New Anti-CD24 Antibody

Abstract

The present disclosure provides anti-CD24 antibodies and anti-CD24 / CD47 bispecific antibody molecules, isolated polynucleotides encoding them, pharmaceutical compositions containing them and uses thereof.

Description

[Technical Field]

[0001] The present disclosure relates generally to novel anti-CD24 antibodies. [Background technology]

[0002] Cluster of differentiation 24 (CD24, UniProt:P25063), also known as heat-stable antigen (HSA) or small cell lung cancer cluster 4 antigen, is a 32-amino acid mucin-like glycosylphosphatidylinositol (GPI)-anchored molecule that is highly glycosylated and anchored to the cell surface via GPI linkages. CD24 can interact with sialic acid-binding Ig-like lectin 10 (Siglec-10), which is expressed on innate immune cells (e.g., macrophages) to reduce harmful inflammatory responses to infection, sepsis, liver injury, and chronic graft-versus-host disease (Pirruccello et al., J. Immunol. 136, 3779-3784 (1986); Chen et al., Glycobiology 9, 800-806 (2014); Chen et al., Cell 152(3), 467-478; Chen GY et al. Nature Biotechnology 29, 428-435 (2011); Chen GY et al. Science 323(5922), 1722-1725 (2009); and Toubai T et al. Blood 123(22), 3512-3513 (2014)). CD24 binding to Siglec-10 induces an inhibitory signaling cascade mediated by SHP-1 and / or SHP-2 phosphatases that associate with two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic tail of Siglec-10, thereby blocking the cytoskeletal rearrangements required for toll-like receptor (TLR)-mediated inflammation and phagocytosis by macrophages (Crocker PR et al., Nature Reviews Immunology. 7, 255-266 (2007); Abram CL et al., J. Leuko.c Biol. 102 (3), 657-675 (2017) and Dietrich J et al., J. Immunol. 166 (4), 2514-2521 (2001) 12-14).

[0003] CD24 overexpression has been observed in a variety of cancer cells, based on immunohistochemical studies, including, but not limited to, breast cancer (85%), rectal cancer (84%), ovarian cancer (83%), pancreatic cancer (72%), bladder cancer (62%), cholangiocarcinoma (51%), prostate cancer (48%), and small cell lung cancer (45%). A meta-analysis of 28 studies revealed that CD24 is overexpressed in 68% of human cancers, and CD24 expression correlates with higher self-renewal capacity, more metastasis, and poor prognosis (Krisiansen G. et al., Am J Pathol, 161:1215-1221, 2002; Krisiansen G. et al., Clin cancer Res, 9:4906-4913, 2003; Krisiansen G. et al., Br J Cancer, 88:231-236, 2003; Krisiansen G. et al., Prostate, 58:182-192, 2004; Hocob J. et al., Pancreatology, 4:454-460, 2004; Samuel ED, BioMed Central, 3:3-15, 2004; Min-Cheng S. et al., Cancer letter, 1-6, 2005 and Lee et al., Oncol. Rep. 2009, 22, 1149-1156). Recent studies have shown that CD24-mediated inhibition of the innate immune system can be used by cancer cells as a mechanism to evade clearance by Siglec-10-expressing macrophages (Amira et al., Nature. 2019 August;572(7769):392-396).

[0004] Despite the development of therapeutic agents targeting CD24, there is a great need for novel anti-CD24 antibodies. Summary of the Invention

[0005] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody.

[0006] The present disclosure provides novel anti-CD24 antibody molecules, their amino acid and nucleotide sequences, and uses thereof.

[0007] In one aspect, the present disclosure provides the nucleic acids of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 14, 16, 17, 18, 19, 21, 23, 25, 27, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 95, 96, 97, 104, 105, 106, 113, 114, 115, 126, 127, 128, 136, 137, 150, 151, 152, 160, 161, 162, 168, 169 and / or one, two or three light chain CDR sequences selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 13, 15, 20, 22, 24, 26, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 98, 99, 100, 107, 108, 116, 117, 129, 130, 131, 138, 139, 140, 145, 153, 154, 155, 163, 171, 172, 173, 184, 185, 186, 187, 188, 225, 226 and 227.

[0008] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein a) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; b) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 11; c) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 11; d) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; e) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23; f) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 28; g) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 28; h) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36; i) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42; j) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48; k) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97; l) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106; m) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115; n) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 126, SEQ ID NO: 127 and SEQ ID NO: 128; o) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 136 and SEQ ID NO: 137; p) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 150, SEQ ID NO: 151 and SEQ ID NO: 152; q) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 162; r) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 168, SEQ ID NO: 169 and SEQ ID NO: 170; and s) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 222, SEQ ID NO: 223 and SEQ ID NO: 224; The heavy chain variable region comprises a heavy chain variable region selected from the group consisting of:

[0009] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein a) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; b) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12; c) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 15; d) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24; e) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 26, SEQ ID NO: 22 and SEQ ID NO: 24; f) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 29, SEQ ID NO: 22 and SEQ ID NO: 31; g) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 37; h) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43; i) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; j) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100; k) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 37; l) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 116 and SEQ ID NO: 117; m) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 131; n) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 138, SEQ ID NO: 139 and SEQ ID NO: 140; o) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 29, SEQ ID NO: 145 and SEQ ID NO: 24; p) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 153, SEQ ID NO: 154 and SEQ ID NO: 155; q) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 163, SEQ ID NO: 154 and SEQ ID NO: 155; r) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 171, SEQ ID NO: 172 and SEQ ID NO: 173; s) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 184, SEQ ID NO: 108 and SEQ ID NO: 37; t) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 108 and SEQ ID NO: 37; u) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 186, SEQ ID NO: 108 and SEQ ID NO: 37; v) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 187, SEQ ID NO: 108 and SEQ ID NO: 37; w) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 108 and SEQ ID NO: 37; and x) a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 225, SEQ ID NO: 226 and SEQ ID NO: 227. The light chain variable region comprises a light chain variable region selected from the group consisting of:

[0010] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein a) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; b) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11 and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12; c) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 7, SEQ ID NO: 14 and SEQ ID NO: 11 and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 15; d) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 15; e) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23 and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24; f) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 27 and SEQ ID NO: 28 and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 26, SEQ ID NO: 22 and SEQ ID NO: 24; g) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 28 and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 29, SEQ ID NO: 22 and SEQ ID NO: 31; h) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36 and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 37; i) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 38, SEQ ID NO: 40 and SEQ ID NO: 42 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 39, SEQ ID NO: 41 and SEQ ID NO: 43; j) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; k) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100; l) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 37; m) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 116 and SEQ ID NO: 117; n) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 126, SEQ ID NO: 127 and SEQ ID NO: 128 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 131; o) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 136 and SEQ ID NO: 137 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 138, SEQ ID NO: 139 and SEQ ID NO: 140; p) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 28 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 29, SEQ ID NO: 145 and SEQ ID NO: 24; q) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 150, SEQ ID NO: 151 and SEQ ID NO: 152 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 153, SEQ ID NO: 154 and SEQ ID NO: 155; r) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 162 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 163, SEQ ID NO: 154 and SEQ ID NO: 155; s) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 168, SEQ ID NO: 169 and SEQ ID NO: 170 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 171, SEQ ID NO: 172 and SEQ ID NO: 173; t) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 184, SEQ ID NO: 108 and SEQ ID NO: 37; u) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 108 and SEQ ID NO: 37; v) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 186, SEQ ID NO: 108 and SEQ ID NO: 37; w) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 187, SEQ ID NO: 108 and SEQ ID NO: 37; x) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 108 and SEQ ID NO: 37; or y) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 222, SEQ ID NO: 223 and SEQ ID NO: 224 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 225, SEQ ID NO: 226 and SEQ ID NO: 227. Includes:

[0011] In some embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region selected from the group consisting of SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, SEQ ID NO:66, SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:78, SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:101, SEQ ID NO:109, SEQ ID NO:118, SEQ ID NO:122, SEQ ID NO:132, SEQ ID NO:141, SEQ ID NO:146, SEQ ID NO:156, SEQ ID NO:164, SEQ ID NO:174, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, and homologous sequences sharing at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereto.

[0012] In some embodiments, an antibody or antigen-binding fragment thereof provided herein comprises a light chain variable region selected from the group consisting of SEQ ID NO:52, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:120, SEQ ID NO:124, SEQ ID NO:134, SEQ ID NO:143, SEQ ID NO:148, SEQ ID NO:158, SEQ ID NO:166, SEQ ID NO:176, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:221, and homologous sequences sharing at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity thereof.

[0013] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein a) a heavy chain variable region comprising SEQ ID NO: 50 and a light chain variable region comprising SEQ ID NO: 52; b) a heavy chain variable region comprising SEQ ID NO: 54 and a light chain variable region comprising SEQ ID NO: 56; c) a heavy chain variable region comprising SEQ ID NO: 58 and a light chain variable region comprising SEQ ID NO: 64; d) a heavy chain variable region comprising SEQ ID NO: 62 and a light chain variable region comprising SEQ ID NO: 64; e) a heavy chain variable region comprising SEQ ID NO: 66 and a light chain variable region comprising SEQ ID NO: 68; f) a heavy chain variable region comprising SEQ ID NO: 70 and a light chain variable region comprising SEQ ID NO: 72; g) a heavy chain variable region comprising SEQ ID NO: 74 and a light chain variable region comprising SEQ ID NO: 76; h) a heavy chain variable region comprising SEQ ID NO: 78 and a light chain variable region comprising SEQ ID NO: 80; i) a heavy chain variable region comprising SEQ ID NO: 82 and a light chain variable region comprising SEQ ID NO: 84; j) a heavy chain variable region comprising SEQ ID NO: 86 and a light chain variable region comprising SEQ ID NO: 88; k) a heavy chain variable region comprising SEQ ID NO: 101 and a light chain variable region comprising SEQ ID NO: 103; l) a heavy chain variable region comprising SEQ ID NO: 109 and a light chain variable region comprising SEQ ID NO: 111; m) a heavy chain variable region comprising SEQ ID NO: 118 and a light chain variable region comprising SEQ ID NO: 120 or SEQ ID NO: 221; n) a heavy chain variable region comprising SEQ ID NO: 122 and a light chain variable region comprising SEQ ID NO: 124; o) a heavy chain variable region comprising SEQ ID NO: 132 and a light chain variable region comprising SEQ ID NO: 134; p) a heavy chain variable region comprising SEQ ID NO: 141 and a light chain variable region comprising SEQ ID NO: 143; q) a heavy chain variable region comprising SEQ ID NO: 146 and a light chain variable region comprising SEQ ID NO: 148; r) a heavy chain variable region comprising SEQ ID NO: 156 and a light chain variable region comprising SEQ ID NO: 158; s) a heavy chain variable region comprising SEQ ID NO: 164 and a light chain variable region comprising SEQ ID NO: 166; t) a heavy chain variable region comprising SEQ ID NO: 174 and a light chain variable region comprising SEQ ID NO: 176; u) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 180; v) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 180; w) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 180; x) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 181; y) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 181; z) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 181; aa) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 182; bb) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 182; cc) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 182; dd) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 183; ee) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 183; ff) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 183; gg) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 195; hh) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 196; ii) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 197; jj) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 198; kk) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 199; ll) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 200; mm) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 201; nn) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 202; oo) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 203; pp) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 200; qq) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 201; rr) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 202; ss) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 203; tt) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 200; uu) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 201; vv) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 202; ww) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 203; xx) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 200; yy) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 201; zz) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 202; aaa) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 203; bbb) a heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 200; ccc) a heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 201; ddd) a heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 202; or eee) a heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 203 Includes:

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more amino acid residue substitutions or modifications, but retain specific binding affinity for human CD24 or cynomolgus CD24.

[0015] In some embodiments, substitutions are in one or more CDR sequences and / or in one or more of the VH or VL sequences, but not in any of the CDR sequences.

[0016] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin constant region, optionally the constant region of a human Ig, or optionally the constant region of a human IgG.

[0017] In some embodiments, the constant region comprises a human IgG1, IgG2, IgG3, or IgG4 constant region.

[0018] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are chimeric or humanized.

[0019] In some embodiments, an antibody or antigen-binding fragment thereof provided herein is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabody (ds diabody), single-chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single domain antibody, nanobody, domain antibody, and bivalent domain antibody.

[0020] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are bispecific.

[0021] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a first and a second epitope of CD24, or are capable of specifically binding to CD24 and a second antigen.

[0022] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein bind to a second antigen via a Fab or scFv, and optionally bind to CD24 via a Fab and to the second antigen via an scFv.

[0023] In some embodiments, the bispecific antibody molecule comprises a heavy chain in the format of VH(anti-CD24)-CH1-hinge-CH2-CH3-spacer-second antigen-binding scFv, associated with a light chain in the format of VL(anti-CD24)-CL.

[0024] In some embodiments, the second antigen is optionally an immune-related target selected from the group consisting of PD-L1, PD-L2, PD-1, CLTA-4, TIM-3, LAG3, CD160, 2B4, TGF beta, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, ICAM-1, NKG2C, SLAMF7, NKp80, CD160, B7-H3, LFA-1, ICOS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, SIRPα, Siglec10, LILRB2, Clever-1, Macro, LILRB4, Siglec15, CSF1R, PSGL-1, VSIG-4, B2M, and CD83.

[0025] In some embodiments, the second antigen comprises a tumor antigen.

[0026] In some embodiments, the tumor antigen is CA-125, gangliosides G(D2), G(M2) and G(D3), CD19, CD20, CD33, CD47, CD52, Ep-CAM, CEA, CLDN18.2, bombesin-like peptide, PSA, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, CD44v9, Ki-67, cancer-associated mucin, VEGF, These include VEGFRs (e.g., VEGFR3), estrogen receptors, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, IL-2R, CDH6, CEA, FOLR1, TROP2, PTK7, SLITRK6, CD142, NECTIN-4, ROR1, ROR2, CD142, CD123, CD22, CD79b, DLL3, SLC family, or CO17-1A.

[0027] In some embodiments, the second antigen is an immunosuppressive molecule selected from the group consisting of PD-L1, SIRPα, CD47, and B2M.

[0028] In some embodiments, the tumor antigen is CD47.

[0029] In some embodiments, the antibody or antigen-binding fragment is linked to one or more conjugates, optionally covalently attached either directly or via a linker.

[0030] In some embodiments, the conjugate comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent or other anti-cancer drug.

[0031] In some embodiments, the antibody or antigen-binding fragment is capable of specifically binding to CD24, optionally wherein the CD24 is derived from a human or a cynomolgus monkey, and optionally wherein the CD24 is recombinant CD24 or CD24 expressed on a cell surface.

[0032] In one aspect, the present disclosure provides antibodies or antigen-binding fragments thereof that compete with the antibodies or antigen-binding fragments thereof provided herein for the same epitope.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof provided herein competes for the same epitope with an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 107, 184, 185, 186, 187, and 188, LCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 37.

[0034] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein compete for the same epitope with an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively.

[0035] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein selectively bind to CD24 expressed on cancer cells over non-cancer cells.

[0036] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof provided herein and a pharmaceutically acceptable carrier.

[0037] In some embodiments, the pharmaceutical compositions provided herein further comprise a second therapeutic agent.

[0038] In some embodiments, the second therapeutic agent is an antagonist to one or more immunosuppressive molecules.

[0039] In some embodiments, the second therapeutic agent is a CD47 antagonist.

[0040] In some embodiments, the CD47 antagonist is a SIRPα-Fc fusion protein or a variant thereof, or an anti-CD47 antibody or an antigen-binding fragment thereof.

[0041] In one aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein.

[0042] In some embodiments, the isolated polynucleotide provided herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 102, 110, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, and 220.

[0043] In one aspect, the present disclosure provides a vector comprising an isolated polynucleotide provided herein.

[0044] In one aspect, the present disclosure provides a host cell comprising a vector provided herein.

[0045] In one aspect, the present disclosure provides a method of expressing an antibody or antigen-binding fragment thereof provided herein, the method comprising culturing a host cell provided herein under conditions in which a vector provided herein is expressed.

[0046] In one aspect, the present disclosure provides a method of treating a disease or condition in a subject that would benefit from modulation of CD24 activity, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a pharmaceutical composition provided herein.

[0047] In some embodiments, the disease or condition is a CD24-associated disease or condition.

[0048] In some embodiments, the disease or condition is cancer, an adaptive immune disease, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0049] In some embodiments, the cancer is lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, adenocarcinoma, leukemia, myeloma and lymphoma. In some embodiments, the cancer is chemotherapy-resistant cancer.

[0050] In some embodiments, the disease or condition is a B-cell lymphoma, e.g., a hematological cancer selected from Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), marginal zone B-cell lymphoma (MZL), mantle cell lymphoma (MCL), Richter's syndrome, Burkitt's lymphoma, or follicular lymphoma.

[0051] In some embodiments, the subject is a human.

[0052] In some embodiments, the methods provided herein include administering to a subject a therapeutically effective amount of a second therapeutic agent, in some embodiments, the second therapeutic agent is an antagonist to one or more immunosuppressive molecules.

[0053] In some embodiments, the second therapeutic agent is a CD47 antagonist.

[0054] In some embodiments, the CD47 antagonist is a SIRPα-Fc fusion protein or a variant thereof, or an anti-CD47 antibody or an antigen-binding fragment thereof.

[0055] In some embodiments, administration is by oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.

[0056] In one aspect, the present disclosure provides a method of modulating CD24 activity in a cell expressing CD24, the method comprising exposing the cell expressing CD24 to an antibody or antigen-binding fragment thereof provided herein.

[0057] In one aspect, the present disclosure provides a method for detecting the presence or amount of CD24 in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of CD24 in the sample.

[0058] In one aspect, the present disclosure provides a method of diagnosing a CD24-related disease or condition in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; c) determining the presence or amount of CD24 in the sample; and d) correlating the presence or amount of CD24 with the presence or status of a CD24-related disease or condition in the subject.

[0059] In one aspect, the disclosure provides use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD24-associated disease or condition in a subject.

[0060] In some embodiments, the medicament further comprises a second therapeutic agent, hi some embodiments, the second therapeutic agent is an antagonist to one or more immunosuppressive molecules.

[0061] In some embodiments, the second therapeutic agent is a CD47 antagonist, hi some embodiments, the CD47 antagonist comprises a SIRPα-Fc fusion protein or variant thereof and an anti-CD47 antibody or antigen-binding fragment thereof.

[0062] In one aspect, the present disclosure provides use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a diagnostic reagent for diagnosing a CD24-associated disease or condition.

[0063] In one aspect, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein useful in detecting CD24, optionally recombinant CD24, cell surface-expressed CD24, or cells expressing CD24.

[0064] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CD24 and comprises an antigen-binding fragment provided herein. In some embodiments, the antigen-binding fragment is a Fab or scFv.

[0065] In some embodiments, the CARs provided herein are bispecific.

[0066] In some embodiments, the CAR is capable of further specifically binding to a second antigen other than CD24 or a second epitope on CD24, hi some embodiments, the second antigen is a tumor antigen.

[0067] In some embodiments, the TCR signaling domain is selected from the group consisting of the intracellular signal region sequences of CD3ζ, FccRIγ, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or combinations thereof.

[0068] In some embodiments, the transmembrane region comprises the transmembrane region of CD3, CD4, CD8, or CD28.

[0069] In one aspect, the present disclosure provides a nucleic acid sequence encoding the chimeric antigen receptor (CAR) provided herein. In one aspect, the present disclosure provides a cell comprising the nucleic acid sequence provided herein. In one aspect, the present disclosure provides a vector comprising the nucleic acid sequence provided herein.

[0070] In one aspect, the present disclosure provides a cell genetically modified to express a CAR provided herein.

[0071] In some embodiments, the cell is an immune cell, optionally the immune cell is a T lymphocyte, an NK cell, a monocyte, a macrophage, or an NKT lymphocyte.

[0072] In one aspect, the present disclosure provides a method for stimulating a T cell-mediated immune response in a mammal against cells or tissues expressing CD24, the method comprising administering to the mammal an effective amount of cells genetically modified to express a CAR provided herein.

[0073] In one aspect, the present disclosure provides a method of treating a mammal having a CD24-associated disease or condition, the method comprising administering to the mammal an effective amount of a cell provided herein, thereby treating the mammal.

[0074] In some embodiments, the cells are autologous T cells.

[0075] In some embodiments, the CD24-associated disease or condition is cancer.

[0076] In some embodiments, the mammal is a human subject.

[0077] In one aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering an anti-CD24 antibody or antigen-binding fragment thereof that binds only to glycosylated or sialylated CD24, preferably that binds only to sialic acid of CD24.

