and the binding activity of the affinity-matured CD24 antibody was significantly higher than that of the chimeric antibody G14.

[0185] As shown in Figures 4A and 4B and Table 5, both the chimeric antibody G14 and the affinity-matured CD24 antibody were able to specifically bind to the CD24 protein on the surface of MDA-MB-468 cells, and the binding activity of the affinity-matured CD24 antibody was significantly higher than that of the chimeric antibody G14. The above results demonstrate that the antibodies of the present invention can effectively bind to CD24 on the surface of tumor cells. [Table 4] [Table 5]

[0186] Example 9 - Biacore affinity determination of chimeric and affinity matured antibodies against CD24 protein Biotinylated CD24 protein (ACRO, Cat. No. CD4-H82E9) was coupled to a CM5 biosensor chip (Cytiva, BR100530), and then two-fold dilutions of CD24 antibody at different concentrations were passed over the surface of the chip. The reaction signals were detected in real time using a Biacore (Cytiva, Biacore 8K) to obtain association (ka) and dissociation (kd) curves, and then the affinity (K D ) values ​​were obtained by fitting, as shown in Table X below. The results show that the binding affinities of affinity-matured antibodies huG14-234, huG14-235, and huG14-301 to CD24 protein (ACRO, catalog number CD4-H82E9) were significantly higher than that of the chimeric antibody G14. [Table 20]

[0187] Example 10 - Evaluation of ADCC activity Examples 10-17 of the present invention involve three CD24 antibodies for comparison, the full-length sequences of which are set forth as follows: SWA11 (light chain: SEQ ID NO: 601, heavy chain: SEQ ID NO: 602), P6373 (light chain: SEQ ID NO: 603, heavy chain: SEQ ID NO: 604) and IMM47H (light chain: SEQ ID NO: 605, heavy chain: SEQ ID NO: 606).

[0188] Jurkat cells containing the FcγRIIIA luciferase reporter gene and target cells NALM6 or MDA-MB-468 were seeded into 96-well plates at an effector-target ratio of 6:1, and then 3-fold gradient dilutions of the antibody to be tested and its isotype (anti-HEL IgG1 isotype control antibody) were added at a starting concentration of 20 μg / ml. After 5 hours of incubation at 37°C, luciferase substrate was added, the reaction was allowed to proceed for 5 minutes, and the fluorescence intensity was detected by a microplate reader.

[0189] Figures 5A, 5B, 5C and Table 6 show that antibodies huG14-124, huG14-125, huG14-131, huG14-216, huG14-217, huG14-230, huG14-232, huG14-234, huG14-235, huG14-301 all have ADCC activity against NALM6 and MDA-MB-468. [Table 6]

[0190] Example 11 - Evaluation of ADCP activity Monocytes were isolated from PBMCs and induced to differentiate into M2 macrophages with M-CSF, TGFβ, and IL-10 for further use. NALM6 and MDA-MB-468 tumor cells were labeled with CFSE and mixed with M2 macrophages at a 1:1 ratio. A 5-fold gradient dilution of the anti-CD24 antibody G14 and its affinity-matured sequence, starting at 5 μg / ml, was then added. After incubation for 4 hours at room temperature, the cells were washed and subjected to CD11b flow staining. The ratio of each cell subpopulation was detected by flow cytometry. Phagocytosis efficiency = CFSE+CD11b+ / CD11b+.

[0191] Figures 6A, 6B, 6C, 6D and Table 7 show that all antibodies tested exhibited tumor phagocytic activity against NALM6 and MDA-MB-468. [Table 7]

[0192] Example 12 - Described CD24 antibodies exhibit reduced binding to B cells and activated T cells CD24 protein is expressed on both normal B cells and activated T cells. Flow cytometry was used to study the binding profile of CD24 antibody to B cells and activated T cells. Serial dilutions of CD24 antibody were incubated with normal primary B cells or activated T cells, which were activated with CD3 or CD28, respectively, for 3 days at 4°C for 1 hour, and then washed three times with PBS. A 1:200 dilution of fluorescent secondary antibody, goat anti-human IgG(Fc)-PE, was then added to each well and incubated for 30 minutes at 4°C. After washing three times with PBS, the cells were resuspended and subjected to flow cytometry detection using a CytoFLEX.