[0078] In some embodiments, the anti-CD24 antibody or antigen-binding fragment thereof selectively binds to CD24 expressed on cancer cells over non-cancer cells.

[0079] In some embodiments, the anti-CD24 antibody or antigen-binding fragment thereof competes for the same epitope with an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 107, 184, 185, 186, 187, and 188, LCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 37, or competes for the same epitope with an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively.

[0080] In some embodiments, the anti-CD24 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 184, 185, 186, 187, and 188, LCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 37; or a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97, respectively, and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100, respectively. [Brief explanation of the drawings]

[0081] [Figure 1A] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on 293F blank cells. [Figure 1B] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on 293F-hCD24 cells. [Figure 1C] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on MCF7 cells. [Figure 1D] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on MDA-MB-468 cells. [Figure 1E] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on Nalm1 cells. [Figure 1F] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on CHOK1-hCD24 cells. [Figure 1G] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on HT-29 cells. [Figure 1H] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on NCI H1975 cells. [Figure 1I] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on A549 cells. [Figure 1J] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein on Nalm6 cells. [Figure 1K] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on SU-DHL6 cells. [Figure 1L]FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein on human B cells. [Figure 1M] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on DoHH2 cells. [Figure 1N] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on HT29 cells. [Figure 1O] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein on Huh7 cells. [Figure 1P] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on MCF7 cells. [Figure 1Q] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on a549 cells. [Figure 1R] 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and the benchmark antibody SN3 on NCI-H1975 cells. [Figure 2A] FIG. 1 shows the phagocytic index of chimeric anti-CD24 antibodies and the reference antibody SN3 on 293F-hCD24 cells. [Figure 2B] FIG. 1 shows the phagocytic index of chimeric anti-CD24 antibodies and the reference antibody SN3 on HT-29 cells. [Figure 3A] FIG. 1 shows the phagocytic index of combined treatment with anti-CD47 antibody hu5F9 and chimeric anti-CD24 antibody chAb110D4G4 on HT29 colorectal tumor cells. [Figure 3B] FIG. 1 shows the phagocytic index of combined treatment of anti-CD47 antibody hu5F9 and chimeric anti-CD24 antibody chAb111F3A2 on HT29 colorectal tumor cells. [Figure 3C] FIG. 1 shows the phagocytic index of combined treatment of anti-CD47 antibody hu5F9 and chimeric anti-CD24 antibody chAb81A1F8 on HT29 colorectal tumor cells. [Figure 4A]Fig. 1 shows the MFI of hIgG4 alone, ch81A1F8-IgG4 alone, the bispecific antibody (ch81A1F8-IgG4+Hu5F9-derived scFv), and the benchmark antibody SN3 on HT29 cells. [Figure 4B] FIG. 1 shows the phagocytic index of hIgG4 alone, hu5F9 alone, ch81A1F8-IgG4 alone, bispecific antibody (ch81A1F8-IgG4+Hu5F9-derived scFv), and the combination of anti-CD24 antibody ch81A1F8-IgG4 and anti-CD47 antibody Hu5F9 ("Combo") on HT29 cells. [Figure 5A] FIG. 1 shows the MFI of chimeric anti-CD24 antibody 81A1F8 and humanized 81A1F8 antibody on HT-29. [Figure 5B] FIG. 1 shows the MFI of chimeric anti-CD24 antibody 81A1F8 and humanized 81A1F8 antibody on Nalm6. [Figure 5C] FIG. 1 shows the MFI of chimeric anti-CD24 antibody 101H9G9A2 and humanized 101H9G9A2 antibody on HT-29. [Figure 5D] FIG. 1 shows the KD of chimeric anti-CD24 antibody 81A1F8 and humanized 81A1F8 antibody measured using the Gatorprime system. [Figure 6A] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and / or benchmark antibody SN3 on cells digested with neuraminidase or deglycosylation. [Figure 6B] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and / or benchmark antibody SN3 on cells digested with neuraminidase or deglycosylation. [Figure 6C] FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and / or benchmark antibody SN3 on cells digested with neuraminidase or deglycosylation. [Figure 6D]FIG. 1 shows the MFI of chimeric anti-CD24 antibodies provided herein and / or benchmark antibody SN3 on cells digested with neuraminidase or deglycosylation. [Figure 7A] FIG. 1 shows the KD of chimeric anti-CD24 antibodies provided herein and the reference antibody SWA11 on mFc-tagged human CD24 as measured by BIAcore. [Figure 7B] FIG. 1 shows the KD of chimeric anti-CD24 antibody 81A1F8 and the humanized 81A1F8 antibody provided herein on mFc-tagged human CD24 as measured by BIAcore. [Figure 8] FIG. 1 shows the normalized OD450 of different concentrations of chimeric anti-CD24 antibody 81A1F8 on mFc-tagged human CD24 as measured by ELISA. [Figure 9A] FIG. 1 shows the phagocytic index of chimeric anti-CD24 antibodies, reference antibody SWA11, and anti-CD47 antibody hu5F9-IgG4 on HT-29 cells. [Figure 9B] FIG. 1 shows the phagocytic index of chimeric anti-CD24 antibodies, reference antibody SWA11, and anti-CD47 antibody hu5F9-IgG4 on Nalm6 cells. [Figure 9C] FIG. 1 shows the phagocytic index of the 81A1F8-LALAPG antibody with rare Fc-dependent functions alone or in combination with rituximab on SU-DHL-6 cells. [Figure 9D] FIG. 1 shows the phagocytic index of the anti-CD24 antibody 81A1F8 alone, the anti-CD47 antibody Hu5F9 alone, and the combination of the anti-CD24 antibody 81A1F8 and the anti-CD47 antibody Hu5F9 on HT-29 cells. [Figure 10A] FIG. 1 shows the ADCC effect induced by chimeric anti-CD24 antibody 81A1F8 and humanized 81A1F8 antibody on HT-29 cells. [Figure 10B] FIG. 1 shows the CDC effect induced by chimeric anti-CD24 antibody 81A1F8 and humanized 81A1F8 antibody on hCD24-MCF7 cells. [Figure 11A]FIG. 1 shows tumor volume following administration of chimeric anti-CD24 antibody 81A1F8 alone, oxaliplatin alone, and the combination of chimeric anti-CD24 antibody 81A1F8 and oxaliplatin in a C57BL / 6J syngeneic mouse model. [Figure 11B] FIG. 1 shows body weight following administration of chimeric anti-CD24 antibody 81A1F8 alone, oxaliplatin alone, and the combination of chimeric anti-CD24 antibody 81A1F8 and oxaliplatin in a C57BL / 6J syngeneic mouse model. [Figure 11C] FIG. 1 shows a summary of an in vivo efficacy study of the chimeric anti-CD24 antibody 81A1F8 alone, oxaliplatin alone, and the combination of the chimeric anti-CD24 antibody 81A1F8 and oxaliplatin in a C57BL / 6J syngeneic mouse model. [Figure 11D] FIG. 1 shows the CD8 / CD4 and M1 / M2 macrophage ratios induced by combined treatment with chimeric anti-CD24 antibody 81A1F8 and oxaliplatin in a C57BL / 6J syngeneic mouse model. [Figure 12A] FIG. 1 shows tumor volume following administration of chimeric anti-CD24 antibody 81A1F8-mIgG2a at different dosages in the MC38-hCD24 syngeneic model. [Figure 12B] FIG. 1 shows tumor volumes following administration of chimeric anti-CD24 antibody 81A1F8-mIgG2a and chimeric anti-CD24 antibody 81A1F8-mIgG2a at different dosages after re-challenge in the MC38-hCD24 syngeneic model. [Figure 12C] FIG. 1 shows tumor growth curves for individual mice following administration of chimeric anti-CD24 antibody 81A1F8-mIgG2a at different dosages in the MC38-hCD24 syngeneic model. [Figure 12D] FIG. 1 shows a summary of the in vivo efficacy of chimeric anti-CD24 antibody 81A1F8-mIgG2a at different dosages. [Figure 13A] FIG. 1 shows tumor growth curves after administration of single agents (81A1F8-2141-mIgG2a, oxaliplatin or atezolizumab) or combination strategies. [Figure 13B]FIG. 1 shows tumor growth curves for each mouse in the treatment groups. [Figure 13C] FIG. 1 shows an overview of the in vivo efficacy of single agent (81A1F8-2141-mIgG2a, oxaliplatin or atezolizumab) or combination strategies. [Figure 14A] FIG. 1 shows tumor growth curves after administration of single agents (101H9G9A2-mIgG2a, 81A1F8-2141-mIgG2a, oxaliplatin or atezolizumab) or combination strategies. [Figure 14B] FIG. 1 shows the body weight of mice after administration of single agents (101H9G9A2-mIgG2a, 81A1F8-2141-mIgG2a, oxaliplatin or atezolizumab) or combination strategies. [Figure 14C] FIG. 1 shows an overview of the in vivo efficacy of single agent (101H9G9A2-mIgG2a, 81A1F8-2141-mIgG2a, oxaliplatin or atezolizumab) or combination strategies. [Figure 15] FIG. 1 shows the MFI of anti-cmCD24 antibodies provided herein on cmCD24-293F cells. [Figure 16A] FIG. 1 shows the phagocytic index of anti-cmCD24 antibodies provided herein on cynoCD24-293F cells. [Figure 16B] FIG. 1 shows the ADCC effect induced by the anti-cmCD24 antibodies provided herein on cmCD24-293F cells. [Figure 16C] FIG. 1 shows the CDC effect induced by the anti-cmCD24 antibodies provided herein on cynoCD24-293F cells. DETAILED DESCRIPTION OF THE INVENTION

[0082] The following description of the present disclosure is intended merely to illustrate various embodiments of the present disclosure. As such, the specific modifications discussed should not be construed as limitations on the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, modifications, and alterations can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0083] definition The term "antibody," as used herein, includes any immunoglobulin, monoclonal, polyclonal, multivalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A naturally occurring, intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and each heavy chain comprises a variable region (V H ) and a first, second, and third constant region (C H1 , C H2 , C H3 ), mammalian light chains are classified as λ or κ, and each light chain consists of a variable region (V L) and constant regions. Antibodies have a "Y" shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y contains the variable region and first constant region of a single heavy chain bound to the variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three hypervariable loops called complementarity-determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3; heavy chain CDRs including HCDR1, HCDR2, and HCDR3). The CDR boundaries of the antibodies and antigen-binding domains disclosed herein may be determined according to the Kabat, IMGT, AbM, Chothia, or Al-Lazikani convention (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J. Mol. Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); NR Whitelegg et al., Protein Engineering, v13(12), 819-824(2000); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al, Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)).The three CDRs are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major antibody classes or isotypes are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0084] The term "antibody molecule" as used herein refers to an antigen-binding protein or polypeptide comprising at least one antibody fragment (such as a CDR and / or variable region sequence). Antibody molecules include, for example, monoclonal antibodies, antibody fragments or domains, fusion proteins comprising antibody fragments or domains, and polypeptide complexes comprising antibody fragments or domains.

[0085] The term "antigen-binding domain" (e.g., CD24-binding domain), as used herein, refers to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding domains include, but are not limited to, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), diabodies, multispecific antibodies, camelized single-domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding domain is capable of binding to the same antigen as bound by the parent antibody. In certain embodiments, an antigen-binding domain may comprise one or more CDRs from a particular human antibody grafted onto framework regions from one or more different human antibodies. More detailed formats for antigen-binding domains are described in Spiess et al., 2015 and Brinkman et al., mAbs, 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.

[0086] "Fab," with respect to an antibody, refers to the portion of an antibody that consists of a single light chain (both variable and constant regions) connected by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0087] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0088] "F(ab')2" refers to a Fab' dimer.

[0089] "Fragment difficult (Fd)" with respect to antibodies refers to the amino-terminal half of a heavy chain fragment that can combine with a light chain to form Fab. For example, an Fd fragment can consist of the VH and CH1 domains.

[0090] With respect to antibodies, "Fv" refers to the smallest antibody fragment that contains a complete antigen-binding site. An Fv fragment consists of a single light chain variable region linked to a single heavy chain variable region. Numerous Fv designs are available, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond, and scFv, which can be formed using a peptide linker to link the two domains together as a single polypeptide. Fv constructs have also been created that contain the variable domains of the corresponding immunoglobulin heavy or light chains linked to constant domains. Fvs have also been multimerized to form diabodies and triabodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).

[0091] A "single-chain Fv antibody" or "scFv" refers to a genetically engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)).

[0092] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" comprises three peptide chains: two Vs linked by a peptide linker (e.g., a long flexible linker) via disulfide bridges. L Two V's bonded to the moiety H In some embodiments, dsFv-dsFv' are bispecific, in which each disulfide-paired heavy and light chain has a different antigen specificity.

[0093] "Fc," with respect to an antibody, refers to the portion of an antibody consisting of the second and third constant regions of a first heavy chain linked via disulfide bonds to the second and third constant regions of a second heavy chain. The Fc portion of an antibody is involved in various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0094] "Camelized single domain antibodies," "heavy chain antibodies," or "HCAbs" are antibodies that combine two V H It refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10;231(1-2):25-38(1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302(2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies were originally derived from the Camelidae family (camels, dromedaries, and llamas). Camelized antibodies lack light chains but have a robust antigen-binding repertoire (Hamers-Casterman C. et al., Nature. June 3; 363(6428):446-8(1993); Nguyen VK. et al. "Heavy-chain antibodies in Camelidae: a case of evolutionary innovation," Immunogenetics. April; 54(1):39-47(2002); Nguyen VK. et al. Immunology. May; 109(1):93-101(2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. November; 21(13):3490-8. Epub 2007 Jun 15 (2007)).

[0095] "Nanobody" refers to an antibody fragment consisting of a VHH domain and two constant domains, CH2 and CH3, derived from a heavy chain antibody.

[0096] A "diabody" or "dAb" comprises a small antibody fragment with two antigen-binding sites, the fragments arranged in the same polypeptide chain as V L V connected to the domain H Domain (V H -V L or V L -V H ) (see, for example, Holliger P. et al., Proc Natl Acad Sci US A. Jul 15;90(14):6444-8(1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes).

[0097] In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0098] In certain embodiments, an "scFv dimer" refers to a dimer of different V H -V L V dimerized with the moiety H -V L (linked by a peptide linker), such that one part V H But the other part of V L and form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes).

[0099] In other embodiments, the "scFv dimer" is L1 -V H2 V associated with (linked by a peptide linker) H1 -V L2(also linked by a peptide linker), so that V H1 and V L1 cooperated, V H2 and V L2 These are bispecific diabodies in which each pair cooperates with another pair and each pair has a different antigen specificity.

[0100] A "domain antibody" refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In particular examples, two or more V H The domains are covalently linked with peptide linkers to create bivalent or multivalent domain antibodies. H The domains may target the same or different antigens.

[0101] The term "chimeric" as used herein refers to an antibody or antigen-binding domain having a portion of the heavy and / or light chain derived from one species and the remainder of the heavy and / or light chain derived from another species. In an illustrative example, a chimeric antibody may contain a constant region derived from a human and a variable region derived from a non-human species, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0102] The term "humanized" as used herein means that an antibody or antigen-binding domain comprises CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, constant regions derived from a human.

[0103] The term "operably linked" or "operably linked" refers to the juxtaposition of two or more biological sequences of interest, with or without a spacer or linker or intervening sequence, in a relationship that allows them to function in their intended manner. When used in reference to a polypeptide, it is intended to mean that the polypeptide sequences are linked in a manner that allows the linked product to have its intended biological function. For example, an antibody variable region can be operably linked to a constant region to provide a stable product with antigen-binding activity. As another example, an antigen-binding domain can be operably linked to another antigen-binding domain via an intervening sequence between them, which may be a spacer or may comprise a much longer sequence such as an antibody constant region. This term can also be used in reference to polynucleotides. As an example, when a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequences are linked in a manner that allows regulated expression of the polypeptide from the polynucleotide.

[0104] The terms "fusion" or "fused," when used in reference to an amino acid sequence (e.g., a peptide, polypeptide, or protein), refer to the joining of two or more amino acid sequences into a single non-naturally occurring amino acid, for example, by chemical conjugation or recombinant means. A fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and expressed by introducing a construct containing the recombinant polynucleotide into a host cell.

[0105] "Antigen," as used herein, refers to a compound, composition, peptide, polypeptide, protein, or substance capable of stimulating antibody production or a T-cell response in a cell culture or an animal, and includes compositions (such as those containing cancer-specific proteins) added to cell cultures (such as hybridomas) or injected or absorbed into animals. Antigens react with specific products of humoral or cellular immunity (such as antibodies), including those induced by heterologous antigens.

[0106] The term "CD24," as used herein, is intended to encompass any form of CD24, e.g., 1) the native, unprocessed CD24 molecule, a "full-length" CD24 chain, or a naturally occurring variant of CD24, including, e.g., a splice variant or allelic variant; 2) any form of CD24 that results from processing in the cell; or 3) full-length, fragmented (e.g., truncated forms, extracellular / transmembrane domain) or modified (e.g., mutated, glycosylated / PEGylated, His-tagged / immunofluorescence fused) forms of a CD24 subunit produced by recombinant methods.

[0107] The terms "anti-CD24 antibody," "anti-CD24 binding domain," or "CD24 binding domain" refer to an antibody that binds to CD24 (e.g., human, mouse, or cynomolgus CD24). "Antibody" refers to an antibody or antigen-binding domain capable of specifically binding to

[0108] The terms "specific binding" or "specifically bind," as used herein, refer to a non-random binding reaction between two molecules, such as, for example, between an antibody and an antigen.

[0109] The binding of an antibody to recombinant CD24 or CD24 expressed on the surface of a cell can also be expressed by a "half maximal effective concentration" (EC50) value, which refers to the concentration of antibody at which 50% of its maximal effect (e.g., binding or inhibition) is observed. EC 50The binding level can be measured by methods known in the art, for example, sandwich assays such as ELISA, Western blot, flow cytometry assays and other binding assays. In certain embodiments, the antibodies and fragments thereof provided herein have a binding level of 0.05nM or less, 0.06nM or less, 0.07nM or less, 0.08nM or less, 0.09nM or less, 0.1nM or less, 0.2nM or less, 0.3nM or less, 0.4nM or less, 0.5nM or less, 0.6nM or less, 0.7nM or less, 0.8nM or less, 0.9nM or less, 1nM or less, 2nM or less, 3nM or less, 4nM or less, 5nM or less, 6nM or less, 7nM or less, 8nM or less, 9nM or less, 10nM or less, 20nM or less, 25nM or less, Specifically binds to recombinant human CD24 with an EC50 (i.e., 50% binding concentration) of 30 nM or less, 35 nM or less, 40 nM or less, 50 nM or less, 60 nM or less, 70 nM or less, 80 nM or less, 90 nM or less, 100 nM or less, 110 nM or less, 120 nM or less, 122 nM or less, 124 nM or less, 126 nM or less, 128 nM or less, 130 nM or less, 132 nM or less, 134 nM or less, 136 nM or less, 138 nM or less, 140 nM or less, 200 nM or less, 210 nM or less, 250 nM or less, 300 nM or less, or 400 nM or less.

[0110] The ability to "block binding" or "compete for the same epitope" as used herein refers to the ability of an antibody or antigen-binding domain to inhibit the binding interaction between two molecules (e.g., human CD24 and Siglec-10) to any detectable degree. In certain embodiments, an antibody or antigen-binding domain that blocks binding between two molecules inhibits the binding interaction between the two molecules by at least 85% or at least 90%. In certain embodiments, this inhibition may be greater than 85% or greater than 90%.

[0111] The term "epitope" as used herein refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. Epitopes can be formed from both contiguous amino acids (also called linear or continuous epitopes) or noncontiguous amino acids juxtaposed by tertiary folding of a protein (also called conformational or conformational epitopes). Epitopes formed from contiguous amino acids are typically arranged linearly along the primary amino acid residues on a protein, and small segments of contiguous amino acids can be digested from antigens bound to major histocompatibility complex (MHC) molecules or retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically contains at least three, more usually at least five, about seven, or about eight to ten amino acids in a unique spatial conformation. Two antibodies may bind to the same or closely related epitopes within an antigen if they exhibit competitive binding for that antigen. For example, an antibody or antigen-binding domain can be considered to bind to the same / closely related epitope as the reference antibody if it blocks binding of the reference antibody to the antigen by at least 85%, or at least 90%, or at least 95%.