[0193] Figures 7A, 7B, 7C, 7D and Table 8 show that huG14-124, huG14-125, huG14-131, huG14-216, huG14-217, huG14-230, huG14-232, huG14-234, huG14-235, and huG14-301 did not bind to primary B cells and activated T cells. However, IMM47H and SWA11 showed significant binding to B cells and P6373, and to activated T cells. This is potentially important because the antibodies of the present invention may have significant safety advantages over previously described anti-CD34 antibodies. [Table 8]

[0194] Example 13 – Tumor cell-specific elimination To evaluate the tumor-specific elimination activity of anti-CD24 antibodies, 1 × 10 5 CFSE-labeled NALM6 cells were mixed with human PBMCs at a 1:7 ratio. Anti-CD24 antibodies G14, SWA11, IMM47H, and P6373 were then added to the NALM6 / PBMC mixture at a final concentration of 10 μg / ml. After 4 hours of incubation at 37°C and 5% CO2, the cell mixture was stained with PE-labeled anti-human CD19 antibody for 30 minutes at 4°C in the dark. The cells were washed three times with 200 μl staining buffer (1×PBS + 1% BSA), resuspended in 50 μl staining buffer (1×PBS + 1% BSA), and placed in a Beckman cyto FLFX for detection and data analysis. NALM6 exclusion rate (%) = 1 - (CFSE+CD19+ cells in anti-CD24-treated group / CFSE+CD19+ cells in isotype control group) × 100%, B cell exclusion rate (%) = 1 - (percentage of CD19+CFSE- B cells in anti-CD24-treated group / percentage of CD19+CFSE- B cells in isotype control group) × 100%.

[0195] As shown in Table 9, huG14-301 and P6373 can selectively eliminate tumor cells (NALM6) and spare normal B cells in PBMCs. However, the reference antibodies SWA11, IMM47H, kill both NALM6 and normal B cells. These results indicate that G14 may have a better safety profile than previously described anti-CD24 antibodies. [Table 9]

[0196] Example 14 - Glycosyl Selectivity CD24 is a highly glycosylated protein, and the affinity of antibody to CD24 protein with different glycosylation levels varies.Research and literature have shown that the glycosylation of CD24 in tumor cells is different from that in normal cells.Usually, the glycosylation of CD24 in tumor cells is reduced to a certain extent, thereby exposing the amino acid residues of the protein (for such literature, see, for example, WO2019222082A1).

[0197] The binding ability of the antibodies to CD24 with different glycosylation levels was determined by ELISA. The method was as follows: 1) digesting CD24(27-56aa)-GST(NEOX-4) with N-glycanase, neuraminidase, or N-glycanase+neuraminidase, respectively; 2) diluting the CD24 to 1 μg / ml with PBS before and after digestion, and then coating a 96-well plate with the CD24 overnight at 4°C; 3) after blocking with BSA, adding 0.78 μg / ml, 0.19 μg / ml, or 0.05 μg / ml of G14 antibody and incubating at 37°C for 1 hour; 4) adding a secondary antibody: washing the plate three times with PBS, diluting HRP-labeled anti-human IgG Fc antibody (Jackson, catalog number 109-035-098) 5000-fold, adding 100 μl of it to each well, and incubating at 37°C for 1 hour; 5) Color development: Wash the plate with PBS six times, add 50 μl TMB (Beijing Zhizhou Biotechnology Co., Ltd., Catalog No. ZZ180001) to each well, place the plate in the dark at room temperature for 10 minutes, and then add 50 μl of 2M H2SO4 to terminate the color development reaction.

[0198] FIG. 8 shows that the affinity of G14 for NEOX-4 protein digested with N-glycanase, neuraminidase, or N-glycanase plus neuraminidase was higher than that before digestion.

[0199] Cell preparation: The above cells (NALM6, MDA-MB-468, primary B cells) were counted (cell viability 98%), centrifuged, resuspended in buffer, and plated in 5x10 cells into a 96-well conical-bottom plate. 5The cells were aliquoted at 200 μl / well. After centrifugation, the supernatant was removed, and the cells were resuspended in blank medium. N-glycanase, neuraminidase, or N-glycanase + neuraminidase was added, respectively, and then placed in an incubator for 3 hours at 37°C. The cells were washed three times with PBS, resuspended in PBS containing 2 μg / ml of huG14-235 or huG14-301, and then incubated at room temperature for 1 hour. After washing three times with PBS, fluorescent secondary antibody staining was performed, and the fluorescence intensity was detected by CytoFLEX.

[0200] Figures 9A, 9B, and 9C show that huG14-235 or huG14-301 strongly bound to NALM6 and MDA-MB-468, and antibody binding to NALM6 and MDA-MB-468 was significantly enhanced when tumor cells were treated with N-glycanase, neuraminidase, or N-glycanase plus neuraminidase. Although huG14-235 or huG14-301 did not bind to primary B cells, they could bind to primary B cells after deglycosylation by enzymatic digestion.

[0201] Example 15 - Analysis of CD24 antibody binding epitopes We synthesized truncations of the CD24 extracellular segment (NEOX43-NEOX47) overlapping various portions of the segment to investigate the precise epitope of the CD24 antibody binding to the antigen. First, a 96-well plate was coated with 1 μg / mg of CD24 antigen overnight at 4°C and blocked with BSA for 1 hour at room temperature. The CD24 antibodies were then mixed with 10 μg / ml of each of the different truncations (NEOX43-NEOX47) and added to the 96-well plate for 1 hour of incubation at room temperature. After washing three times with PBS, an anti-hFc HRP secondary antibody was added for incubation, and its reaction substrate was added for detection. The results, shown in Figure 10, demonstrate that both G14 and P6373 bound to the NEOX46:SNSGLAPNPT peptide segment.

[0202] To further investigate the specific amino acid site bound by CD24 antibodies, a series of single point mutations (NEOX52-NEOX61) were designed in NEOX46:SNSGLAPNPT, and then the same method was used to study the binding of the antibodies to different peptide segments. The results, shown in Figure 11, indicate that antibodies such as G14 and P6373 differed in the specific amino acid binding site of the epitope peptide segment, SNSGLAPNPT. G14 bound to CD24 at 44S, 45G, 46L, 47A, and 48P, whereas P6373 bound at 43N, 45G, 46L, 47A, and 48P (residue numbering is based on the full-length CD24 sequence with the signal peptide, as shown in SEQ ID NO: 502). For further details, see Figure 12 (binding epitope comparison).

[0203] Example 16 - Pharmacodynamic study of CD24 antibodies in a colon cancer mouse model hSiglec10 transgenic mice, 1 × 10 6 MC38-hCD24 tumor cells were inoculated subcutaneously, and the tumor volume was 150 mm 3 When the tumor volume reached 90%, they were treated with CD24 antibody intraperitoneally at a dose of 20 mg / kg twice a week for two consecutive weeks. The tumor volume was measured twice a week, and the tumor growth curve was plotted. The results in Figure 13 show that the G14 antibody exhibited significant tumor-inhibitory activity with a tumor inhibition rate of 90%.

[0204] Example 17 - Pharmacodynamic studies of CD24 antibodies in hematological tumor models 5 × 10 6 NALM6 tumor cells were inoculated subcutaneously, and tumor volumes were 150 mm 3When the tumor volume reached 100%, the mice were intraperitoneally administered different doses of CD24 antibody for treatment. The mice were administered twice a week for three consecutive weeks. The tumor volume was measured twice a week, and the tumor growth curve was plotted. The results in Figures 14A and 14B show that both low-dose and high-dose G14, huG14-235, and huG14-301 antibodies showed significant tumor inhibition activity, with tumor inhibition rates of more than 90%.

[0205] Example 18 - Pharmacodynamic studies of CD24 antibodies in breast cancer 1 × 10 7 Triple-negative breast cancer MDA-MB-468 cells were inoculated subcutaneously, and tumor volume was 150 mm 3 When the tumor size reached 100 μg / mL, the mice were intraperitoneally administered with CD24 antibody for treatment. The mice were administered twice a week for four consecutive weeks (FIG. 15A) or three consecutive weeks (FIG. 15B). The tumor volumes were measured twice a week, and the tumor growth curves were plotted. The results in FIGS. 15A and 15B show that the G14, huG14-235, and huG14-301 antibodies exhibited significant tumor-suppressing activity (G14 exhibited 66% tumor-suppressing activity).

[0206] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0207] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the incorporated text are excluded to the extent they contradict definitions in this disclosure.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CD24, wherein said antibody does not bind to T lymphocytes.