[0112] " Conservative substitution " refers to the substitution of an amino acid residue with a different amino acid residue that has a side chain with similar physicochemical properties in terms of amino acid sequence.For example, conservative substitution can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr and Phe).As is known in the art, conservative substitution usually does not cause significant changes in the conformational structure of protein, and therefore can retain the biological activity of protein.

[0113] The terms "homologue" and "homologous," as used herein, refer to a nucleic acid sequence (or its complementary strand) or amino acid sequence that is interchangeable and has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to another sequence when optimally aligned.

[0114] "Percent (%) sequence identity" is defined, with respect to an amino acid sequence (or nucleic acid sequence), as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available at the US National Center for Biotechnology Information (NCBI) website; see also Altschul S F et al, J. Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available at the European Bioinformatics Institute website; see also Higgins D Get al, Methods in Enzymology, 266:383-402 (1996); Larkin M A et al, Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize the parameters as needed for the alignment, such as by selecting a suitable algorithm.

[0115] As used herein, "effector function" refers to the biological activity resulting from the binding of the Fc region of an antibody to its effector, such as the C1 complex, Fc receptor, and effector cell (e.g., macrophage). Exemplary effector functions include complement-dependent cytotoxicity (CDC), which is induced by the interaction of an antibody with C1q on the C1 complex; antibody-dependent cell-mediated cytotoxicity (ADCC), which is induced by the binding of the Fc region of an antibody to an Fc receptor on an effector cell; and antibody-dependent cellular phagocytosis (ADCP), which is induced by the binding of the Fc region of an antibody to a phagocytic cell. It is well established that specific glycan structures associated with the conserved biantennary glycan in the Fc-CH2 domain can strongly influence the interaction with FcyR, which mediates ADCC and ADCP, as well as the interaction with Clq binding, the initial binding event that leads to CDC (see Reusch D, Tejada ML. Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology 2015;25:1325-34).

[0116] "Treating" or "treatment" of a condition, as used herein, includes preventing or alleviating the condition, slowing the onset or rate of occurrence of the condition, reducing the risk of occurrence of the condition, preventing or delaying the onset of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, causing complete or partial regression of the condition, curing the condition, or some combination thereof.

[0117] The terms "subject" or "individual" or "animal" or "patient," as used herein, refer to a human or non-human animal, including a mammal or primate, in need of diagnosis, prognosis, amelioration, prevention, and / or treatment of a disease or disorder. Mammalian subjects include humans, domestic animals, livestock, and zoo, sport, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cows, and bears.

[0118] The term "vector" as used herein refers to a vehicle into which a polynucleotide encoding a protein can be operably inserted to cause expression of that protein. Vectors can be used to transform, transduce, or transfect host cells to cause expression of the genetic elements they carry within the host cell. Examples of vectors include plasmids, phagemids, cosmids, and artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as λ phage or M13 phage, and animal viruses. Categories of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors can contain various elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, vectors can contain an origin of replication. The vector may also contain materials to aid its entry into the cell, including, but not limited to, a viral particle, liposome, or protein coating. The vector may be an expression vector or a cloning vector.

[0119] The term "host cell," as used herein, refers to a cell into which an exogenous polynucleotide and / or vector has been introduced.

[0120] "CD24-associated disease or condition," as used herein, refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of CD24. In some embodiments, the CD24-associated condition includes an immune-related disorder, such as cancer, an autoimmune disease, an inflammatory disease, or an infectious disease.

[0121] "Cancer," as used herein, refers to any medical condition characterized by malignant cell growth or neoplasia, abnormal proliferation, invasion, or metastasis, and includes both solid tumors and non-solid cancers (hematologic malignancies), such as leukemia. As used herein, "solid tumor" refers to a solid mass of neoplastic and / or malignant cells. Examples of cancers or tumors include hematologic malignancies, oral cancer (e.g., lip, tongue, or pharynx), digestive tract (e.g., esophagus, stomach, small intestine, colon, large intestine, or rectum), peritoneum, liver and biliary tract, pancreas, respiratory system (small cell and non-small cell), such as larynx, or lung, bone, connective tissue, skin (e.g., melanoma), breast, reproductive organs (fallopian tubes, uterus, cervix, testes, ovaries, or prostate), urinary tract (e.g., bladder or kidney), brain, and endocrine glands, such as the thyroid gland. In certain embodiments, the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma, and colorectal cancer. In certain embodiments, the cancer is selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

[0122] The term "chemoresistant cancer" as used herein refers to a type of cancer that does not respond to the effects of chemotherapy. For example, a cancer that has been responding to chemotherapy or a combination of different chemotherapy drugs may suddenly begin to grow, which may be referred to as a chemotherapy-resistant cancer.

[0123] The term "pharmaceutically acceptable" indicates that the specified carrier, vehicle, diluent, excipient(s) and / or salt is generally chemically and / or physically compatible with the other ingredients comprising the formulation and physiologically compatible with the recipient thereof.

[0124] Anti-CD24 antibody The present disclosure provides anti-CD24 antibodies and antigen-binding fragments thereof that include one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of anti-CD24 antibodies 33E7E12, 71A4F12, 56A7E3, 58F10-1F6, 73H5-1G2, 110D4G4, 81A1F8, 100F2E3, 111F3A2, 81A7A10, 107D10D11, 101H9G9A2, 173B1C1, 107D10E11, 109G10A6, 94G12D11, 92F5B8, 185B11E3, 168B11E2, or 188H6D3. In certain embodiments, the anti-CD24 antibodies and antigen-binding fragments thereof provided herein are capable of specifically binding to CD24. Optionally, the CD24 is derived from a human or a cynomolgus monkey. In certain embodiments, the CD24 is recombinant CD24 or CD24 expressed on the cell surface.

[0125] "33E7E12" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:50 and a light chain variable region of SEQ ID NO:52.

[0126] "71A4F12" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:54 and a light chain variable region of SEQ ID NO:56.

[0127] "56A7E3" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:58 and a light chain variable region of SEQ ID NO:64.

[0128] "58F10-1F6" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:62 and a light chain variable region of SEQ ID NO:64.

[0129] "73H5-1G2" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:66 and a light chain variable region of SEQ ID NO:68.

[0130] "110D4G4" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:70 and a light chain variable region of SEQ ID NO:72.

[0131] "81A1F8" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:74 and a light chain variable region of SEQ ID NO:76.

[0132] "100F2E3" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:78 and a light chain variable region of SEQ ID NO:80.

[0133] "111F3A2" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:82 and a light chain variable region of SEQ ID NO:84.

[0134] "81A7A10" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:86 and a light chain variable region of SEQ ID NO:88.

[0135] "107D10D11" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:101 and a light chain variable region of SEQ ID NO:103.

[0136] "101H9G9A2" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:109 and a light chain variable region of SEQ ID NO:111.

[0137] "173B1C1" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:118 and a light chain variable region of SEQ ID NO:120 or SEQ ID NO:221.

[0138] "107D10E11" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:122 and a light chain variable region of SEQ ID NO:124.

[0139] "109G10A6" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:132 and a light chain variable region of SEQ ID NO:134.

[0140] "94G12D11" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:141 and a light chain variable region of SEQ ID NO:143.

[0141] "92F5B8" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:146 and a light chain variable region of SEQ ID NO:146.

[0142] "185B11E3" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:156 and a light chain variable region of SEQ ID NO:158.

[0143] "168B11E2" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:164 and a light chain variable region of SEQ ID NO:166.

[0144] "188H6D3" as used herein refers to a murine monoclonal antibody having a heavy chain variable region of SEQ ID NO:174 and a light chain variable region of SEQ ID NO:176.

[0145] Table 1 shows the CDR sequences of these 20 anti-CD24 antibodies according to IMGT numbering. The heavy and light chain variable region sequences are also provided below.

[0146] [Table 1-1] [Table 1-2] [Table 1-3]

[0147] The heavy or light chain variable region sequences of the 33E7E12, 71A4F12, 56A7E3, 58F10-1F6, 73H5-1G2, 110D4G4, 81A1F8, 100F2E3, 111F3A2, 81A7A10, 107D10D11101H9G9A2, 173B1C1, 107D10E11, 109G10A6, 94G12D11, 92F5B8, 185B11E3, 168B11E2 or 188H6D3 antibodies are provided below.

[0148] 33E7E12-VH Amino acid sequence (SEQ ID NO:50): EVKLEESGGGLVQPGGSMKLSCFVSGFTFSNSWMNWVRQSPEKGLEWVAQI QLKSDNYATRYVESVRGRFIISRDDDSKSSVSLQMNNLRTEDTGIYYCSSGTDY WGQGTTLTVSS Nucleic acid sequence (SEQ ID NO:51): GAAGTGAAGCTTGAGGAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGAT CCATGAAACTCTCCTGTTTTGTCTCTGGATTCACTTTCAGTAACTCCTGGA TGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTCAG ATTCAATTGAAATCTGATAATTATGCAACACGTTATGTGGAGTCTGTGAG AGGGAGGTTCATCATCTCAAGAGATGATTCCAAAAGTAGTGTCTCCCTGC AAATGAACAACTTAAGGACTGAAGACACTGGAATTTATTACTGTTCCTCT GGGACCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

[0149] 33E7E12-VL Amino acid sequence (SEQ ID NO:52): DVVMTQTPLASVIIGQPASISCKSSQSLLFSNGKTYLNWLLQRPGQSPKRLIY LVSNLDSGVPDRFTGSGSGTDFTLKIGRVEAEDLGVYYCVQGTHFPYTFGGG TKLEIK Nucleic acid sequence (SEQ ID NO:53): GATGTTGTGATGACCCAGACTCCACTCGCTTTGTCGGTTATCATTGGACA ACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATTTAGTAATG GAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAG CGCCTAATCTATCTGGTGTCTAATCTGGACTCTGGAGTCCCTGACAGGTT CACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCGGCAGAGTGG AGGCTGAAGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCA TACACGTTCGGTGGGGGGACCAAGTTGGAAAATAAAA

[0150] 71A4F12-VH Amino acid sequence (SEQ ID NO:54): EVQLQQSGPELVKPGASVKIPCKASGYTFTAYNMDWVKQSHGMSLEWIGDI NPNNGDTVYNQNFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARRGSY YYGSSHYALDFWGQGTSVSVSS Nucleic acid sequence (SEQ ID NO: 55): GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC AGTGAAGATACCCTGCAAGGCTTCTGGATACACATTCACTGCCTACAACA TGGACTGGGTGAAGCAGAGCCATGGAATGAGCCTTGAGTGGATTGGAGAT ATTAATCCTAACAATGGTGATACTGTCTACAACCAGAATTTCAAGGGCAA GGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAACTCC GCAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGAAGGGGG TCCTATTACTACGGTAGTAGTCACTATGCTCTGGACTTCTGGGGTCAAGG AACCTCAGTCTCCGTCTCCTCA

[0151] 71A4F12-VL Amino acid sequence (SEQ ID NO: 56): DIQMTQITSSLSASLGNRVTISCRASQDIRNYLNWYQQKPDGTVKLLIYYTSR LLSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQDNSLPRTFGGGTRLEIR Nucleic acid sequence (SEQ ID NO: 57): GATATCCAGATGACACAGATTACATCCTCCCTGTCTGCCTCTCTGGGAAA CAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGGAATTATTTAA ACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTAC ACATCAAGATTACTCTCAGGAGTCCCATCACGGTTCAGTGGCAGTGGGTC TGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTG CCACTTACTTTTGCCAACAGGATAATTCGCTTCCTCGGACGTTCGGTGGA GGCACCAGGCTGGAAATCAGA

[0152] 56A7E3-VH Amino acid sequence (SEQ ID NO:58): EVQLQQSGPELVKPGASVKIPCKASGYTFTAYNMDWVKQSHGMSLEWIGDI NPINGGTIYNQNFEGKATLTVDKSSSTAYLELRSLTSEDTAVYYCARRGSYYY GSSHYALDFWGQGTSVSVSS Nucleic acid sequence (SEQ ID NO:59): GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC AGTGAAGATACCCTGCAAGGCTTCTGGATACACATTCACTGCCTACAACA TGGACTGGGTGAAGCAGAGCCATGGAATGAGCCTTGAGTGGATTGGAGAT ATTAATCCTATCAATGGTGGTACTATCTACAACCAGAATTTCGAGGGCAA GGCCACATTGACTGTAGACAAGTCTTCCAGCACAGCCTACTTGGAACTCC GCAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGAAGGGGG TCCTATTACTACGGTAGTAGCCACTATGCTTTGGACTTCTGGGGTCAAGG AACCTCAGTCTCCGTCTCCTCA

[0153] 56A7E3-VL Amino acid sequence (SEQ ID NO: 64): DIQMTQITSSLSASLGDRVTITCRASQDISNYLNWYQQKPDGTVKLLIYYTSRL LSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQDHTLPRTFGGGTKLEIR Nucleic acid sequence (SEQ ID NO:61): GATATCCAGATGACACAGATTACATCCTCCCTGTCTGCCTCTCTGGGAGA CAGAGTCACCATCACTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAA ACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTAC ACATCAAGATTACTCTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTC TGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTG CCACTTACTTTTGCCAACAGGATCATACGCTTCCTCGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAGA

[0154] 58F10-1F6-VH Amino acid sequence (SEQ ID NO: 62): EVQLRQSGPELVKPGAAVKISCKASGYSFTGYHMNWVKQSPEKSLEWIGEIN PITSGITYNQIFKAKATLTVDKSSSTAYLQLKSLTSEDSAVYYCTRRDYGTSLD YWGQGTTLTVSS Nucleic acid sequence (SEQ ID NO: 63): GAGGTCCAGCTGCGGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTGC GGTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCACTGGCTACCACA TGAACTGGGTGAAGCAAAGTCCTGAAAAGAGCCTTGAGTGGATTGGAGA GATTAATCCTATCACTAGTGGTATTACCTACAACCAGATTTTCAAGGCCAA GGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACTTGCAGCTCA AGAGCCTGACATCTGAGGACTCTGCAGCTCTATTACTGTACAAGAAGGGAC TACGGTACTAGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTC CTCA

[0155] 58F10-1F6-VL Amino acid sequence (SEQ ID NO: 64): DIQMTQITSSLSASLGDRVTITCRASQDISNYLNWYQQKPDGTVKLLIYYTSRL LSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQDHTLPRTFGGGTKLEIR Nucleic acid sequence (SEQ ID NO: 65): GATATCCAGATGACACAGATTACATCCTCCCTGTCTGCCTCTCTGGGAGA CAGAGTCACCATCACTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAA ACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTAC ACATCAAGATTACTCTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTC TGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTG CCACTTACTTTTGCCAACAGGATCATACGCTTCCTCGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAGA

[0156] 73H5-1G2-VH Amino acid sequence (SEQ ID NO: 66): QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWIRQSPGKGLEWLGVIW RGGSTDYNAAFMSRLSITKDNSKSQVFFQMNSLQAADTAIYYCANNYGYDC FAYWGQGTLVTVSA Nucleic acid sequence (SEQ ID NO: 67): CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAG CCTGTCCATAACCTGCACAGTCTCTGGTTTCTCATTAACTAACTATGGTG TACACTGGATTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTG ATATGGAGAGGTGGAAGCACAGACTACAATGCAGCTTTCATGTCCAGACT GAGCATCACCAAGGACAACTCCAAGAGCCAAGTTTTCTTTCAAATGAACA GTTCTGCAAGCTGCTGACACTGCCATATATTACTGTGCCAACAACTATGGT TACGACTGCTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGC A

[0157] 73H5-1G2-VL Amino acid sequence (SEQ ID NO: 68): DVQITQSPSYLAASPGETITINCRASKSIIKYLAWYQEKPGKTNKLLIYSGSTLQ SGIPSRFSGSGSGTDFTLTITSLEPEDFAMYYCQQHNEYPLTFGAGTRLELK Nucleic acid sequence (SEQ ID NO:69): GATGTCCAAATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGA AACCATTACTATTAATTGCAGGGCAAGTAAGAGTATTATCAAATATTTAG CCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCT GGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATC TGGTACAGATTTCACTCTCACCATCACTAGCCTGGAGCCTGAAGATTTTG CAATGTATTACTGTCAACAGCATAATGAATACCCGCTCACGTTCGGTGCT GGGACCAGGCTGGAGCTGAAA

[0158] 110D4G4-VH Amino acid sequence (SEQ ID NO:70): EVQLQQSGPGLVKPGASVKISCRASGYLFTGYYMNWVKQSPEKSLEWIGEIN PNTGDPRYDQKFKAKATLTVDRSSSTAYMHLKSLTSEDSAVYFCARRDYGTS LDYWGQGTTLTVTS Nucleic acid sequence (SEQ ID NO:71): GAGGTTCAGCTGCAGCAGTCTGGACCTGGGCTGGTGAAGCCTGGGGCTTC AGTGAAGATATCCTGCAGGGCTTCTGGTTACCTATTCACTGGCTACTACA TGAACTGGGTGAAGCAAAGTCCTGAAAAGAGCCTTGAGTGGATTGGAGA GATTAATCCTAACACTGGTGATCCTAGATACGACCAGAAGTTTAAGGCCA AGGCCACATTGACTGTAGACAGATCCTCCAGCACAGCCTACATGCACCTC AAGAGCCTGACATCTGAAGACTCTGCAGCTCTATTTCTGTGCCAGGAGGGA CTACGGTACTAGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCA CCTCA

[0159] 110D4G4-VL Amino acid sequence (SEQ ID NO:72): DVQITQSPSYLAASPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTL QSGIPSRFSGSGSGSDFTLTISSLEPEDFAMYYCQQHNEYPLTFGAGTKLELK Nucleic acid sequence (SEQ ID NO: 73): GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGA AACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAGCAAATATTTAG CCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCT GGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATC TGGTTCAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTG CAATGTATTACTGTCAACAGCATAATGAATATCCGTCACGTTCGGTGCT GGGACCAAGCTGGAGCTGAAA

[0160] 81A1F8-VH Amino acid sequence (SEQ ID NO:74): EVQLQQSGPELVKPGASVKISKATGYLFTGYYMNWVKQSPEKSLEWIGEIN PNTGDTNNNQKFKAKATMTVDRSSSTAYMQLKRLTSEDSAVYYCVRRDYG TSLDYWGQGTTLTVSS Nucleic acid sequence (SEQ ID NO: 75): GAGGTTCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTC AGTGAAGATATCCTGCAAGGCTACTGGTTACTTATTCACTGGCTACTACA TGAACTGGGTGAAACAAAGTCCTGAAAAGAGCCTTGAGTGGATTGGAGA GATCAATCCTAACACTGGTGATACTAACAACAACCAGAAGTTCAAGGCCA AGGCCACAATGACTGTTGACAGATCCTCCAGCACAGCCTACATGCAGCTC AAGAGGCTGACATCTGAGGACTCTGCAGTCTATTATTGTGTAAGAAGGGA CTACGGTACTAGTCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTC CTCA

[0161] 81A1F8-VL Amino acid sequence (SEQ ID NO: 76): DVQITQSPSYLAASPGETITINCRASKSINKYLAWYQEKPGKTNKLLIHSGSTL QSGIPSRFSGSRSGTDFTLTISNLEPEDFAMYYCQQHNEYPITFGAGTKLELK Nucleic acid sequence (SEQ ID NO: 77): GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGA AACCATTACTATTAATTGCAGGGCAAGTAAGAGTATTAACAAATATTTAG CCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCCACTCT GGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTAGATC TGGAACAGATTTCACTCTCACCATCAGTAACCTGGAGCCTGAAGATTTTG CAATGTATTACTGTCAACAACATAATGAATATCCGATCACGTTCGGTGCT GGGACCAAGCTGGAGCTGAAA

[0162] 100F2E3-VH Amino acid sequence (SEQ ID NO:78): QVQLQQPGAELVRPGSSVKLSCKTSDYTFTSYWMDWVKQRPGQGLEWIGNI YPSDSKTHSNQKFRDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARRGGY YGYAMDYWGQGTSVTVSS Nucleic acid sequence (SEQ ID NO:79): CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAGGCCTGGGTCTTC AGTGAAGCTGTCCTGCAAGACTTCTGACTACACCTTCACCAGCTACTGGA TGGATTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAATGGATTGGTAAC ATTTACCCTTCTGATAGTAAAACTCACTCCAATCAAAAGTTCAGGGACAA GGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCA ACAGCCTGACATCTGAAGACTCTGCGGTCTATTACTGTGCAAGAAGAGGT GGTTACTACGGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCAC CGTCTCCTCA