2. The antibody of claim 1 , wherein the antibody does not bind to activated T lymphocytes.

3. The antibody of claim 1 or 2, wherein the antibody does not bind to B lymphocytes.

4. The antibody was administered to CD24 at a concentration of 1×10 －８ Less than K Ｄ The antibody according to any one of claims 1 to 3, which binds to

5. The antibody was administered to CD24 at a concentration of 1×10 －９ Less than K Ｄ The antibody according to any one of claims 1 to 3, which binds to

6. The antibody according to any one of claims 1 to 5, wherein the antibody is a humanized antibody or a chimeric antibody.

7. The antibody may be Fab, F(ab) ２ 7. The antibody of claim 1, which is a single-chain variable fragment (scFv).

8. The antibody of any one of claims 1 to 6, wherein the antibody is a heavy chain antibody or an antigen-binding fragment thereof.

9. The antibody according to any one of claims 1 to 6, wherein the antibody is an IgG antibody.

10. The antibody is IgG １ The antibody of claim 9, which is an isotype.

11. The antibody is IgG ４ The antibody of claim 9, which is an isotype.

12. The antibody of any one of claims 1 to 11, wherein the antibody binds to serine at residue 18 of SEQ ID NO:

500.

13. The antibody of any one of claims 1 to 11, wherein the antibody requires the presence of serine at residue 18 of SEQ ID NO:500 for binding.

14. The antibody according to any one of claims 1 to 13, wherein the antibody binds to an epitope consisting of the amino acid sequence of SEQ ID NO:

501.

15. An antibody or antigen-binding fragment thereof that binds to CD24, wherein the antibody or antigen-binding fragment thereof is a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:401; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:403; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:405; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:407; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:411 An antibody or antigen-binding fragment thereof comprising:

16. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:401; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:403; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:405; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:407; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:411 The antibody according to any one of claims 1 to 15, comprising:

17. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:402; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:403; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:406; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:408; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:412 The antibody according to any one of claims 1 to 15, comprising:

18. The antibody a. light chain complementarity determining region 1 (LCDR1) set forth in SEQ ID NO: 402; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:403; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:406; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:408; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 409; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:412 The antibody according to any one of claims 1 to 15, comprising:

19. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 23, 33, 43, 53, 63, 73, 83, 703, 713, 723, 733, and 743; b. a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 24, 34, 44, 54, 64, 74, 84, 704, 714, 724, 734, and 744; c. a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 25, 35, 45, 55, 65, 75, 85, 705, 715, 725, 735, and 745; d. a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76, 86, 706, 716, 726, 736, and 746; e. a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 27, 37, 47, 57, 67, 77, 87, 707, 717, 727, 737, and 747; and / or f. A heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 28, 38, 48, 58, 68, 78, 88, 708, 718, 728, 738, and 748. The antibody according to any one of claims 1 to 15, comprising:

20. the antibody comprises a combination of LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, and HCDR3, the combination comprising: (i) LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 14, LCDR3 comprising SEQ ID NO: 15, HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, HCDR3 comprising SEQ ID NO: 18; (ii) LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ ID NO: 24, LCDR3 comprising SEQ ID NO: 25, HCDR1 comprising SEQ ID NO: 26, HCDR2 comprising SEQ ID NO: 27, HCDR3 comprising SEQ ID NO: 28; (iii) LCDR1 comprising SEQ ID NO: 33, LCDR2 comprising SEQ ID NO: 34, LCDR3 comprising SEQ ID NO: 35, HCDR1 comprising SEQ ID NO: 36, HCDR2 comprising SEQ ID NO: 37, HCDR3 comprising SEQ ID NO: 38; (iv) LCDR1 comprising SEQ ID NO: 43, LCDR2 comprising SEQ ID NO: 44, LCDR3 comprising SEQ ID NO: 45, HCDR1 comprising SEQ ID NO: 46, HCDR2 comprising SEQ ID NO: 47, HCDR3 comprising SEQ ID NO: 48; (v) LCDR1 comprising SEQ ID NO: 53, LCDR2 comprising SEQ ID NO: 54, LCDR3 comprising SEQ ID NO: 55, HCDR1 comprising SEQ ID NO: 56, HCDR2 comprising SEQ ID NO: 57, HCDR3 comprising SEQ ID NO: 58; (vi) LCDR1 comprising SEQ ID NO: 63, LCDR2 comprising SEQ ID NO: 64, LCDR3 comprising SEQ ID NO: 65, HCDR1 comprising SEQ ID NO: 66, HCDR2 comprising SEQ ID NO: 67, HCDR3 comprising SEQ ID NO: 68; (vii) LCDR1 comprising SEQ ID NO: 73, LCDR2 comprising SEQ ID NO: 74, LCDR3 comprising SEQ ID NO: 75, HCDR1 comprising SEQ ID NO: 76, HCDR2 comprising SEQ ID NO: 77, HCDR3 comprising SEQ ID NO: 78; (viii) LCDR1 comprising SEQ ID NO: 83, LCDR2 comprising SEQ ID NO: 84, LCDR3 comprising SEQ ID NO: 85, HCDR1 comprising SEQ ID NO: 86, HCDR2 comprising SEQ ID NO: 87, HCDR3 comprising SEQ ID NO: 88; (ix) LCDR1 comprising SEQ ID NO: 703, LCDR2 comprising SEQ ID NO: 704, LCDR3 comprising SEQ ID NO: 705, HCDR1 comprising SEQ ID NO: 706, HCDR2 comprising SEQ ID NO: 707, HCDR3 comprising SEQ ID NO: 708; (x) LCDR1 comprising SEQ ID NO: 713, LCDR2 comprising SEQ ID NO: 714, LCDR3 comprising SEQ ID NO: 715, HCDR1 comprising SEQ ID NO: 716, HCDR2 comprising SEQ ID NO: 717, HCDR3 comprising SEQ ID NO: 718; (xi) LCDR1 comprising SEQ ID NO: 723, LCDR2 comprising SEQ ID NO: 724, LCDR3 comprising SEQ ID NO: 725, HCDR1 comprising SEQ ID NO: 726, HCDR2 comprising SEQ ID NO: 727, HCDR3 comprising SEQ ID NO: 728; (xii) an LCDR1 comprising SEQ ID NO: 733, an LCDR2 comprising SEQ ID NO: 734, an LCDR3 comprising SEQ ID NO: 735, an HCDR1 comprising SEQ ID NO: 736, an HCDR2 comprising SEQ ID NO: 737, and an HCDR3 comprising SEQ ID NO: 738; and (xiii) LCDR1 comprising SEQ ID NO: 743, LCDR2 comprising SEQ ID NO: 744, LCDR3 comprising SEQ ID NO: 745, HCDR1 comprising SEQ ID NO: 746, HCDR2 comprising SEQ ID NO: 747, and HCDR3 comprising SEQ ID NO: 748 The antibody according to any one of claims 1 to 15, selected from the group consisting of:

21. the antibody comprises a combination of LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, and HCDR3, the combination comprising: (i) LCDR1 as set forth in SEQ ID NO: 13, LCDR2 as set forth in SEQ ID NO: 14, LCDR3 as set forth in SEQ ID NO: 15, HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18; (ii) LCDR1 as set forth in SEQ ID NO:23, LCDR2 as set forth in SEQ ID NO:24, LCDR3 as set forth in SEQ ID NO:25, HCDR1 as set forth in SEQ ID NO:26, HCDR2 as set forth in SEQ ID NO:27, HCDR3 as set forth in SEQ ID NO:28; (iii) LCDR1 as set forth in SEQ ID NO: 33, LCDR2 as set forth in SEQ ID NO: 34, LCDR3 as set forth in SEQ ID NO: 35, HCDR1 as set forth in SEQ ID NO: 36, HCDR2 as set forth in SEQ ID NO: 37, HCDR3 as set forth in SEQ ID NO: 38; (iv) LCDR1 as set forth in SEQ ID NO: 43, LCDR2 as set forth in SEQ ID NO: 44, LCDR3 as set forth in SEQ ID NO: 45, HCDR1 as set forth in SEQ ID NO: 46, HCDR2 as set forth in SEQ ID NO: 47, HCDR3 as set forth in SEQ ID NO: 48; (v) LCDR1 as set forth in SEQ ID NO: 53, LCDR2 as set forth in SEQ ID NO: 54, LCDR3 as set forth in SEQ ID NO: 55, HCDR1 as set forth in SEQ ID NO: 56, HCDR2 as set forth in SEQ ID NO: 57, HCDR3 as set forth in SEQ ID NO: 58; (vi) LCDR1 as set forth in SEQ ID NO: 63, LCDR2 as set forth in SEQ ID NO: 64, LCDR3 as set forth in SEQ ID NO: 65, HCDR1 as set forth in SEQ ID NO: 66, HCDR2 as set forth in SEQ ID NO: 67, HCDR3 as set forth in SEQ ID NO: 68; (vii) LCDR1 as set forth in SEQ ID NO: 73, LCDR2 as set forth in SEQ ID NO: 74, LCDR3 as set forth in SEQ ID NO: 75, HCDR1 as set forth in SEQ ID NO: 76, HCDR2 as set forth in SEQ ID NO: 77, HCDR3 as set forth in SEQ ID NO: 78; (viii) LCDR1 as set forth in SEQ ID NO: 83, LCDR2 as set forth in SEQ ID NO: 84, LCDR3 as set forth in SEQ ID NO: 85, HCDR1 as set forth in SEQ ID NO: 86, HCDR2 as set forth in SEQ ID NO: 87, HCDR3 as set forth in SEQ ID NO: 88; (ix) LCDR1 as set forth in SEQ ID NO:703, LCDR2 as set forth in SEQ ID NO:704, LCDR3 as set forth in SEQ ID NO:705, HCDR1 as set forth in SEQ ID NO:706, HCDR2 as set forth in SEQ ID NO:707, HCDR3 as set forth in SEQ ID NO:708; (x) LCDR1 as set forth in SEQ ID NO:713, LCDR2 as set forth in SEQ ID NO:714, LCDR3 as set forth in SEQ ID NO:715, HCDR1 as set forth in SEQ ID NO:716, HCDR2 as set forth in SEQ ID NO:717, HCDR3 as set forth in SEQ ID NO:718; (xi) LCDR1 as set forth in SEQ ID NO:723, LCDR2 as set forth in SEQ ID NO:724, LCDR3 as set forth in SEQ ID NO:725, HCDR1 as set forth in SEQ ID NO:726, HCDR2 as set forth in SEQ ID NO:727, HCDR3 as set forth in SEQ ID NO:728; (xii) LCDR1 as set forth in SEQ ID NO:733, LCDR2 as set forth in SEQ ID NO:734, LCDR3 as set forth in SEQ ID NO:735, HCDR1 as set forth in SEQ ID NO:736, HCDR2 as set forth in SEQ ID NO:737, HCDR3 as set forth in SEQ ID NO:738; and (xiii) LCDR1 as set forth in SEQ ID NO: 743, LCDR2 as set forth in SEQ ID NO: 744, LCDR3 as set forth in SEQ ID NO: 745, HCDR1 as set forth in SEQ ID NO: 746, HCDR2 as set forth in SEQ ID NO: 747, HCDR3 as set forth in SEQ ID NO: 748 The antibody according to any one of claims 1 to 15, selected from the group consisting of:

22. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 16; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 17; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 18 The antibody according to any one of claims 1 to 15, comprising:

23. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:24; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:25; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:26; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 27; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:28 The antibody according to any one of claims 1 to 15, comprising:

24. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 33; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 34; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 35; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 36; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 37; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 38 The antibody according to any one of claims 1 to 15, comprising:

25. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:43; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:44; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:45; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:46; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 47; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:48 The antibody according to any one of claims 1 to 15, comprising:

26. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO:53; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:54; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO:55; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:56; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 57; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:58 The antibody according to any one of claims 1 to 15, comprising:

27. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 63; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO:64; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 65; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:66; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 67; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:68 The antibody according to any one of claims 1 to 15, comprising:

28. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 73; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 74; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 75; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 76; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 77; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:78 The antibody according to any one of claims 1 to 15, comprising:

29. The antibody a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 83; b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 84; c. a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 85; d. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 86; e. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 87; and / or f. Heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 88 The antibody according to any one of claims 1 to 15, comprising:

30. 30. The antibody of any one of claims 1-29, wherein the antibody comprises an immunoglobulin light chain amino acid variable region sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.

31. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain amino acid variable region sequence identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.