[0163] 100F2E3-VL Amino acid sequence (SEQ ID NO:80): DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQDNYLAWYQQKPGQPPKL LIYGASIRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPYTFGG GTKLEIK Nucleic acid sequence (SEQ ID NO:81): GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGA GAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAATAGTGGAA ATCAAGACAACTACTTGGCTGGTACCAGCAGAAACCAGGGCAGCCTCCT AAACTGTTGATCTACGGGGCATCCATTAGGGAATCTGGGGTCCCTGATCG CTTCACAGGCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGTAGTG TGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAAAATGATCATAGTTAT CCGTACACGTTCGGAGGGGGGACCAAGTTGGAAAATAAAA

[0164] 111F3A2-VH Amino acid sequence (SEQ ID NO:82): QVQLKESGPGLVAPSQSLSITCTVSGFSLITYGVSWVRQPPGKGLEWLGVIWG DGTTNYHSALKPRLTISKDDSKSQVLLKLNSLQTDDTATYYCANYYGYPFFA YWGQGTLVTVSA Nucleic acid sequence (SEQ ID NO:83): CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAG CCTGTCCATCACATGCACTGTCTCAGGATTCTCATTAATCACCTATGGTG TAAGCTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTA ATATGGGGTGACGGGACCACAAATTATCATTCAGCTCTCAAACCCAGACT GACCATCAGCAAGGATGACTCCAAGAGCCAAGTCCTCTTAAAATTGAACA GTTCTGCAAACTGATGACACAGCCACGTACTACTGTGCCAACTACTATGGT TACCCCTTTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGC A

[0165] 111F3A2-VL Amino acid sequence (SEQ ID NO:84): DIQMTQSSSSFSVSLGDRVTITCKASEDIYNRLAWYQQKPGSAPGVLISGATN LVTGIPSRFSGSGSGKDYTLTITSLQTEDVATYYCQQYWSSSPPTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO:85): GACATCCAGATGACACAATCTTCATCCTCCTTTTCTGTATCTCTAGGAGAC AGAGTCACCATTACTTGCAAGGCAAGTGAGGACATATATAATCGGTTAGC CTGGTATCAACAGAAACCAGGAAGTGCTCCTGGGGTCTTAATATCTGGTG CAACCAATTTGGTAACTGGGATTCCTTCAAGATTCAGTGGCAGTGGATCT GGAAAGGATTACACTCTCACCATTACCAGTCTTCAGACTGAAGATGTTGC TACTTATTACTGTCAACAGTATTGGAGTAGTCCTCCGACGTTCGGTGGAGG CACCAAGCTGGAAATCAAA

[0166] 81A7A10-VH Amino acid sequence (SEQ ID NO:86): QVQLKESGPGLVAPLQSLSITCTVSGFSLSSYGVSWVRQLPGKGLEWLGVIW GDGSTDYHSTLTSRLSFSKDNSESQVFLKLNSLQTDDTATYYCAIFYGYDEGF AYWGQGTLVTVSA Nucleic acid sequence (SEQ ID NO:87): CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTTACAGAG CCTGTCCATCACATGCACTGTCTCAGGGTTCTCATTAAGCAGTTATGGTGT AAGCTGGGTTCGCCAGCTTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAA TATGGGGTGACGGGAGCACAGATTATCATTCAACTCTCACATCCAGACTG AGCTTCAGCAAGGATAATTCCGAGAGCCAAGTTTTCTTAAAACTGAATAG TCTGCAAACTGATGACACAGCCACATACTACTGTGCCATTTTCTATGGTTA CGACGAAGGGTTTGCTTATTGGGGCCAAGGGACTCTGGTCACTGTCTCTG CA

[0167] 81A7A10-VL Amino acid sequence (SEQ ID NO:88): ENVLTQSPAIMSAFLGEKVTMSCRASSTVNYMYWYQQKSDASPKVWIYYTS RLAPGVPARFSGSGSGNSFSLTISSMEGEDAATYYCQQFTSSPLTFGAGTKLEL R Nucleic acid sequence (SEQ ID NO:89): GAAAATGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATTTCTAGGGGA GAAGGTCACCATGAGCTGCAGGGCCAGCTCAACTGTAAATTACATGTACT GGTACCAGCAGAAGTCAGATGCCTCCCCCAAAGTTTGGATTTATTACACA TCCAGGTTGGCTCCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGG GAACTCTTTTTTCTCTCACAATCAGCAGCATGGAGGGTGAAGATGCTGCCA CTTATTACTGCCAGCAGGTTTACTAGTTCCCCGCTCACGTTCGGTGCTGGGA CCAAGCTGGAGCTGAGA

[0168] 107D10D11-VH Amino acid sequence (SEQ ID NO: 101): QIQLKQSGPGLVQPSQSLSITCTVSGFSLSSYGVNWVRQSPGKGLEWLGMIWS GGNTDYNAAFISRVSFTKDNSKSQVFFTMNSLQADDTAIYYCARIWYYGMD YWGQGTSVTVSS Nucleic acid sequence (SEQ ID NO: 102): CAGATACAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAG TCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAAGTAGTTATGGTGT AAATTGGGTTCGCCAGGTCTCCAGGAAAGGGTCTGGAATGGCTGGGAATGA TATGGAGTGGTGGAAACACAGACTATAATGCAGCTTTCATATCCAGAGTG AGCTTCACCAAGGACAATTCCAAGAGCCAAGTTTTCTTTACAATGAACAG TCTGCAAGCTGATGACACAGCCATATATTACTGTGCCAGAATATGGTACT ATGGTATGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA

[0169] 107D10D11-VL Amino acid sequence (SEQ ID NO: 103): DIVMTQSQKFMSTTVGDRVSITCKASQNVGTAVAWFQQKPGQSPKILIYSASI RYPGVPDRFTGSGSGTDFTLTTSNVQSEDLADYFCLQYITFPRTFGGGTKLYIK Nucleic acid sequence (SEQ ID NO: 110): GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACAACAGTAGGAGA CAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTGTAG CCTGGTTTCAACAGAAACCAGGACAATCTCCTAAAATACTGATTTATTCA GCATCCATTCGCTACCCTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCT GGGACAGATTTCACTCTCACCACTAGCAATGTGCAGTCTGAAGACCTGGC AGATTATTTCTGTCTGCAATATATCACCTTTCCTCGGACGTTCGGTGGAGGG CACCAAGCTGTATATCAAA

[0170] 101H9G9A2-VH Amino acid sequence (SEQ ID NO: 109): QVQLLQPGAELVRPGSSVKLSCKASDYTFTSYWMDWVKQRPGQGLEWIGNI YPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARRGGY YAYAMDYWGQGTSVTVSS Nucleic acid sequence (SEQ ID NO:204): CAGGTGCAGCTGCTGCAGCCCGGCGCTGAGCTGGTGAGACCCGGAAGCA GCGTGAAGCTGAGCTGCAAAGCCAGCGACTACACATTCACAAGCTACTGG ATGGACTGGGTGAAACAGAGACCTGGCCAGGGACTGGAATGGATTGGAA ACATCTACCCCAGTGACAGCAAGACCCACTACAACCAGAAGTTCAAGGAC AAAGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGCAGCT GAACAGCCTGACCAGCGAAGACAGCGCCGTGTACTACTGCGCCAGAAGA GGCGGCTACTACGCCTATGCCATGGACTACTGGGGGCAGGGAACCAGCGT GACCGTGAGCAGC

[0171] 101H9G9A2-VL Amino acid sequence (SEQ ID NO: 111): DIVMTQSPSSLSVSAGEKVTMRCKSSQSLLNSGNQDNYLAWYQQKPGQPPKL LLYGARIRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPYTFG GGTKVEIK Nucleic acid sequence (SEQ ID NO: 205): GACATTGTGATGACCCAGAGCCCCAGCAGCCTGAGCGTGAGCGCCGGAG AAAAAGTGACCATGAGATGCAAGAGCAGCCAGAGCCTGCTGAACAGCGG GAACCAGGACAACTACCTGGCCTGGTATCAACAGAAGCCCGGCCAGCCCC CCAAACTGCTGCTGTATGGAGCCAGAATCAGAGAGAGCGGCGTGCCCGAC AGATTCATCGGCAGCGGCAGCGGAACCGACTTCACCCTGACCATCAGCAG CGTGCAGGCTGAGGACCTGGCCGTGTACTACTGCCAGAACGACCACAGCT ACCCCTATACCTTTGGAGGCGGCACCAAGGTGGAGATCAAA

[0172] 173B1C1-VH Amino acid sequence (SEQ ID NO: 118): QVQLQQSGAELARPGASVKMSCKASGYTFTSNTMHWVKQRPGQGLEWIGYI NPGSAYTNYNQKFNDKATLTADKSSSTASMQLSSLTSEDSAVYYCARLATYY DNDGYAMDYWGQGTSVTVSS Nucleic acid sequence (SEQ ID NO:206): CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGTGCCTC AGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCAACACGA TGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATAC ATTAATCCTGGCAGTGCTTATACTAACTACAATCAGAAGTTCAATGACAA GGCCACATTGACTGCAGACAAGTCCTCCAGCACAGCCTCCATGCAACTGA GCAGCCTGACATCTGAGGACTCTGCAGCTCTATTACTGTGCAAGACTCGCG ACCTACTATGATAACGACGGATATGCTATGGACTATTGGGGTCAAGGAAC CTCAGTCACCGTCTCCTCCA

[0173] 173B1C1-VL(1) Amino acid sequence (SEQ ID NO: 120): DIQMTQSTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSK LHSGVPSRFSGSGSGTDFSLTITNLEQEDFATYFCQQGHTLPWTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO:207): GACATCCAGATGACACAGTCTACATCCTCCCTGTCTGCCTCTCTGGGAGAC AGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAATTATTTAAA CTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACA CATCAAAATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCT GGAACAGATTTTTCTCTCACCATTACCAACCTGGAGCAAGAAGATTTTGCC ACTTACTTTTGCCAACAGGGTCATACGCTTCCGTGGACGTTCGGTGGAGGC ACCAAACTGGAGATCAAA

[0174] 173B1C1-VL(2) Amino acid sequence (SEQ ID NO: 221): DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSK LHSGVPSRFSGSGSGTDFSLTITNLEQEDFATYFCQQGHTLPWTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO:208): GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGAC AGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAATTATTTAAA CTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACA CATCAAAATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCT GGAACAGATTTTTCTCTCACCATTACCAACCTGGAGCAAGAAGATTTTGCC ACTTACTTTTGCCAACAGGGTCATACGCTTCCGTGGACGTTCGGTGGAGGC ACCAAACTGGAGATCAAA

[0175] 107D10E11-VH Amino acid sequence (SEQ ID NO: 122): EVQLQQSGTGLVKPGTSVKLSCKASGYSFTSYWITWVKQRPGQGLEWIGDIS PAGGGRNYNERFKNKATLTVDASSSTAYMQLSSLTSEDSAVYYCVRGDSTV DLDYWGQGTTLTVSS Nucleic acid sequence (SEQ ID NO:228): GAGGTTCAGCTGCAGCAGTCTGGGACTGGACTTGTGAAGCCTGGGACTTC AGTGAAGTTGTCCTGCAAGGCTTCTGGCTACAGCTTCACCAGCTACTGGAT AACCTGGGTGAAACAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAT ATTTCTCCTGCTGGTGGTGGTCGTAACTACAATGAGAGATTCAAGAACAA GGCCACACTGACTGTAGACGCATCCTCCAGCACAGCCTACATGCAGCTCA GCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGTAAGAGGTGAT AGTACGGTAGACTTAGACTACTGGGGCCAAGGCACCACTCTCACAGTCTC CTCA

[0176] 107D10E11-VL Amino acid sequence (SEQ ID NO: 124): DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIY RVSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGG TKLEIK Nucleic acid sequence (SEQ ID NO: 229): GATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAG TCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGC AACACTTACTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTC CTGATATATCGGGTGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAG TGGCAGTGGGTCAGGGACTGCTTTCACACTGAGAATCAGTAGAGTGGAGG CTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCGTACA CGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA

[0177] 109G10A6-VH Amino acid sequence (SEQ ID NO: 132): EVLLQQSGPELVKPGASVKIPCKASGYGFTDYNMDWIKQSHGKSLEWIGDIN PHNGDTIYNQKFKGKAKLTVDKSSTTAYMELRSLASEDTAVYYCARRGAYY YGSSHYALDFWGQGTSVTVSS Nucleic acid sequence (SEQ ID NO:209): GAGGTCCTGCTGCAACAGTCTGGACCTGAACTGGTGAAGCCTGGGGCTTC AGTGAAGATACCCTGCAAGGCTTCTGGATACGGATTCACTGACTACAACA TGGACTGGATAAAACAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGA TATTAATCCTCACAATGGTGATACTATCTACAACCAGAAGTTCAAGGGCA AGGCCAAATTGACTGTAGACAAGTCCTCCACCACTGCCTACATGGAACTC CGCAGCCTGGCATCTGAGGACACCGCAGTCTATTACTGTGCAAGGAGGGG GGCATATTACTACGGTAGTAGCCACTATGCTCTGGACTTCTGGGGTCAAG GAACCTCAGTCACCGTCTCCTCA

[0178] 109G10A6-VL Amino acid sequence (SEQ ID NO: 134): DIQMTQTTSSLSASLGDRVTISCRASQDITNYLNWYQQKPDGTVKLLIYYTSR LFSGVPSRFSGSGSGTDYSLTISNLKREDIATYFCQQGNTLPWTFGGGTKLEIK Nucleic acid sequence (SEQ ID NO: 210): GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGAC AGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTACCAATTATTTAAA CTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACA CATCAAGATTATTCTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCT GGAACAGATTATTCTCTCACCATTAGCAACCTGAAACGAGAAGATATTGC CACTTACTTTTGCCAACAGGGTAATACGCTTCCGTGGACGTTCGGTGGAG GCACCAAGCTGGAAATCAAA

[0179] 94G12D11-VH Amino acid sequence (SEQ ID NO: 141): EVQLQQSGPELVKPGASVKISKASGYLFTGYYMNWVKQSPEKSLEWIGEIN PNKGDSNLNQNFKAKATLTVDKSSSTAYMQFKSLTFEDSAVYFCVRRDYGTS LDHWGQGTTLTVSS Nucleic acid sequence (SEQ ID NO:211): GAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC AGTGAAGATATCCTGCAAGGCCTCTGGTTACTTATTCACTGGCTACTACAT GAACTGGGTGAAACAAAGTCCTGAAAAGAGTCTTGAGTGGATTGGAGAG ATTAATCCTAACAAAGGTGATAGTAATCTCAACCAGAATTTCAAGGCCAA GGCCACATTGACTGTTGACAAATCCTCCAGCACAGCCTACATGCAGTTCA AGAGCCTGACATTTGAGGACTCTGCAGCTCTATTTCTGTGTAAGAAGGGAC TACGGCACTAGTCTTGACCACTGGGGCCAAGGCACGACTCTCACAGTCTC CTCA

[0180] 94G12D11-VL Amino acid sequence (SEQ ID NO: 143): DVQITQSPSYLATSPGETITINCRASKSISKYLAWYQEKPGKTKKLLIYSGSTL HTAIPSRFSGRGFGTDFTLTISSLEPEDFAMYYCQQHNEYPITFGGGTKLELK Nucleic acid sequence (SEQ ID NO:212): GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTACATCTCCTGGAGAA ACCATTACTATTAATTGCAGGGCAAGTAAGAGTATTAGCAAATATTTAGC CTGGTATCAAGAGAAACCTGGGAAAACTAAGAAGCTTCTTATCTACTCTG GATCCACTTTGCATACTGCAATTCCATCAAGGTTCAGTGGCCGTGGATTTG GTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAA TGTATTACTGTCAACAGCATAATGAATACCCAATCACATTCGGTGGTGGG ACCAAGCTGGAGCTGAAA

[0181] 92F5B8-VH Amino acid sequence (SEQ ID NO: 146): EVQLQQSGPELVKPGASVKISKATGYLFTGYYMNWVKQSPEKSLEWIGEIN PNTGDTNNNQKFKAKATMTVDRSSSTAYMQLKSLTSEDSAVYYCVRRDYGT SLDYWGQGTTLTVSS Nucleic acid sequence (SEQ ID NO:213): GAGGTCCAGCTGCAGCAGTCTGGACCTGAGTTGGTGAAGCCTGGGGCTTC AGTGAAGATATCCTGCAAGGCTACTGGTTACTTATTCACTGGCTACTACAT GAACTGGGTGAAGCAAAGTCCTGAAAAGAGCCTTGAGTGGATTGGAGAG ATCAATCCTACACTGGTGATACTAACAACAACCAGAAGTTCAAGGCCAA GGCCACAATGACTGTTGACAGATCCTCCAGTACAGCCTACATGCAGCTCA AGAGCCTGACATCTGAGGACTCTGCAGCTCTATTATTGTGTAAGAAGGGAC TACGGTACTAGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTC CTCA

[0182] 92F5B8-VL Amino acid sequence (SEQ ID NO: 148): DVQITQSPSYLAASPGETITINCRASKSINKYLAWYQEKPGKTNKLLIHSGSTL HSGIPSRFSGSRSGTDFTLTISNLEPEDFAMYYCQQHNEYPLTFGAGTKLELK Nucleic acid sequence (SEQ ID NO:214): GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAA ACCATTACTATTAATTGCAGGGCAAGTAAGAGTATTAACAAATATTTAGC CTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCCACTCTG GATCCACTTTGCACTCTGGAATTCCATCAAGGTTCAGTGGCAGTAGATCTG GAACAGATTTCACTCTCACCATCAGTAACCTGGAGCCTGAAGATTTTGCA ATGTATTACTGTCAACAACATAATGAATACCCGTCACGTTCGGTGCTGG GACCAAGCTGGAGCTGAAA

[0183] 185B11E3-VH Amino acid sequence (SEQ ID NO: 156): QVQLQQSGAELARPGASVKMSCKASGYTFTSNTMHWVKQRPGQGLEWIGYI NPGSAYTNYNQKFNDKATLTADKSSSTASMQLSSLTSEDSAVYYCARLATYY DNDGYAMDYWGQGTSVTVSS Nucleic acid sequence (SEQ ID NO:215): CAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGCTAGACCCGGAGCTTC CGTGAAGATGAGCTGCAAAGCCAGCGGCTACACATTCACATCCAACACAA TGCACTGGGTGAAACAGAGGCCTGGCCAGGGACTGGAGTGGATTGGGTA CATCAACCCCGGCAGCGCCTACACCAACTACAACCAGAAATTCAATGACA AAGCCACACTGACCGCCGACAAAAGCAGCAGCACAGCCAGCATGCAGCT GAGCAGCCTGACCTCCGAGGACAGCGCTGTGTACTACTGCGCCAGACTGG CCACCTACTACGACAACGACGGATACGCCATGGATTACTGGGGACAGGGA ACCAGCGTGACCGTGAGCAGC

[0184] 185B11E3-VL Amino acid sequence (SEQ ID NO: 158): ENVLTQSPAIMSASPGEKVTMTCSASSSVNYMYWYQQRSSTSPKLWIYGTSK LASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPYTFGGGTKLEI K Nucleic acid sequence (SEQ ID NO: 216): GAGAATGTGCTGACCCAGAGCCCCGCCATCATGAGTGCCAGCCCAGGAGA GAAGGTGACAATGACATGCAGCGCCAGCAGCAGCGTGAATTACATGTACT GGTACCAGCAGAGAAGCAGCACCAGCCCCAAGCTGTGGATCTACGGCAC CAGCAAGCTGGCCAGCGGAGTGCCCGGCAGATTCTCCGGAAGTGGAAGC GGAAATAGCTACAGCCTGACTATCAGCAGCATGGAGGCCGAAGATGTTGC TACCTACTACTGCTTTCAGGGCAGCGGCTACCCCTACACCTTCGGCGGCGG CACCAAGCTGGAGATCAAG

[0185] 168B11E2-VH Amino acid sequence (SEQ ID NO: 164): QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPPGKGLEWLGVIW GDGNTNYHSALISRLSISKDNSKSQVFLKLNSLQTDDTATYYCANLYVLFAY WGQGTLVTVSA Nucleic acid sequence (SEQ ID NO:217): CAGGTGCAGCTGAAGGAAAGCGGCCCCGGCCTGGTGGCCCCATCTCAGAG TCTGAGCATCACCTGCACAGTGAGCGGCTTCAGCCTGACAAGCTACGGCG TGAGCTGGGTGAGACAGCCCCCCGGAAAGGGCTGGAATGGCTGGGCGT GATTTGGGGCGACGGCAACACAAACTACCACAGCGCCCTGATCAGCAGAC TGAGCATCAGCAAGGACAATAGCAAGAGCCAGGTGTTCCTGAAGCTGAA CAGCCTGCAGACCGACGACACCGCCACCTACTACTGCGCCAACCTGTACG TGCTGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCT

[0186] 168B11E2-VL Amino acid sequence (SEQ ID NO: 166): ENVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSSTSPKLWIYGTSK LASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQGSGYPYTFGGGTKLEI K Nucleic acid sequence (SEQ ID NO:218): GAGAATGTGCTGACCCAGAGCCCCGCCATCATGAGTGCCAGCCCAGGAGA GAAGGTGACAATGACATGCAGCGCCAGCAGCAGCGTGAGTTACATGCACT GGTACCAGCAGAAGAGCAGCACCAGCCCCAAACTGTGGATCTACGGGAC CAGCAAGCTGGCCAGCGGAGTGCCCGGAAGATTCAGTGGCAGCGGAAGC GGCAATAGCTACAGCCTGACTATCAGCAGCATGGAGGCCGAAGATGTTGC TACCTACTACTGTTTTCAGGGAAGCGGCTACCCATACACCTTCGGGGGGG GGACCAAGCTGGAAATCAAG

[0187] 188H6D3-VH Amino acid sequence (SEQ ID NO: 174): QVQLQQPGTELVKPGASVKLSCKASGYTFTNYWIHWVKQRPGQGLEWIGDI NPTNGGTNYNEKFKTKAILTVDRSSSTAYMQVSSLTSEDSAVYYCARSGGYD FDYWGQGTTLTVSS Nucleic acid sequence (SEQ ID NO:219): CAGGTGCAGCTGCAGCAGCCAGGCACAGAGCTGGTGAAGCCCGGCGCCA GCGTGAAACTGAGCTGCAAAGCCAGCGGCTACACCTTCACAAACTACTGG ATTCATTGGGTGAAGCAGAGGCCTGGCCAGGGACTGGAGTGGATCGGCG ACATTAACCCCACAAACGGAGGCACAAACTACAACGAGAAATTCAAGAC CAAAGCCATCCTGACCGTGGACAGAAGCAGTAGCACAGCCTACATGCAG GTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTATTGCGCCAGAAG CGGCGGCTACGACTTCGACTACTGGGGCCAGGGCACCACCCTGACCGTGA GCAGC

[0188] 188H6D3-VL Amino acid sequence (SEQ ID NO: 176): DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLI YLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTYFPLTFGA GTKLELK Nucleic acid sequence (SEQ ID NO: 220): GACGTGGTGATGACCCAGACCCCCCTGACACTGAGCGTGACCATCGGACA GCCCGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGGACAGCGAC GGAAAAACATATCTGAATTGGCTGCTGCAGAGGCCTGGCCAGAGCCCCAA AAGACTGATCTACCTGGTGAGCAAGCTGGACAGCGGGGTGCCCGACAGAT TCACCGGCAGCGGAAGCGGCACCGACTTCACCCTGAAGATCTCCAGAGTG GAAGCCGAGGACCTGGGCGTGTACTACTGCTGGCAGGGAACCTACTTCCC CCTGACCTTTGGAGCCGGCACCAAGCTGGAACTGAAG

[0189] Although the CDRs are known to be involved in antigen binding, it is understood that not all six CDRs are essential or invariant. In other words, it is possible to replace, alter, or modify one or more CDRs in the anti-CD24 antibodies 33E7E12, 71A4F12, 56A7E3, 58F10-1F6, 73H5-1G2, 110D4G4, 81A1F8, 100F2E3, 111F3A2, 81A7A10, 107D10D11, 101H9G9A2, 173B1C1, 107D10E11, 109G10A6, 94G12D11, 92F5B8, 185B11E3, 168B11E2, or 188H6D3 while substantially retaining specific binding affinity for CD24.

[0190] In certain embodiments, the anti-CD24 antibodies and antigen-binding fragments provided herein comprise the heavy chain CDR3 sequence of one of the anti-CD24 antibodies 33E7E12, 71A4F12, 56A7E3, 58F10-1F6, 73H5-1G2, 110D4G4, 81A1F8, 100F2E3, 111F3A2, 81A7A10, 107D10D11, 101H9G9A2, 173B1C1, 107D10E11, 109G10A6, 94G12D11, 92F5B8, 185B11E3, 168B11E2, or 188H6D3. In certain embodiments, the anti-CD24 antibodies and antigen-binding fragments provided herein comprise a heavy chain CDR3 sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 14, 16, 17, 18, 19, 21, 23, 25, 27, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 97, 104, 105, 106, 113, 114, 115, 126, 127, 128, 136, 137, 150, 151, 152, 160, 161, 162, 168, 169, 170, 222, 223, and 224. The heavy chain CDR3 region is centrally located in the antigen-binding site and is therefore thought to make the most contacts with the antigen and provide the most free energy for the affinity of the antibody for the antigen. Furthermore, heavy chain CDR3 is considered to be by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition, and conformation through multiple diversification mechanisms (Tonegawa S. Nature. 302:575-81). The diversity of heavy chain CDR3 is sufficient to confer most antibody specificities (Xu JL, Davis MM. Immunity. 13:37-45) and desirable antigen-binding affinities (Schier R, et al. J Mol Biol. 263:551-67).

[0191] In certain embodiments, the antibodies and / or antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and / or antigen-binding fragments thereof are capable of specifically binding to CD24. The CDR sequences provided in Table 1 are derived from mouse antibodies, but they can be grafted into any suitable FR sequences of any suitable species, e.g., mouse, human, rat, rabbit, among others, using suitable methods known in the art, e.g., recombinant techniques.

[0192] In certain embodiments, the antibodies and / or antigen-binding fragments thereof provided herein are humanized. Humanized antibodies or antigen-binding fragments are desirable for their reduced immunogenicity in humans. Humanized antibodies are chimeric in their variable regions because non-human CDR sequences are grafted onto human or substantially human FR sequences. Humanization of antibodies or antigen-binding fragments can essentially be achieved by substituting non-human (such as mouse) CDR genes with the corresponding human CDR genes in human immunoglobulin genes (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0193] Methods known in the art can be used to select suitable human heavy and light chain variable domains to achieve this goal. In an illustrative example, a "best-fit" approach can be used, in which a non-human (e.g., rodent) antibody variable domain sequence is screened or BLASTed against a database of known human variable domain sequences to identify the closest human sequence to the non-human query sequence and use it as a human scaffold onto which the non-human CDR sequences are grafted (see, e.g., Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, frameworks derived from the consensus sequences of all human antibodies may be used for grafting non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623). In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein are composed of substantially all human sequences, except for the non-human CDR sequences. In some embodiments, the variable region FRs and, if present, the constant region are derived entirely or substantially from human immunoglobulin sequences. The human FR sequences and human constant region sequences, e.g., the FR sequences from one human antibody and the constant region from another human antibody, may be derived from different human immunoglobulin genes.

[0194] The present disclosure also provides humanized anti-CD24 antibodies 81A1F8-VH11 / VL11, 81A1F8-VH21 / VL11, 81A1F8-VH31 / VL11, 81A1F8-VH11 / VL21, 81A1F8-VH21 / VL21, 81A1F8-VH31 / VL21, 81A1F8-VH11 / VL31, 81A1F8-VH21 / VL31, 81A1F8-VH31 / VL31, 81A1F8-VH11 / VL41, 81A1F8-VH21 / VL41 or 81A1F8-VH31 / VL41; or 101H9G9A2-mVH / mVL-V1, 101H9G9A2-mVH / mVL-V2, 101H9G9A2-mVH / mVL-V3, 101H9G9A 2-mVH / mVL-V4, 101H9G9A2-mVH / mVL-V5, 101H9G9A2-hVH1 / hVL1, 101H9G9A2-hVH1 / hVL2, 101H9G9A2-hVH1 / hVL3, 101H9 G9A2-hVH1 / hVL4, 101H9G9A2-hVH2 / hVL1, 101H9G9A2-hVH2 / hVL2, 101H9G9A2-hVH2 / hVL3, 101H9G9A2-hVH2 / hVL4, 101H 9G9A2-hVH3 / hVL1, 101H9G9A2-hVH3 / hVL2, 101H9G9A2-hVH3 / hVL3, 101H9G9A2-hVH3 / hVL4, 101H9G9A2-hVH4 / hVL1, 101 Anti-CD24 antibodies and antigen-binding fragments thereof are provided, which comprise one or more (e.g., 1, 2, 3, 4, 5, or 6) CDR sequences of H9G9A2-hVH4 / hVL2, 101H9G9A2-hVH4 / hVL3, 101H9G9A2-hVH4 / hVL4, 101H9G9A2-hVH5 / hVL1, 101H9G9A2-hVH5 / hVL2, 101H9G9A2-hVH5 / hVL3, or 101H9G9A2-hVH5 / hVL4.

[0195] "81A1F8-VH21 / VL11" as used herein refers to a humanized antibody based on 81A1F8 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO:177 and the light chain variable region of SEQ ID NO:180.

[0196] "81A1F8-VH21 / VL11" as used herein refers to a humanized antibody based on 81A1F8 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO:178 and the light chain variable region of SEQ ID NO:180.

[0197] "81A1F8-VH31 / VL11" as used herein refers to a humanized antibody based on 81A1F8 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO:179 and the light chain variable region of SEQ ID NO:180.

[0198] "81A1F8-VH11 / VL21" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:177 and the light chain variable region of SEQ ID NO:181.

[0199] "81A1F8-VH21 / VL21" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:178 and the light chain variable region of SEQ ID NO:181.

[0200] "81A1F8-VH31 / VL21" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:179 and the light chain variable region of SEQ ID NO:181.

[0201] "81A1F8-VH11 / VL31" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:177 and the light chain variable region of SEQ ID NO:182.

[0202] "81A1F8-VH21 / VL31" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:178 and the light chain variable region of SEQ ID NO:182.

[0203] "81A1F8-VH31 / VL31" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:179 and the light chain variable region of SEQ ID NO:182.

[0204] "81A1F8-VH11 / VL41" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:177 and the light chain variable region of SEQ ID NO:183.

[0205] "81A1F8-VH21 / VL41" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:178 and the light chain variable region of SEQ ID NO:183.

[0206] "81A1F8-VH31 / VL41" as used herein refers to the 81A1F8 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:179 and the light chain variable region of SEQ ID NO:183.

[0207] "101H9G9A2-mVH / mVL-V1" as used herein refers to a 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:189 and the light chain variable region of SEQ ID NO:195.

[0208] "101H9G9A2-mVH / mVL-V2" as used herein refers to a humanized antibody based on 101H9G9A2 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO:189 and the light chain variable region of SEQ ID NO:196.

[0209] "101H9G9A2-mVH / mVL-V3" as used herein refers to a humanized antibody based on 101H9G9A2 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO:189 and the light chain variable region of SEQ ID NO:197.

[0210] "101H9G9A2-mVH / mVL-V4" as used herein refers to a humanized antibody based on 101H9G9A2 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO:189 and the light chain variable region of SEQ ID NO:198.

[0211] "101H9G9A2-mVH / mVL-V5" as used herein refers to a humanized antibody based on 101H9G9A2 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO:189 and the light chain variable region of SEQ ID NO:199.

[0212] "101H9G9A2-hVH1 / hVL1" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:190 and the light chain variable region of SEQ ID NO:200.

[0213] "101H9G9A2-hVH1 / hVL2" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:190 and the light chain variable region of SEQ ID NO:201.

[0214] "101H9G9A2-hVH1 / hVL3" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:190 and the light chain variable region of SEQ ID NO:202.

[0215] "101H9G9A2-hVH1 / hVL4" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO: 190 and the light chain variable region of SEQ ID NO: 203.

[0216] "101H9G9A2-hVH2 / hVL1" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:191 and the light chain variable region of SEQ ID NO:200.

[0217] "101H9G9A2-hVH2 / hVL2" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:191 and the light chain variable region of SEQ ID NO:201.

[0218] "101H9G9A2-hVH2 / hVL3" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:191 and the light chain variable region of SEQ ID NO:202.

[0219] "101H9G9A2-hVH2 / hVL4" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO: 191 and the light chain variable region of SEQ ID NO:203.

[0220] "101H9G9A2-hVH3 / hVL1" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:192 and the light chain variable region of SEQ ID NO:200.

[0221] "101H9G9A2-hVH3 / hVL2" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO: 192 and the light chain variable region of SEQ ID NO:201.

[0222] "101H9G9A2-hVH3 / hVL3" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO: 192 and the light chain variable region of SEQ ID NO:202.

[0223] "101H9G9A2-hVH3 / hVL4" as used herein refers to a humanized antibody based on 101H9G9A2 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO: 192 and the light chain variable region of SEQ ID NO: 203.

[0224] "101H9G9A2-hVH4 / hVL1" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:193 and the light chain variable region of SEQ ID NO:200.

[0225] "101H9G9A2-hVH4 / hVL2" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO: 193 and the light chain variable region of SEQ ID NO:201.

[0226] "101H9G9A2-hVH4 / hVL3" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:193 and the light chain variable region of SEQ ID NO:202.

[0227] "101H9G9A2-hVH4 / hVL4" as used herein refers to a humanized antibody based on 101H9G9A2 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO: 193 and the light chain variable region of SEQ ID NO:203.

[0228] "101H9G9A2-hVH5 / hVL1" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:194 and the light chain variable region of SEQ ID NO:200.

[0229] "101H9G9A2-hVH5 / hVL2" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO: 194 and the light chain variable region of SEQ ID NO:201.

[0230] "101H9G9A2-hVH5 / hVL3" as used herein refers to the 101H9G9A2 murine / chimeric-based humanized antibody comprising the heavy chain variable region of SEQ ID NO:194 and the light chain variable region of SEQ ID NO:202.

[0231] "101H9G9A2-hVH5 / hVL4" as used herein refers to a humanized antibody based on 101H9G9A2 murine / chimeric, comprising the heavy chain variable region of SEQ ID NO: 194 and the light chain variable region of SEQ ID NO: 203.

[0232] Table 2 shows the CDR sequences of these 37 humanized anti-CD24 antibodies according to IMGT numbering. The heavy and light chain variable region sequences are also provided below.

[0233] [Table 2(1)-1] [Table 2(1)-2]

[0234] The heavy or light chain variable region sequences of these humanized antibodies are provided below.

[0235] 81A1F8-VH11 / VL11 VH amino acid sequence (SEQ ID NO: 177): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSISTAYMELSRLRSDDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 180): DVQITQSPSSLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0236] 81A1F8-VH21 / VL11 VH amino acid sequence (SEQ ID NO: 178): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSTSTAYMELSSLRSEDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 180): DVQITQSPSSLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0237] 81A1F8-VH31 / VL11 VH amino acid sequence (SEQ ID NO: 179): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQRLEWMG EINPNTGDTNNNQKFKARVTITVDRSASTAYMELSSLRSEDTAVYYCVRRDY GTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 180): DVQITQSPSSLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0238] 81A1F8-VH11 / VL21 VH amino acid sequence (SEQ ID NO: 177): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSISTAYMELSRLRSDDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 181): DVQITQSPSFLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTEFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0239] 81A1F8-VH21 / VL21 VH amino acid sequence (SEQ ID NO: 178): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSTSTAYMELSSLRSEDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 181): DVQITQSPSFLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTEFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0240] 81A1F8-VH31 / VL21 VH amino acid sequence (SEQ ID NO: 179): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQRLEWMG EINPNTGDTNNNQKFKARVTITVDRSASTAYMELSSLRSEDTAVYYCVRRDY GTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 181): DVQITQSPSFLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTEFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0241] 81A1F8-VH11 / VL31 VH amino acid sequence (SEQ ID NO: 177): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSISTAYMELSRLRSDDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 182): DVQITQSPSSLSASVGDRVTITCRASKSINKYLAWYQQKPGKVPKLLIHSGSTL QSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0242] 81A1F8-VH21 / VL31 VH amino acid sequence (SEQ ID NO: 178): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSTSTAYMELSSLRSEDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 182): DVQITQSPSSLSASVGDRVTITCRASKSINKYLAWYQQKPGKVPKLLIHSGSTL QSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0243] 81A1F8-VH31 / VL31 VH amino acid sequence (SEQ ID NO: 179): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQRLEWMG EINPNTGDTNNNQKFKARVTITVDRSASTAYMELSSLRSEDTAVYYCVRRDY GTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 182): DVQITQSPSSLSASVGDRVTITCRASKSINKYLAWYQQKPGKVPKLLIHSGSTL QSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHNEYPITFGQGTKLEIK

[0244] 81A1F8-VH11 / VL41 VH amino acid sequence (SEQ ID NO: 177): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSISTAYMELSRLRSDDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 183): DVQITQSPSTLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTEFLTISSLQPDDFATYYCQQHNEYPITFGQGTKLEIK

[0245] 81A1F8-VH21 / VL41 VH amino acid sequence (SEQ ID NO: 178): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQGLEWMG EINPNTGDTNNNQKFKARVTMTVDRSTSTAYMELSSLRSEDTAVYYCVRRD YGTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 183): DVQITQSPSTLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTEFLTISSLQPDDFATYYCQQHNEYPITFGQGTKLEIK

[0246] 81A1F8-VH31 / VL41 VH amino acid sequence (SEQ ID NO: 179): QVQLVQSGAEVKKPGASVKVSCKASGYLFTGYYMNWVRQAPGQRLEWMG EINPNTGDTNNNQKFKARVTITVDRSASTAYMELSSLRSEDTAVYYCVRRDY GTSLDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 183): DVQITQSPSTLSASVGDRVTITCRASKSINKYLAWYQQKPGKAPKLLIHSGSTL QSGVPSRFSGSRSGTEFLTISSLQPDDFATYYCQQHNEYPITFGQGTKLEIK

[0247] 101H9G9A2-mVH / mVL-V1 VH amino acid sequence (SEQ ID NO: 189): QVQLLQPGAELVRPGSSVKLSCKASDYTFTSYWMDWVKQRPGQGLEWIGNI YPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARRGGY YAYAMDYWGQGTSVTVSS VL amino acid sequence (SEQ ID NO: 195): DIVMTQSPSSLSVSAGEKVTMRCKSSQSLLQSGNQDNYLAWYQQKPGQPPKL LLYGARIRESGVPDRFIGSGSGTFTLTISSVQAEDLAVYYCQNDHSYPYTFG GGTKVEIK

[0248] 101H9G9A2-mVH / mVL-V2 VH amino acid sequence (SEQ ID NO: 189): QVQLLQPGAELVRPGSSVKLSCKASDYTFTSYWMDWVKQRPGQGLEWIGNI YPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARRGGY YAYAMDYWGQGTSVTVSS VL amino acid sequence (SEQ ID NO: 196): DIVMTQSPSSLSVSAGEKVTMRCKSSQSLLNAGNQDNYLAWYQQKPGQPPK LLLYGARIRESGVPDRFIGSGSGTFTLTISSVQAEDLAVYYCQNDHSYPYTFG GGTKVEIK

[0249] 101H9G9A2-mVH / mVL-V3 VH amino acid sequence (SEQ ID NO: 189): QVQLLQPGAELVRPGSSVKLSCKASDYTFTSYWMDWVKQRPGQGLEWIGNI YPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARRGGY YAYAMDYWGQGTSVTVSS VL amino acid sequence (SEQ ID NO: 197): DIVMTQSPSSLSVSAGEKVTMRCKSSQSLLSSGNQDNYLAWYQQKPGQPPKL LLYGARIRESGVPDRFIGSGSGTFTLTISSVQAEDLAVYYCQNDHSYPYTFG GGTKVEIK

[0250] 101H9G9A2-mVH / mVL-V4 VH amino acid sequence (SEQ ID NO: 189): QVQLLQPGAELVRPGSSVKLSCKASDYTFTSYWMDWVKQRPGQGLEWIGNI YPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARRGGY YAYAMDYWGQGTSVTVSS VL amino acid sequence (SEQ ID NO: 198): DIVMTQSPSSLSVSAGEKVTMRCKSSQSLLYSGNQDNYLAWYQQKPGQPPKL LLYGARIRESGVPDRFIGSGSGTFTLTISSVQAEDLAVYYCQNDHSYPYTFG GGTKVEIK

[0251] 101H9G9A2-mVH / mVL-V5 VH amino acid sequence (SEQ ID NO: 189): QVQLLQPGAELVRPGSSVKLSCKASDYTFTSYWMDWVKQRPGQGLEWIGNI YPSDSKTHYNQKFKDKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARRGGY YAYAMDYWGQGTSVTVSS VL amino acid sequence (SEQ ID NO: 199): DIVMTQSPSSLSVSAGEKVTMRCKSSQSLLASGNQDNYLAWYQQKPGQPPKL LLYGARIRESGVPDRFIGSGSGTFTLTISSVQAEDLAVYYCQNDHSYPYTFG GGTKVEIK

[0252] 101H9G9A2-hVH1 / hVL1 VH amino acid sequence (SEQ ID NO: 190): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 200): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQDNYLAWYQQKPGQPPKL LIYGARIRESGVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQNDHSYPYTFGG GTKVEIK

[0253] 101H9G9A2-hVH1 / hVL2 VH amino acid sequence (SEQ ID NO: 190): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 201): DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGNQDNYLAWYLQKPGQSPQLLI YGARIRESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQNDHSYPYTFGGG TKVEIK

[0254] 101H9G9A2-hVH1 / hVL3 VH amino acid sequence (SEQ ID NO: 190): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 202): EIVMTQSPATLSVSPGERATLSCKSSQSLLNSGNQDNYLAWYQQKPGQAPRL LIYGARIRESGIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQNDHSYPYTFGGG TKVEIK

[0255] 101H9G9A2-hVH1 / hVL4 VH amino acid sequence (SEQ ID NO: 190): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 203): DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQDNYLAWYQQKPGKAPKL LIYGARIRESGVPSRFSGSGSGTDFLTISSLQPEDLATYYCQNDHSYPYTFGG GTKVEIK

[0256] 101H9G9A2-hVH2 / hVL1 VH amino acid sequence (SEQ ID NO: 191): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQRLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSASTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 200): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQDNYLAWYQQKPGQPPKL LIYGARIRESGVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQNDHSYPYTFGG GTKVEIK

[0257] 101H9G9A2-hVH2 / hVL2 VH amino acid sequence (SEQ ID NO: 191): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQRLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSASTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 201): DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGNQDNYLAWYLQKPGQSPQLLI YGARIRESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQNDHSYPYTFGGG TKVEIK

[0258] 101H9G9A2-hVH2 / hVL3 VH amino acid sequence (SEQ ID NO: 191): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQRLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSASTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 202): EIVMTQSPATLSVSPGERATLSCKSSQSLLNSGNQDNYLAWYQQKPGQAPRL LIYGARIRESGIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQNDHSYPYTFGGG TKVEIK

[0259] 101H9G9A2-hVH2 / hVL4 VH amino acid sequence (SEQ ID NO: 191): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQRLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSASTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 203): DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQDNYLAWYQQKPGKAPKL LIYGARIRESGVPSRFSGSGSGTDFLTISSLQPEDLATYYCQNDHSYPYTFGG GTKVEIK

[0260] 101H9G9A2-hVH3 / hVL1 VH amino acid sequence (SEQ ID NO: 192): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSISTAYMELSRLRSDDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 200): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQDNYLAWYQQKPGQPPKL LIYGARIRESGVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQNDHSYPYTFGG GTKVEIK

[0261] 101H9G9A2-hVH3 / hVL2 VH amino acid sequence (SEQ ID NO: 192): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSISTAYMELSRLRSDDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 201): DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGNQDNYLAWYLQKPGQSPQLLI YGARIRESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQNDHSYPYTFGGG TKVEIK

[0262] 101H9G9A2-hVH3 / hVL3 VH amino acid sequence (SEQ ID NO: 192): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSISTAYMELSRLRSDDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 202): EIVMTQSPATLSVSPGERATLSCKSSQSLLNSGNQDNYLAWYQQKPGQAPRL LIYGARIRESGIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQNDHSYPYTFGGG TKVEIK

[0263] 101H9G9A2-hVH3 / hVL4 VH amino acid sequence (SEQ ID NO: 192): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSISTAYMELSRLRSDDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 203): DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQDNYLAWYQQKPGKAPKL LIYGARIRESGVPSRFSGSGSGTDFLTISSLQPEDLATYYCQNDHSYPYTFGG GTKVEIK

[0264] 101H9G9A2-hVH4 / hVL1 VH amino acid sequence (SEQ ID NO: 193): QVQLVQSGAEVKKPGSSVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 200): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQDNYLAWYQQKPGQPPKL LIYGARIRESGVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQNDHSYPYTFGG GTKVEIK

[0265] 101H9G9A2-hVH4 / hVL2 VH amino acid sequence (SEQ ID NO: 193): QVQLVQSGAEVKKPGSSVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 201): DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGNQDNYLAWYLQKPGQSPQLLI YGARIRESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQNDHSYPYTFGGG TKVEIK

[0266] 101H9G9A2-hVH4 / hVL3 VH amino acid sequence (SEQ ID NO: 193): QVQLVQSGAEVKKPGSSVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 202): EIVMTQSPATLSVSPGERATLSCKSSQSLLNSGNQDNYLAWYQQKPGQAPRL LIYGARIRESGIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQNDHSYPYTFGGG TKVEIK

[0267] 101H9G9A2-hVH4 / hVL4 VH amino acid sequence (SEQ ID NO: 193): QVQLVQSGAEVKKPGSSVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTITVDKSTSTAYMELSSLRSEDTAVYYCARRGG YYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 203): DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQDNYLAWYQQKPGKAPKL LIYGARIRESGVPSRFSGSGSGTDFLTISSLQPEDLATYYCQNDHSYPYTFGG GTKVEIK

[0268] 101H9G9A2-hVH5 / hVL1 VH amino acid sequence (SEQ ID NO: 194): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELRSLRSDDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 200): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQDNYLAWYQQKPGQPPKL LIYGARIRESGVPDRFSGSGSGTDFTLTISSLQAEDLAVYYCQNDHSYPYTFGG GTKVEIK

[0269] 101H9G9A2-hVH5 / hVL2 VH amino acid sequence (SEQ ID NO: 194): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELRSLRSDDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 201): DIVMTQSPLSLPVTPGEPASISCKSSQSLLNSGNQDNYLAWYLQKPGQSPQLLI YGARIRESGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCQNDHSYPYTFGGG TKVEIK

[0270] 101H9G9A2-hVH5 / hVL3 VH amino acid sequence (SEQ ID NO: 194): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELRSLRSDDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 202): EIVMTQSPATLSVSPGERATLSCKSSQSLLNSGNQDNYLAWYQQKPGQAPRL LIYGARIRESGIPARFSGSGSGTEFTLTISSLQSEDLAVYYCQNDHSYPYTFGGG TKVEIK

[0271] 101H9G9A2-hVH5 / hVL4 VH amino acid sequence (SEQ ID NO: 194): QVQLVQSGAEVKKPGASVKVSCKASDYTFTSYWMDWVRQAPGQGLEWMG NIYPSDSKTHYNQKFKDRVTMTVDKSTSTAYMELRSLRSDDTAVYYCARRG GYYAYAMDYWGQGTLVTVSS VL amino acid sequence (SEQ ID NO: 203): DIQMTQSPSSLSASVGDRVTITCKSSQSLLNSGNQDNYLAWYQQKPGKAPKL LIYGARIRESGVPSRFSGSGSGTDFLTISSLQPEDLATYYCQNDHSYPYTFGG GTKVEIK

[0272] In certain embodiments, the anti-CD24 antibodies and fragments thereof provided herein further comprise an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region comprises a heavy chain and / or a light chain constant region. The heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region. In certain embodiments, the heavy chain constant region comprises an Fc region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ.

[0273] In some embodiments, the anti-CD24 antibodies and antigen-binding fragments thereof provided herein have the constant region of an immunoglobulin (Ig), optionally a human Ig, optionally a human IgG. In some embodiments, the anti-CD24 antibodies and antigen-binding fragments thereof provided herein comprise the constant region of a human IgG1, IgG2, IgG3, or IgG4.

[0274] Human IgG isotypes (subclasses of mature gamma globulin class G antibodies; IgG1, IgG2, IgG3, and IgG4) exhibit different abilities to mobilize effector functions. For example, ADCC is promoted by IgG1 and IgG3, ADCP is promoted by IgG1, IgG2, IgG3, and IgG4, and CDC is promoted by IgG1 and IgG3. Isotype-specific involvement of these effector functions is based on selectivity for Fc receptors on different immune cells and their ability to bind C1q, thereby activating membrane attack complex (MAC) assembly. Among the various isotypes, IgG1 and IgG3 have higher relative affinity for Fcγ receptors, including FcγRI, FcγRIIa / b / c, and FcγRIIIa / b. However, IgG2 has significantly lower affinity for Fcγ receptors, except for the FcγRIIa H131 polymorphism, and IgG4 only has measurable affinity for FcγRI.

[0275] In certain embodiments, the anti-CD24 antibodies and antigen-binding fragments provided herein comprise a constant region of an IgG1 isotype capable of inducing ADCC, CDC, or ADCP, or a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function. Effector functions such as ADCC and CDC can cause cytotoxicity against CD24-expressing cells. Effector function can be assessed using various assays, such as Fc receptor binding assays, C1q binding assays, and cytolytic assays.

[0276] Antibody variants The present disclosure also encompasses various variants of the antibodies and / or antigen-binding fragments thereof provided herein. In certain embodiments, the present disclosure encompasses various types of variants of the exemplary antibodies provided herein, namely, 33E7E12, 71A4F12, 56A7E3, 58F10-1F6, 73H5-1G2, 110D4G4, 81A1F8, 100F2E3, 111F3A2, 81A7A10, or 107D10D11.

[0277] In certain embodiments, antibody variants comprise one or more modifications or substitutions in one or more CDR sequences, as provided in Table 1, one or more FR sequences, heavy or light chain variable region sequences and / or constant regions (e.g., Fc regions) provided herein. Such variants retain the specific binding affinity of their parent antibody for CD24 but have one or more desirable properties conferred by the modification(s) or substitution(s). For example, antibody variants may have improved antigen-binding affinity, improved glycosylation pattern, reduced risk of glycosylation, reduced deamination, reduced or eliminated effector function(s), improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues).

[0278] Using methods known in the art, e.g., "alanine scanning mutagenesis," parent antibody sequences may be screened to identify suitable or preferred residues for modification or substitution (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and the modified antibodies are produced and screened for interesting properties. If substitution at a particular amino acid position demonstrates an interesting functional change, that position can be identified as a promising residue for modification or substitution. The identified promising residues may be further evaluated by substituting them with different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0279] Affinity mutants Affinity variants may contain modifications or substitutions in one or more CDR sequences, such as those provided in Table 1, one or more FR sequences, or heavy or light chain variable region sequences provided herein. Affinity variants retain the specific binding affinity for CD24 of their parent antibody, or may further have improved specific binding affinity for CD24 over that of the parent antibody. In certain embodiments, at least one (or all) substitutions in a CDR sequence, FR sequence, or variable region sequence comprise conservative substitutions.

[0280] Those skilled in the art will understand that one or more amino acid residues may be substituted in the CDR and FR sequences provided in Table 1, while the resulting antibody or antigen-binding fragment still retains or even has improved binding affinity to CD24. Various methods known in the art can be used to achieve this goal. For example, phage display technology can be used to generate and express a library of antibody variants (such as Fab or scFv variants), which can then be screened for binding affinity to human CD24. For another example, computer software can be used to substantially simulate the binding of an antibody to human CD24 and identify amino acid residues on the antibody that form the binding interface. Such residues may be avoided for substitution to prevent a reduction in binding affinity, or may be targeted for substitution to provide stronger binding.

[0281] In certain embodiments, the humanized antibodies or antigen-binding fragments provided herein contain one or more amino acid residue substitutions in one or more CDR sequences and / or one or more FR sequences. In certain embodiments, the affinity variants contain a total of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in the CDR and / or FR sequences.

[0282] In certain embodiments, the anti-CD24 antibodies and antigen-binding fragments thereof comprise one, two, or three CDR sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to those(s) listed in Table 1, while retaining binding affinity for CD24 at a similar or even higher level than that of its parent antibody.

[0283] In certain embodiments, anti-CD24 antibodies and antigen-binding fragments thereof comprise one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to those(s) listed in Table 1, while retaining similar or even higher levels of binding affinity for CD24 as the parent antibody. In some embodiments, a total of 1 to 10 amino acids have been substituted, inserted, or deleted in a sequence selected from those(s) listed in Table 1. In some embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs).

[0284] Glycosylation variants The anti-CD24 antibodies and antigen-binding fragments provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment.

[0285] An antibody or antigen-binding fragment thereof may contain one or more amino acid residues having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue in a tripeptide sequence, such as asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline). O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine. Removal of native glycosylation sites can be conveniently achieved, for example, by altering the amino acid sequence to substitute one of the above tripeptide sequences (for N-linked glycosylation sites) or a serine or threonine residue present in the sequence (for O-linked glycosylation sites). New glycosylation sites can be created in an analogous manner by introducing such tripeptide sequences or serine or threonine residues.

[0286] Cysteine-engineered mutants The anti-CD24 antibodies and antigen-binding fragments provided herein also include cysteine ​​engineered variants, which contain one or more introduced free cysteine ​​amino acid residues.

[0287] Free cysteine ​​residue is not part of disulfide bridge.The mutants that cysteine ​​is engineered are useful for, for example, conjugation with cytotoxic and / or imaging compounds, labels or radioisotopes at engineered cysteine ​​site, for example, via maleimide or haloacetyl.The method of engineering antibody or antigen-binding fragment to introduce free cysteine ​​residue is known in the art, for example, see WO2006 / 034488.

[0288] Fc variants The anti-CD24 antibodies and antigen-binding fragments provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions in the Fc region and / or hinge region.

[0289] In certain embodiments, the anti-CD24 antibody or antigen-binding fragment contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants may have an extended pharmacokinetic half-life because they bind to FcRn at acidic pH, allowing them to escape degradation in lysosomes and then be translocated and released outside the cell. Methods for genetically engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, e.g., Vaughn, D. et al, Structure, 6(1):63-73, 1998; Kontermann, R. et al, Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al, Cancer Research, 70:3269-3277(2010); and Hinton, P. et al, J. Immunology, 176:346-356(2006).

[0290] In certain embodiments, the anti-CD24 antibody or antigen-binding fragment contains one or more amino acid substitutions that alter antibody-dependent cellular cytotoxicity (ADCC). Certain amino acid residues in the CH2 domain of the Fc region can be substituted to provide enhanced ADCC activity. Alternatively, or in addition, carbohydrate structures on the antibody can be altered to enhance ADCC activity. Methods for altering ADCC activity by antibody engineering have been described in the art, e.g., Shields RL et al., J Biol Chem. 2001, 276(9):6591-604; Idusogie EE et al., J Immunol. 2000, 164(8):4178-84; Steurer W et al., J Immunol. 1995, 155(3):1165-74; Idusogie EE et al., J Immunol. 2001, 166(4):2571-5; Lazar GA et al., PNAS, 2006, 103(11):4005-4010; Ryan MC et al., Mol. Cancer Ther., 2007, 6:3009-3018; Richards JO et al., Mol. Cancer Ther. 2008, 7(8):2517-27; Shields R. Let al, J. Biol. Chem, 2002, 277:26733-26740; Shinkawa T. et al, J. Biol. Chem, 2003, 278:3466-3473.

[0291] In certain embodiments, the anti-CD24 antibody or antigen-binding fragment comprises one or more amino acid substitution(s) that alter complement dependent cytotoxicity (CDC), e.g., by improving or reducing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821) and WO94 / 29351 for other examples of Fc region variants.

[0292] In certain embodiments, the anti-CD24 antibody or antigen-binding fragment contains one or more amino acid substitutions at the interface of the Fc region to facilitate and / or promote heterodimerization. These modifications include the introduction of a protuberance in the first Fc polypeptide and a cavity in the second Fc polypeptide, where the protuberance can be positioned within the cavity to promote interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for producing antibodies with these modifications are known in the art and are described, for example, in U.S. Patent No. 5,731,168.

[0293] antigen binding fragment Anti-CD24 antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-CD24 antibodies provided herein, including, for example, the exemplary antibodies whose CDR and FR sequences are shown in Table 1, and various variants thereof (e.g., affinity variants, glycosylation variants, Fc variants, cysteine-engineered variants, etc.).

[0294] In certain embodiments, the anti-CD24 antigen-binding fragment provided herein is a camelized single domain antibody, diabody, single-chain Fv fragment (scFv), scFv dimer, BsFv, dsFv, (dsFv)2, dsFv-dsFv', Fv fragment, Fab, Fab', F(ab')2, diabody, nanobody, domain antibody, single domain antibody, or bivalent domain antibody.

[0295] Various techniques can be used to produce such antigen-binding fragments. Exemplary methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form an F(ab')2 fragment (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0296] In certain embodiments, the antigen-binding fragment is an scFv. The production of scFv is described, for example, in WO93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. The scFv can be fused to an effector protein at either the amino or carboxy terminus to provide a fusion protein (see, for example, Antibody Engineering, ed. Borrebaeck).

[0297] Bispecific antibodies, multivalent antibodies In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. In certain embodiments, the antibodies and / or antigen-binding fragments thereof provided herein are monospecific or bispecific.

[0298] The term "valent" as used herein refers to the presence of a specified number of antigen-binding sites in a given molecule. As such, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen-binding molecule. A bivalent molecule can be monospecific when both binding sites are for specific binding of the same antigen or epitope. Similarly, a trivalent molecule can be bispecific when, for example, two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0299] In certain embodiments, the antibodies or antigen-binding fragments provided herein may be monospecific, but bivalent, trivalent, or tetravalent, having at least two binding sites specific for the same antigen or epitope. In certain embodiments, this provides stronger binding to the antigen or epitope than its monovalent counterpart. In certain embodiments, in bivalent antigen-binding moieties, the first and second valences of the binding sites are structurally identical (i.e., have the same sequence) or structurally different (i.e., have the same specificity but different sequences).

[0300] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein are bispecific. In some embodiments, the bispecific antibodies and antigen-binding fragments thereof provided herein bind to a first and a second epitope of CD24, and the first and second epitopes of CD24 are different.

[0301] In some embodiments, a bispecific antibody or antigen-binding fragment thereof provided herein binds to a first epitope on CD24 and a second epitope on an antigen other than CD24.

[0302] The bispecific antibody molecules provided herein may be in any suitable bispecific format known in the art. Examples of reference bispecific antibody formats known in the art include, but are not limited to, (i) bispecific antibodies with symmetric Fc, (ii) bispecific antibodies with asymmetric Fc, (iii) conventional antibodies with an additional antigen-binding moiety attached, (iv) bispecific antibody fragments, (v) conventional antibody fragments with an additional antigen-binding moiety attached, and (vi) bispecific antibodies with human albumin or a human albumin-binding peptide attached.

[0303] Bispecific IgG-like antibodies (BsIgG) are monovalent for each antigen and can be produced by coexpressing two light chains and two heavy chains in a single host cell. The added IgG is engineered to form a bispecific IgG by adding an additional antigen-binding unit to the amino or carboxyl terminus of the light or heavy chain. The additional antigen-binding unit can be a single domain antibody (unpaired VL or VH) such as a DVD-Ig, a paired antibody variable domain (e.g., Fv or scFv), or an engineered protein scaffold.

[0304] Bispecific antibody fragments are antigen-binding fragments derived from antibodies but lacking some or all of the antibody constant domains. Examples of such bispecific antibody fragments include single domain antibodies, Fvs, Fabs, and diabodies.

[0305] In certain embodiments, the bispecific antibody molecules provided herein are based on "whole" antibody formats, such as whole IgG or IgG-like molecules, and small recombinant formats, such as tandem single-chain variable fragment molecules (taFv), diabodies (Db), single-chain diabodies (scDb), and various other derivatives thereof (see the bispecific antibody formats described in Byrne H. et al. (2013) Trends Biotech, 31(11):621-632). Examples of bispecific antibodies are based on formats including, but not limited to, quadromas, chemically conjugated Fabs (antigen-binding fragments), and BiTEs (bispecific T-cell engagers).

[0306] In certain embodiments, the bispecific antibody molecules provided herein are selected from the group consisting of Triomab; hybrid hybridoma (quadroma); multispecific anticalin platform (Pieris); diabodies; single chain diabodies; tandem single chain Fv fragments; TandAb, trispecific Ab (Affimed); Darts (dual affinity retargeting; Macrogenics); bispecific Xmab (Xencor); bispecific T cell engager (Bites; Amgen; 55 kDa); triplebodies; tribody (Fab-scFv) fusion proteins (CreativeBiolabs) multifunctional recombinant antibody derivatives; Duobody platform (Genmab); Dock and The bispecific formats are selected from: the [lock] platform; the knob-into-hole (KIH) platform; the humanized bispecific IgG antibody (REGN1979) (Regeneron); the Mab2 bispecific antibody (F-Star); the DVD-Ig (dual variable domain immunoglobulin) (Abbvie); the kappa-lambda body; the TBTI (tetravalent bispecific tandem Ig); and the CrossMab.