32. The antibody comprises: (i) a light chain variable region (VL) having an LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a light chain variable region (VL) having an LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 31 16. The antibody of any one of claims 1 to 15, comprising a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 6, 318, and 320; wherein the CDRs are defined according to any one of the following: Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, and North definition.

33. the antibody comprising: (i) a light chain variable region (VL) having an LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a light chain variable region (VL) having an LCDR1, an LCDR2, and an LCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined using any two or three of the following hybrid schemes: Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition, and North definition.

34. 16. The antibody of any one of claims 1-15, wherein the antibody comprises: (i) a light chain variable region (VL) having LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Kabat definition.

35. 16. The antibody of any one of claims 1-15, wherein the antibody comprises: (i) a light chain variable region (VL) having LCDR1, LCDR2, and LCDR3 identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having HCDR1, HCDR2, and HCDR3 identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Chothia definition.

36. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:275, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

276.

37. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 275; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

276.

38. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:277, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

278.

39. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 277; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

278.

40. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 309, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

310.

41. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 309; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

310.

42. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 155, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

156.

43. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 155; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

156.

44. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 157, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

158.

45. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 157; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

158.

46. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 169, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

170.

47. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 169; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

170.

48. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:267, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

268.

49. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 267; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

268.

50. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:271, and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

272.

51. 30. The antibody of any one of claims 1 to 29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NO: 271; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO:

272.

52. 30. The antibody of any one of claims 1-29, wherein the antibody comprises an immunoglobulin light chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the immunoglobulin light chain variable region sequences of Table 1; and an immunoglobulin heavy chain variable region amino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the immunoglobulin heavy chain variable region sequences of Table 1.

53. 30. The antibody of any one of claims 1-29, wherein the antibody comprises an immunoglobulin light chain amino acid sequence identical to any one of the immunoglobulin light chain variable region sequences of Table 1; and an immunoglobulin heavy chain variable region amino acid sequence identical to any one of the immunoglobulin heavy chain variable region sequences of Table 1.

54. The antibody of any one of claims 1 to 53, wherein the antibody comprises an arginine substitution at position 17 of the immunoglobulin light chain according to Kabat numbering.

55. 55. The antibody of any one of claims 1 to 54, wherein the antibody comprises a tyrosine substitution at position 12 of the immunoglobulin light chain according to Kabat numbering.

56. The antibody of any one of claims 1 to 55, wherein the antibody does not bind to T lymphocytes.

57. The antibody of any one of claims 1 to 56, wherein the antibody does not bind to activated T lymphocytes.

58. The antibody of any one of claims 1 to 57, wherein the antibody does not bind to B lymphocytes.

59. 59. The antibody of any one of claims 1 to 58, wherein the antibody comprises an Fc region comprising an amino acid change selected from the list consisting of D356E, L358M, and combinations thereof.

60. A pharmaceutical composition comprising the antibody of any one of claims 1 to 59 and a pharmaceutically acceptable carrier, diluent, or excipient.

61. 61. The pharmaceutical composition of claim 60, formulated for intravenous administration.

62. A nucleic acid encoding the antibody of any one of claims 1 to 59.

63. 63. An expression vector comprising the nucleic acid of claim 62.

64. 64. A eukaryotic cell comprising the expression vector of claim 63.

65. 65. The eukaryotic cell of claim 64, wherein the eukaryotic cell is a CHO cell line.

66. 62. A method for treating cancer or tumor, comprising administering to an individual suffering from said cancer or tumor an antibody of any one of claims 1 to 59 or a pharmaceutical composition of claim 60 or 61.

67. 67. The method of claim 66, wherein the cancer or tumor is a solid tissue cancer.

68. 67. The method of claim 66, wherein the cancer or tumor is a blood cancer.

69. 67. The method of claim 66, wherein the cancer or tumor comprises breast cancer, colon cancer, or ovarian cancer.

70. 70. The method of claim 69, wherein the breast cancer is triple-negative breast cancer.

71. 62. A method for inhibiting CD24 signaling in cancer cells, comprising contacting the cancer cells with an antibody of any one of claims 1 to 59 or a pharmaceutical composition of claim 60 or 61.

72. 72. The method of claim 71, wherein CD24 signaling is not inhibited in non-neoplastic B lymphocytes or non-neoplastic T lymphocytes.

73. 73. The method of claim 71 or 72, wherein the cancer cell is a cancer cell that is in vivo in an individual.

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