[0307] In certain embodiments, the bispecific antibody molecules provided herein are in a bispecific format selected from BsIgG, including CrossMab; DAF (two-in-one); DAF (four-in-one); DutaMab; DT-IgG; knob-in-hole common LC; knob-in-hole assembly; charge pair; Fab arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; kappa-lambda body; and orthogonal Fab. For a detailed description of bispecific antibody formats, see Spiess C., Zhai Q., ​​and Carter PJ (2015) Molecular Immunology 67:95-106, which is incorporated herein by reference in its entirety.

[0308] In certain embodiments, the bispecific antibody molecules provided herein are in a bispecific format selected from an IgG-added antibody having an additional antigen-binding moiety, including DVD-IgG; IgG(H)-scFv; scFv-(H)IgG; IgG(L)-scFv; scFV-(L)IgG; IgG(L,H)-Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; Zybody; and DVI-IgG (four-in-one) (see ibid.).

[0309] In certain embodiments, the bispecific antibody molecules provided herein are in a format selected from bispecific antibody fragments including nanobodies; nanobody-HAS; BiTEs; diabodies; DARTs; TandAbs; sc diabodies; sc-diabody-CH3; diabody-CH3; triplebodies; miniantibodies; minibodies; TriBi minibodies; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F(ab')2; F(ab')2-scFv2; scFv-KIH; Fab-scFv-Fc; tetravalent HCAbs; sc diabody-Fc; diabody-Fc; tandem scFv-Fc; and intrabodies (see ibid.).

[0310] In certain embodiments, the bispecific antibody molecules provided herein are in bispecific formats such as Dock-Lock; ImmTAC; HSAbody; scdiabody-HAS; and tandem scFv-toxin (see ibid.).

[0311] In certain embodiments, the bispecific antibody molecules provided herein are based on a format selected from bispecific antibody conjugates including IgG-IgG; Cov-X-Body; and scFv1-PEG-scFv2 (see ibid.).

[0312] In certain embodiments, the second antigen-binding domain is operably linked to the N-terminus or C-terminus of the CD24-binding domain. In certain embodiments, the CD24-binding domain is operably linked to the N-terminus or C-terminus of the second antigen-binding domain.

[0313] The operable linkage can be a direct chemical bond or an indirect linkage via a spacer or intervening sequence. The term "spacer," as used herein, refers to an artificial amino acid sequence having a length of 1, 2, 3, 4, or 5 amino acid residues, or between 5 and 15, 20, 30, 50, or more, joined by peptide bonds, and is used to link one or more binding domains, such as an scFv and a Fab or IgG. The spacer may be the same as or different from the peptide linker within the scFv. In certain embodiments, the spacer comprises one, two, three, four, or more consecutive or tandem repeats of SEQ ID NOs: 90, 91, 92, and 93. In certain embodiments, the spacer comprises GGGGS (SEQ ID NO: 90). In certain embodiments, the spacer comprises GGGGSGGGGS (SEQ ID NO: 91), GGGSGGGGSGGGGS (SEQ ID NO: 92), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 93). As used herein, the intervening sequence may be any amino acid sequence located between the CD24-binding domain and the second antigen-binding domain, as long as both domains are capable of binding to each other. In the example shown, the intervening sequence may include a heavy chain constant region or a light chain constant region.

[0314] In certain embodiments, the second antigen-binding domain comprises an scFv, and the CD24-binding domain comprises a Fab or IgG. In certain embodiments, the second antigen-binding scFv can be operably linked to the N-terminus or C-terminus of the heavy chain of the CD24-binding Fab or IgG (e.g., the C-terminus of the heavy chain constant region following the CD24-binding Fab), or to the N-terminus or C-terminus of the light chain of the CD24-binding Fab or IgG, or any combination thereof, or vice versa.

[0315] In the example shown, the bispecific antibody molecule can comprise a heavy chain in the format VH(anti-CD24)-CH1-hinge-CH2-CH3-spacer-second antigen-binding scFv or second antigen-binding scFv-spacer-VH(anti-CD24)-CH1-hinge-CH2-CH3 and a light chain in the format VL(anti-CD24)-CL. VH(anti-CD24) and VL(anti-CD24) refer to the heavy and light chain variable domains of the CD24 binding domain, respectively; second antigen-binding scFv refers to the scFv of the second antigen-binding domain; CL refers to the light chain constant region; and CH1-hinge-CH2-CH3 collectively refer to the heavy chain constant region.

[0316] In another illustrative example, the bispecific antibody molecule may also comprise a light chain in the format second antigen-binding scFv-spacer-VL(anti-CD24)-CL or VL(anti-CD24)-CL-spacer-second antigen-binding scFv and a heavy chain in the format VH(anti-CD24)-CH1-hinge-CH2-CH3.

[0317] In certain embodiments, the CD24-binding domain comprises an scFv, and the second antigen-binding domain comprises a Fab or IgG. In certain embodiments, the CD24-binding domain scFv can be operably linked to the N-terminus or C-terminus of the heavy chain of the second antigen-binding Fab or IgG (e.g., the C-terminus of the heavy chain constant region following the CD24-binding Fab), or to the N-terminus or C-terminus of the light chain of the second antigen-binding Fab or IgG, or any combination thereof, or vice versa.

[0318] In an exemplary example, a bispecific antibody molecule may also comprise a heavy chain in the format of second antigen-binding VH-CH1-hinge-CH2-CH3-spacer-scFv(anti-CD24) or scFv(anti-CD24)-spacer-second antigen-binding VH-CH1-hinge-CH2-CH3 and a light chain in the format of second antigen-binding VL-CL. In another exemplary example, a bispecific antibody molecule may also comprise a light chain in the format of scFv(anti-CD24)-spacer-second antigen-binding VL-CL or second antigen-binding VL-CL-spacer-scFv(anti-CD24) and a heavy chain in the format of second antigen-binding VH-CH1-hinge-CH2-CH3.

[0319] In the bispecific antibody molecules provided herein, the second antigen-binding domain may be monovalent (i.e., one scFv or Fab) or multivalent (e.g., more than one scFv or Fab), and / or the CD24-binding domain may be monovalent or multivalent.

[0320] In certain embodiments, the second antigen is an immune-related target, optionally selected from the group consisting of PD-L1, PD-L2, PD-1, CLTA-4, TIM-3, LAG3, CD160, 2B4, TGF beta, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, ICAM-1, NKG2C, SLAMF7, NKp80, CD160, B7-H3, LFA-1, ICOS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, SIRPα, Siglec10, LILRB2, Clever-1, Macro, LILRB4, Siglec15, CSF1R, PSGL-1, VSIG-4, B2M, and CD83.

[0321] In certain embodiments, the second antigen is a tumor-associated antigen or an epitope thereof. The term "tumor-associated antigen" is used interchangeably with the term "tumor antigen" and refers to an antigen that is present or presented on the surface of tumor cells and is located on or within tumor cells. In some embodiments, the tumor-associated antigen can be presented only by tumor cells and not by normal, i.e., non-tumor cells. In some other embodiments, the tumor-associated antigen can be exclusively expressed on tumor cells or can exhibit tumor-specific mutations compared to non-tumor cells. In some other embodiments, the tumor-associated antigen can be found on both tumor cells and non-tumor cells, but is overexpressed in tumor cells compared to non-tumor cells, or is more accessible to antibody binding in tumor cells due to the less compact structure of tumor tissue compared to non-tumor tissue.

[0322] In some embodiments, the tumor antigen is CA-125, gangliosides G(D2), G(M2) and G(D3), CD19, CD20, CD33, CD47, CD52, Ep-CAM, CEA, CLDN18.2, bombesin-like peptide, PSA, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, CD44v9, Ki-67, cancer-associated mucin, VEGF, VEGFR s (e.g., VEGFR3), estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, IL-2R, CDH6, CEA, FOLR1, TROP2, PTK7, SLITRK6, CD142, NECTIN-4, ROR1, ROR2, CD142, CD123, CD22, CD79b, DLL3, SLC family, or CO17-1A.

[0323] In certain embodiments, the second antigen is an immunosuppressive molecule selected from the group consisting of PD-L1, SIRPα, CD47, and B2M.

[0324] In a specific embodiment, the second antigen is CD47.

[0325] In some embodiments, the CD47-binding scFv is operably linked to the C-terminus of the heavy chain constant region following the CD24-binding Fab.

[0326] In certain embodiments, the bispecific antibody molecule comprises a heavy chain in the format of VH(anti-CD24)-CH1-hinge-CH2-CH3-spacer-scFv(anti-CD47), associated with a light chain in the format of VL(anti-CD24)-CL.

[0327] In a specific embodiment, the CD47-binding scFv is a 5F9-derived scFv and comprises the amino acid sequence of SEQ ID NO: 94 (QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMGTIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK).

[0328] Other illustrative examples of tumor-associated antigens include CD10, CD21, CD22, CD25, CD30, CD34, CD37, CD38, CD123, GPRC5D, CD44v6, CD45, CD133, Fms-like tyrosine kinase 3 (FLT-3, CD135), chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), Her2, Her3, IGFR, IL3R, fibroblast activation protein (FAP), CDCP1, Derlin1, tenascin, and frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-α (CD140a), PDGFR-β (CD140b), endoglin, CLEC14, CLEC12a, Tem1-8, and Tie2. Further examples include A33, Cambus-1 (CDw52), carcinoembryonic antigen (CEA), carboanhydrase IX (MN / CA IX), de2-7, EGFRvIII, EpCAM, folate binding protein, G250, c-Kit (CD117), CSF1R (CD115), HLA-DR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate-specific antigen (PSA), and tag-72.

[0329] The bispecific antibodies and antigen-binding fragments provided herein can be produced by any suitable method known in the art. In a conventional approach, two immunoglobulin heavy chain-light chain pairs with different antigen specificities are co-expressed in a host cell to produce a bispecific antibody by recombinant methods (see, for example, Milstein and Cuello, Nature, 305:537 (1983)), which can then be purified by affinity chromatography.

[0330] A recombinant approach may also be used, in which sequences encoding antibody heavy chain variable domains of two specificities are each fused to an immunoglobulin constant domain sequence and then inserted into an expression vector, which is then co-transfected with an expression vector for the light chain sequence into a host cell suitable for recombinant expression of the bispecific antibody (see, e.g., WO94 / 04690; Suresh et al., Methods in Enzymology, 121:210 (1986)). Similarly, scFv dimers can also be recombinantly constructed and expressed from host cells (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)).

[0331] Alternatively, the leucine zipper peptides from the Fos and Jun proteins can be linked to the Fab' portions of two different antibodies by gene fusion. The linked antibodies are reduced at the hinge region to four half antibodies (i.e., monomers) and then reoxidized to form heterodimers (Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)).

[0332] Two antigen-binding domains can be conjugated or cross-linked to form bispecific antibodies or antigen-binding fragments. For example, one antibody can be coupled with biotin, while the other antibody can be coupled with avidin, and the strong association between biotin and avidin will form a complex with the two antibodies to form a bispecific antibody (see, for example, U.S. Pat. No. 4,676,980, WO91 / 00360, WO92 / 00373 and EP03089). For another example, two antibodies or antigen-binding fragments can be cross-linked by conventional methods known in the art, such as those disclosed in U.S. Pat. No. 4,676,980.

[0333] Bispecific antigen-binding fragments may be produced from bispecific antibodies, for example, by proteolytic cleavage or by chemical linkage. For example, an antigen-binding fragment of an antibody (e.g., Fab') may be prepared, converted to a Fab'-thiol derivative, and then mixed and reacted with another converted Fab' derivative having a different antigen specificity to form a bispecific antigen-binding fragment (see, e.g., Brennan et al., Science, 229:81 (1985)).

[0334] In certain embodiments, bispecific antibodies or antigen-binding fragments may be engineered at the interface to form knob-into-hole associations to promote heterodimerization of two distinct antigen-binding sites. "Knobs-into-hole" refers herein to interactions between polypeptides (such as CH3 domains) in which one polypeptide has a protuberance (i.e., "knob") due to the presence of an amino acid residue with a bulky side chain (e.g., tyrosine or tryptophan), and the other polypeptide has a cavity (i.e., "hole") into which a small side chain amino acid residue (e.g., alanine or threonine) and a protuberance can be positioned, facilitating the interaction of the two polypeptides to form a heterodimer or complex. Methods for generating polypeptides with knobs-into-holes are known in the art and are described, for example, in U.S. Pat. No. 5,731,168.

[0335] Conjugates In some embodiments, anti-CD24 antibodies and antigen-binding fragments thereof are linked to one or more conjugates, and optionally, the conjugates are covalently linked either directly or via a linker. A conjugate is a non-proteinaceous moiety that can bind to an antibody or its antigen-binding fragment. It is contemplated that various conjugates may be linked to the antibodies or antigen-binding fragments provided herein (see, for example, "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugates may be linked to antibodies or antigen-binding fragments by, among other methods, covalent bonding, affinity bonding, intercalation, coordinate bonding, complex formation, association, blending, or addition. In some embodiments, the conjugate comprises a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binding agent or other anti-cancer drug.

[0336] In certain embodiments, the antibodies and antigen-binding fragments disclosed herein may be engineered to contain designated sites outside the epitope-binding portion that can be utilized for binding to one or more conjugates. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, to facilitate covalent binding to a conjugate.

[0337] In certain embodiments, the antibody may be indirectly linked to the conjugate or through another conjugate.For example, the antibody or antigen-binding fragment may be conjugated to biotin, and then indirectly conjugated to the second conjugate that is conjugated to avidin.The conjugate may be a toxin (e.g., a chemotherapeutic agent), a detectable label (e.g., a radioisotope, a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label).

[0338] A "toxin" can be any agent that is harmful to cells or can damage or kill cells. Examples of toxins include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and its analogs, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alginate, cyclohexyl 1-phosphate dehydrogenase ... Killing agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agents (e.g., vincristine and vinblastine).

[0339] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes (e.g., 123 I, 124 I, 125 I, 131 I, 35 S, 3 H, 111 In, 112 In, 14 C. 64 Cu, 67 Cu, 86 Y, 88 Y, 90 Y, 177 Lu, 211 At, 186 Re, 188 Re, 153 Sm, 212 Bi and 32 P, other lanthanides, luminescent labels), chromophore moieties, digoxigenin, biotin / avidin, DNA molecules or gold for detection.

[0340] In certain embodiments, the conjugate may be a pharmacokinetic-modifying moiety that serves to increase the half-life of the antibody. Illustrative examples include water-soluble polymers such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and the like. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when more than one polymer is attached, they may be the same molecule or different molecules.

[0341] In certain embodiments, the conjugate may be a purification moiety such as a magnetic bead.

[0342] In certain embodiments, the antibodies and / or antigen-binding fragments thereof provided herein are used as the substrate of a conjugate.

[0343] Polynucleotides and Recombinant Methods The present disclosure provides isolated polynucleotides encoding anti-CD24 antibodies and antigen-binding fragments thereof. In certain embodiments, the isolated polynucleotides encoding the variable regions of the exemplary antibodies provided herein comprise one or more nucleotide sequences as set forth in SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:102, SEQ ID NO:110, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:228, SEQ ID NO:229, and / or SEQ ID NO:220. DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). The encoding DNA can also be obtained by synthetic methods.

[0344] Isolated polynucleotides encoding anti-CD24 antibodies and antigen-binding fragments thereof (e.g., comprising a sequence as set forth in SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:102, SEQ ID NO:110, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:228, SEQ ID NO:229 and / or SEQ ID NO:220) can be inserted into a vector for further cloning (amplification of the DNA) or for expression using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0345] In some embodiments, vector systems include mammalian, bacterial, yeast systems, etc., including but not limited to plasmids such as pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pCMV, pEGFP, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMD18-T, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS420, pLexA, pACT2.2, etc., as well as other laboratory and commercially available vectors. Suitable vectors can include plasmids or viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0346] A vector containing a polynucleotide sequence encoding an antibody or antigen-binding fragment can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or expressing DNA in a vector herein include the prokaryotes, yeast, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example, Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and the like. marcescans and Shigella, and Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Enterobacteriaceae such as Streptomyces.

[0347] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-CD24 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris; pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger, are commonly available and useful herein.

[0348] Suitable host cells for expressing the glycosylated antibodies or antigen fragments provided herein are derived from multicellular organisms. Examples of vertebrate cells include plant and insect cells. Numerous baculovirus strains and mutants have been identified, as well as corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm). Various viral strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses can be used in accordance with the invention herein, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0349] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0350] Host cells are transformed with the above-described expression or cloning vectors to produce anti-CD24 antibodies and cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify the genes encoding the desired sequences. In another embodiment, antibodies can be produced by homologous recombination, as known in the art.

[0351] The host cells used to produce the antibodies or antigen-binding fragments provided herein can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), minimal essential medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM) (Sigma) are suitable for culturing host cells. Furthermore, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90 / 03430; WO87 / 00195; or Reissued U.S. Pat. No. 30,985 can be used as a culture medium for the host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN™), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, will be those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0352] When using recombinant techniques, antibodies can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the culture medium, the supernatant obtained from such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0353] The anti-CD24 antibodies and antigen-binding fragments thereof prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0354] In certain embodiments, solid-phase immobilized protein A is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of protein A as an affinity ligand varies depending on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices, such as controlled-pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation, are also available, depending on the antibody to be recovered.

[0355] After any preliminary purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 and 4.5, preferably performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0356] CAR In another aspect, the present disclosure also provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CD24 and includes the antigen-binding fragments provided herein. In certain embodiments, the antigen-binding fragment is a Fab or scFv. In certain embodiments, the CARs provided herein are bispecific. The CAR can further specifically bind to a second antigen other than CD24 or a second epitope on CD24. In certain embodiments, the second antigen is a tumor antigen, tumor-associated antigen, or immune-related target as described above. The TCR signaling domain can be selected from the group consisting of intracellular signal region sequences of CD3ζ, FccRIγ, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or a combination thereof. The transmembrane region can include the transmembrane region of CD3, CD4, CD8, or CD28. In certain embodiments, the second antigen is an immunosuppressive molecule such as PD-L1, SIRPα, CD47, or B2M.

[0357] In another aspect, the present disclosure also provides nucleic acid sequences encoding the above-described CARs, and cells or vectors comprising such nucleic acid sequences, or cells genetically modified to express the CARs provided herein. The cells can be immune cells, and optionally, the immune cells are T lymphocytes, NK cells, monocytes, macrophages, or NKT lymphocytes.

[0358] Pharmaceutical Composition The present disclosure further provides pharmaceutical compositions comprising an anti-CD24 antibody or antigen-binding fragment thereof and one or more pharmaceutically acceptable carriers.

[0359] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, anesthetics, suspending / dispensing agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other ingredients known in the art, or various combinations thereof.

[0360] Suitable ingredients may include, for example, antioxidants, bulking agents, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, by including one or more antioxidants, such as methionine, in compositions comprising antibodies or antigen-binding fragments and conjugates as provided herein, oxidation of the antibodies or antigen-binding fragments is reduced. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving antibody stability and maximizing shelf life. Thus, in certain embodiments, compositions are provided comprising one or more antibodies or antigen-binding fragments as disclosed herein and one or more antioxidants, such as methionine. Further provided are methods for preventing oxidation, extending the shelf life, and / or improving the efficacy of antibodies or antigen-binding fragments as provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0361] To further illustrate, pharmaceutically acceptable carriers can include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as hydrogenated oils of vegetable origin, coconut oil, corn oil, sesame oil, or peanut oil; antibacterial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as polysorbate 80 (TWEEN-80); Antibacterial agents used as carriers may be added to the pharmaceutical compositions in multi-dose containers, and include phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride.Suitable excipients include, for example, water, saline, dextrose, glycerol, or ethanol.Suitable non-toxic auxiliary substances include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0362] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained-release formulations, or powders. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0363] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition.The injectable pharmaceutical composition can be prepared in any conventional form, such as a liquid solution, suspension, emulsion, or a solid form suitable for preparing a liquid solution, suspension, or emulsion.Preparations for injection can include sterile and / or non-pyrogenic solutions prepared for injection, sterile dry soluble preparations such as lyophilized powders prepared for mixing with a solvent immediately before use, including hypodermic tablets, sterile suspensions prepared for injection, sterile dry insoluble preparations prepared for mixing with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions.The solution can be aqueous or non-aqueous.

[0364] In certain embodiments, the unit dose parenteral preparation is packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile and non-pyrogenic, as known and practiced in the art.

[0365] In certain embodiments, a sterile, lyophilized powder is prepared by dissolving an antibody or antigen-binding fragment as disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at approximately neutral pH. Subsequent sterile filtration of the solution, followed by lyophilization under standard conditions known to those of skill in the art, provides the desired formulation. In one embodiment, the resulting solution is apportioned into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-CD24 antibody or antigen-binding fragment thereof, or composition thereof. Overfilling the vial with a small amount (e.g., about 10%) beyond that required for the dose or set of doses is permissible to facilitate accurate sample withdrawal and accurate dosing. The lyophilized powder may be stored under appropriate conditions, such as at about 4°C to room temperature.

[0366] Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration.In one embodiment, sterile and / or non-pyrogenic water or other liquid suitable carrier is added to the lyophilized powder for reconstitution.The exact amount varies depending on the selected therapy given and can be empirically determined.

[0367] How to use The present disclosure also provides therapeutic methods comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment as provided herein to a subject in need thereof, thereby treating or preventing a CD24-associated disease or condition. In some embodiments, the CD24-associated disease or condition is cancer, an autoimmune disease, an inflammatory disease, an adaptive immune disease, or an infectious disease.

[0368] Examples of cancers include, but are not limited to, non-small cell lung cancer (squamous / non-squamous), small cell lung cancer, renal cell carcinoma, colorectal cancer, colon cancer, ovarian cancer, breast cancer (including basal, ductal, and lobular breast carcinoma), pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, melanoma, myeloma, mycosis fungoides, Merkel cell carcinoma, hepatocellular carcinoma (HCC), and fibrosarcoma. , myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, lymphoid tumors, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic lung carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, classical Hodgkin's lymphoma (CHL), mediastinal primary Large B-cell lymphoma, T-cell / histiocytic-rich B-cell lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera, mast cell-derived tumors, EBV-positive and -negative PTLD and diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma, H These include HV8-associated primary effusion lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia, primary CNS lymphoma, spinal axis tumors, brainstem glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

[0369] In certain embodiments, the cancer is lung cancer, bronchial cancer, bone cancer, liver and bile duct cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, anal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, gastric cancer, vaginal cancer, thyroid cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, adenocarcinoma, leukemia, myeloma and lymphoma. In certain embodiments, the cancer is chemotherapy-resistant.

[0370] In certain embodiments, the disease or condition is a hematological cancer selected from B-cell lymphomas, including, but not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), marginal zone B-cell lymphoma (MZL), mantle cell lymphoma (MCL), Richter's syndrome, Burkitt's lymphoma, or follicular lymphoma.

[0371] Autoimmune diseases include, but are not limited to, acquired immune deficiency syndrome (AIDS, a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behçet's disease, cardiomyopathy, celiac sprue dermatitis herpetiformis; chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, and especially Myocardial infarction, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis , psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (also known as progressive systemic sclerosis (PSS) or systemic sclerosis (SS)), Sjögren's syndrome, stiff man syndrome, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.

[0372] Inflammatory disorders include, for example, chronic and acute inflammatory disorders.Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft-versus-host disease, hemolytic anemia, osteoarthritis, sepsis, stroke, tissue and organ transplantation, vasculitis, diabetic retinopathy and ventilator-induced lung injury.In some embodiments, the CD24-related condition is an inflammatory disease, for example, systemic lupus erythematosus (SLE), intestinal mucosal inflammation, colitis-related wasting disease, multiple sclerosis, viral infection, rheumatoid arthritis, osteoarthritis, Crohn's disease and inflammatory bowel disease, psoriasis, systemic sclerosis, autoimmune diabetes, etc.

[0373] Infectious diseases include, but are not limited to, fungal infections, parasitic / protozoal infections, or chronic viral infections, such as malaria, coccidioiodmycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candidiasis albicans albicans, paracoccidioidomycosis, microsporidiosis, acanthamoeba keratitis, amebiasis, ascariasis, babesiosis, balantidiosis, Baylisascariasis, Chagas disease, clonorchiasis, Cochliomyiasis, cryptosporidiosis, diphyllobothriasis, dracunculiasis, echinococcosis, elephantiasis, enterobiasis, fascioliasis, filariasis, giardiasis, gnathostomiasis, hymenococcosis, isosporiasis, Katayama fever, leishmaniasis, Lyme disease, yokogawa fluke disease, myiasis, onchocerciasis, lice infestation, scabies, schistosomiasis, sleeping sickness, strongyloidiasis, tapeworm disease, toxocariasis , toxoplasmosis, trichinosis, trichuriasis, trypanosomiasis, helminth infection, hepatitis B (HBV), hepatitis C (HCV), herpes virus, Epstein-Barr virus, HIV, cytomegalovirus, herpes simplex virus type I, herpes simplex virus type II, human papillomavirus, adenovirus, human immunodeficiency virus I, human immunodeficiency virus II, Kaposi-West sarcoma-associated herpesvirus infection, Schingling virus (Torquetenovirus), human T-lymphotrophic virus I, human T-lymphotrophic virus II, varicella-zoster, JC virus, or BK virus infection.

[0374] In certain embodiments, the subject is a human.

[0375] In another aspect, provided is a method of treating a disease or condition in a subject that would benefit from modulation of CD24 activity, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment provided herein. The term "disease or condition" may be used interchangeably herein with the term "CD24-associated disease or condition."

[0376] A therapeutically effective amount of an antibody or antigen-binding fragment as provided herein will vary depending on various factors known in the art, such as the subject's weight, age, past medical history, current medications, health status and potential for cross-reactivity, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease development, etc. Dosages may be proportionately reduced or increased by one skilled in the art (e.g., a physician or veterinarian) as indicated by these and other circumstances or requirements.

[0377] In certain embodiments, an antibody or antigen-binding fragment as provided herein may be administered at a therapeutically effective dosage of about 0.01 mg / kg to about 100 mg / kg (e.g., about 0.01 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg). In certain of these embodiments, the antibody or antigen-binding fragment is administered at a dosage of about 50 mg / kg or less, and in certain of these embodiments, the dosage is 10 mg / kg or less, 5 mg / kg or less, 3 mg / kg or less, 1 mg / kg or less, 0.5 mg / kg or less, or 0.1 mg / kg or less. In certain embodiments, the dosage may vary over the course of treatment. For example, in certain embodiments, the initial dosage may be higher than subsequent dosages. In certain embodiments, the dosage may vary over the course of treatment depending on the subject's response.

[0378] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single dose may be administered or several divided doses may be administered over time.

[0379] The antibodies and antigen-binding fragments disclosed herein can be administered by any route known in the art, such as, for example, parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0380] In some embodiments, the antibodies or antigen-binding fragments disclosed herein can be administered alone or in combination with one or more additional therapeutic agents or drugs. In some embodiments, the antibodies or antigen-binding fragments disclosed herein can be administered alone or in combination with a second therapeutic agent. For example, the antibodies or antigen-binding fragments disclosed herein can be administered in combination with a second therapeutic agent, such as a chemotherapeutic agent or anticancer agent. In certain embodiments, the antibodies or antigen-binding fragments disclosed herein can be administered in combination with one or more immunosuppressive molecules, such as antagonists of CD47, SIRPα, PD-L1, or the beta-2 microglobulin subunit of major histocompatibility complex class I (B2M). The term "antagonist" as used herein can refer to any small molecule, small molecule, or microRNA, or antibody or antigen-binding fragment thereof, that blocks or inhibits the binding of CD47, SIRPα, PD-L1, or B2M to their respective binding partners to prevent the induction of immunosuppressive signals. In certain embodiments, the antibodies or antigen-binding fragments disclosed herein are administered in combination with a CD47 antagonist, such as a SIRPα-Fc fusion protein or variant thereof, an anti-CD47 antibody or antigen-binding fragment thereof, etc. In certain embodiments, the antibodies or antigen-binding fragments disclosed herein are administered in combination with a SIRPα antagonist, such as a soluble CD47 or variant thereof, an anti-SIRPα antibody or antigen-binding fragment thereof.

[0381] The term "anti-CD47 antibody" can refer to any known anti-CD47 antibody or antigen-binding fragment thereof, including, but not limited to, Hu5F9, disclosed in US9382320B2. Hu5F9-G4 is also used, which is an anti-human CD47 antibody that is humanized by grafting its complementarity-determining regions (CDRs) into a human IgG4 format to obtain the humanized 5F9 antibody (Hu5F9-G4) (Liu J. et al., PLoS One, 2015, 10:e0137345).

[0382] In certain of these embodiments, an antibody or antigen-binding fragment disclosed herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibody or antigen-binding fragment and the additional therapeutic agent(s) may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment that is administered "in combination with" another therapeutic agent need not be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment that is administered prior to or after another agent is considered to be administered "in combination with" that agent, as that term is used herein, even if the antibody or antigen-binding fragment and the second agent are administered by different routes. Where possible, additional therapeutic agents administered in combination with the antibodies or antigen-binding fragments disclosed herein will be administered according to the schedule listed in the package insert for the additional therapeutic agent, or according to protocols known in the art, such as the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed; Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002)).

[0383] In some embodiments, the present disclosure further provides a method for detecting the presence or amount of CD24 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof and determining the presence or amount of CD24 in the sample.

[0384] In some embodiments, the present disclosure further provides a method for diagnosing a CD24-associated disease or condition in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; c) determining the presence or amount of CD24 in the sample; and d) correlating the presence of CD24 with a CD24-associated disease or condition in the subject.

[0385] In some embodiments, the present disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD24-related disease or condition in a subject, or in the manufacture of a diagnostic reagent for diagnosing a CD24-related disease or condition.

[0386] In another aspect, the present disclosure also provides a method of modulating CD24 activity in a cell expressing CD24, the method comprising exposing the cell expressing CD24 to an antibody or antigen-binding fragment thereof provided herein.

[0387] In another aspect, the present disclosure also provides a method for detecting the presence or amount of CD24 in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of CD24 in the sample.

[0388] In another aspect, the present disclosure also provides a method of diagnosing a CD24-related disease or condition in a subject, the method comprising: a) obtaining a sample from the subject; b) contacting the sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; c) determining the presence or amount of CD24 in the sample; and d) correlating the presence or amount of CD24 with the presence or status of a CD24-related disease or condition in the subject.

[0389] In another aspect, the present disclosure also provides use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating a CD24-related disease or condition in a subject, the medicament further comprising a second therapeutic agent, e.g., a CD47 antagonist, optionally the CD47 antagonist is a SIRPα-Fc fusion protein or variant thereof, or an anti-CD47 antibody or antigen-binding fragment thereof.

[0390] In another aspect, the present disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a diagnostic reagent for diagnosing a CD24-associated disease or condition.

[0391] In another aspect, the present disclosure also provides kits comprising an antibody or antigen-binding fragment thereof provided herein useful in detecting CD24, optionally recombinant CD24, cell surface-expressed CD24, or cells expressing CD24. The term "recombinant," as used herein, refers to the artificial manipulation of one or more biological molecules, such as polynucleotide or polypeptide molecules, using one or more molecular biological techniques to render the biological molecule other than its native state.

[0392] In another aspect, the present disclosure also provides a method for stimulating a T cell-mediated immune response against cells or tissues expressing CD24 in a mammal, comprising administering to the mammal an effective amount of cells genetically modified to express a CAR comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CD24 and comprises an antigen-binding fragment provided herein. In certain embodiments, the antigen-binding fragment is a Fab or scFv. In certain embodiments, the CAR provided herein is bispecific. The CAR can further specifically bind to a second antigen other than CD24 or a second epitope on CD24. In certain embodiments, the second antigen is a tumor antigen as described above. The TCR signaling domain may be selected from the group consisting of intracellular signal region sequences of CD3ζ, FccRIγ, CD27, CD28, CD137, CD134, MyD88, CD40, CD278, TLR, or a combination thereof. The transmembrane region may include the transmembrane region of CD3, CD4, CD8, or CD28. In certain embodiments, the second antigen is an immunosuppressive molecule, such as PD-L1, SIRPα, CD47, or B2M.

[0393] In another aspect, the present disclosure also provides a method for treating a mammal with CD24-related disease or condition, comprising administering to the mammal an effective amount of cells that are genetically modified to express the CAR (e.g., autologous T cells) provided herein, thereby treating the mammal.In certain embodiments, the CD24-related disease or condition is cancer.In certain embodiments, the mammal is a human subject.

[0394] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below fall within the scope of the invention, in whole or in part. These specific compositions, materials, and methods do not limit the invention, but merely exemplify specific embodiments falling within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that numerous variations can be made in the procedures described herein while remaining within the scope of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. [Example]

[0395] [Example 1] Hybridoma Development 1. Method 1.1 Immunization and serum titration 1.1.1 Immunogens and Immunization Strategies Cellular immunization: cells overexpressing human CD24 (HEK293F-hCD24) Genetic immunization: full-length human CD24 expression construct Protein immunization: recombinant human CD24 protein Balb / c and SJL mice were immunized as shown in Table 2. Primary immunization was followed by several boosts until the animals developed sufficient antiserum titers suitable for hybridoma development. All immunization strategies were performed in parallel to compare performance and immune responses at the serum level.

[0396] [Table 2(2)]

[0397] 1.1.2 Immunization schedule

[0398] [Table 3]

[0399] [Table 4]

[0400] [Table 5]

[0401] 1.1.3 Test bleed antiserum analysis Screening: Test breeds were performed and evaluated by testing against the 293F cell line stably overexpressing human CD24 (293F-hCD24) and MCF7 cells using FACS.

[0402] 1.2 Hybridoma generation and screening 1.2.1 Cell fusion and screening Fusion: Splenocyte fusions were performed on mice that responded best to immunization, as determined by test breed FACS. Lymphocytes from the spleen and lymph nodes were fused to the Sp2 / 0-Ag14 cell line using an optimized electrofusion protocol. Multiple fusions were performed to ensure the success of the program.

[0403] Screening and expansion: Fusions were plated in stacks of 96-well plates (10 per well). 4 ~10 5 Plates were monitored for growth and fed weekly. Wells with cell growth were screened by the 10-14 day primary screening assay using Acumen and / or other viable assays. Multiple fusions were performed for each targeting antigen and screened by Acumen. Positive parental clones that showed positive binding to 293F-hCD24 from the primary screen were expanded to 24-well plates for secondary screening.

[0404] Additional antibody screening: Following the primary screen, positive parental clones expanded into 24-well plates were screened again by the assay described in the Hybridoma Screening Funnel below.

[0405] Hybridomas of interest were selected to proceed to subcloning.

[0406] 1.2.2 Subcloning, screening and cryopreservation of hybridomas Subcloning: Parental hybridomas with the desired reactivity and isotype from the above screening funnel were then subcloned by multiple rounds of limiting dilution or single cell sorting until monoclones were obtained.

[0407] Screening and Expansion: Subcloning plates were screened by the Acumen assay, and subclones with good binding ability were expanded to 24 wells for confirmation testing. The specificity and cross-reactivity of these subclones were confirmed by FAC analysis. Briefly, parental 293F cells, 293F-hCD24, breast cancer cells (MCF7, MDA-MB-468), bladder cancer cells (5637), chronic myeloid leukemia cells (Nalm1), CKOK1-hCD24 cells, colorectal cancer cells (HT29), ovarian cancer cells (SKOV3), and lung cancer cells (H1975 and A549) were incubated with the antibodies produced by each subclone. Binding of the primary antibody to the cells was detected using a fluorescent dye-conjugated secondary antibody. Median fluorescence intensity (MFI) was measured by FAC analysis.

[0408] Cryopreservation - Desired subclonal cell lines were sequenced and further expanded in culture flasks for cryopreservation. 0.5–1.0 × 10 7 Four to six vials per cell line were initially cryopreserved at 100 cells / vial. If necessary, a master cell bank and working cell bank could be established for selected, most valuable cell lines.

[0409] 2.Results We discovered a number of antibodies with unique sequences that showed positive binding to 293F cells stably overexpressing human CD24 protein (293F-hCD24). The FACS MFIs of the antibodies staining 293F cells, 293F-hCD24, breast cancer cells (MCF7, MDA-MB-468), bladder cancer cells (5637), chronic myeloid leukemia cells (Nalm1), CKOK1-hCD24 cells, colorectal cancer cells (HT29), ovarian cancer cells (SKOV3), and lung cancer cells (H1975 and A549) are summarized in Table 6 below. Two clones, 110D4G4 and 81A1F8, were also applied to stain DLBCL cells OCI-LY1, OCI-LY7, and SU-DHL-10 cells (Table 7).

[0410] [Table 6]

[0411] [Table 7]

[0412] Compared to SN3, several of our antibodies showed much higher MFI on the tested cells, and most of our antibodies showed stronger signals on CD24-low expressing MCF7 cells.

[0413] [Example 2] Antibody characterization: affinity 1. Method 1.1 Cell-based binding affinity to human CD24 Several antibody sequences from Table 1 were selected to generate and produce human IgG4 chimeric antibodies. The binding affinities of these antibodies and the benchmark antibody SN3 (see, e.g., T Fukukawa et al., Exp Hematol. 1986 Oct;14(9):850-5) to 293F-hCD24 cells, human breast cancer cells (MCF7, MDA-MB-468), bladder cancer cells (5637), chronic myeloid leukemia cells (Nalm1), CKOK1-hCD24 cells, colorectal cancer cells (HT29), ovarian cancer cells (SKOV3), and lung cancer cells (H1975 and A549) were determined by FAC analysis. The FAC analysis protocol is described as follows. a. Cells were digested using TrypLE™ Express Enzyme (1×). The collected cells were centrifuged at 400 g for 5 minutes and the supernatant was discarded. b. Cells were washed twice with cold FACS buffer by centrifugation at 400 g for 5 min and discarding the supernatant. c. Resuspend the cells and add 2*10 5 Cells / well were seeded onto assay plates in 50 μl of FACS buffer, followed by the addition of 50 μl of primary antibody (final primary antibody concentrations: 50.00, 16.67, 5.56, 1.85, 0.62, 0.21, 0.07, 0.02, 0.01, 0.00 μg / ml). Incubate at 40°C for 1 hour. d. Cells were washed twice using the conditions in step b. Cells were resuspended in 100 μl / well of diluted secondary antibody and incubated in the dark at 4° C. for 1 hour. e. Cells were washed twice using the conditions in step b. Cells were resuspended in 100 μl / well of cold FACS buffer. Cells were kept in the dark for FACS analysis.

[0414] 2.Results The binding affinity of the antibodies provided herein was tested for 293F-hCD24 cells and human breast cancer cell lines, including breast cancer cells (MCF7 and MDA-MB-468), bladder cancer cells (5637), chronic myeloid leukemia cells (Nalm1), acute lymphoblastic leukemia cells (Nalm6), CKOK1-hCD24 cells, colorectal cancer cells (HT29), ovarian cancer cells (SKOV3), lung cancer cells (NCI-H1975 and A549), B cell lymphoma cells (SU-DHL6 and DoHH2), human B cells, and hepatocellular carcinoma cells (Huh7). Some were comparable to the benchmark antibody SN3. However, our mAb-110D4G4 and mAb-81A7F8 performed much better than SN3 (Tables 8A-8B).

[0415] Compared with SN3, the maximum MFI (at 50 μg / ml primary antibody concentration) of some antibodies (110D4G4, 81A1F8) on CD24 low-expressing cells MCF7 and A549 was higher than that of SN3 (Tables 8 and 9).

[0416] Some of the present inventors' antibodies (110D4G4-hIgG4, 81A1F8-hIgG4) were more sensitive to the CD24-low expressing cells MCF7 and A549 than SN3 (Figure 1), and had a higher maximum MFI and higher or equivalent binding affinity (Tables 8A-8B, Figure 1).

[0417] [Table 8A]

[0418] [Table 8B]

[0419] [Table 9]

[0420] [Table 10]

[0421] Table 10 shows the binding affinity of the antibodies to 293F cells, 293F-hCD24, breast cancer cells (MCF7, MDA-MB-468), bladder cancer cells (5637), chronic myeloid leukemia cells (Nalm1), CKOK1 cells, CKOK1-hCD24 cells, colorectal cancer cells (HT29), ovarian cancer cells (SKOV3), and lung cancer cells (H1975 and A549). The prefix "ch-" indicates chimeric, hIgG1 represents the heavy chain constant region of the human IgG1 isotype, and hIgG4 represents the heavy chain constant region of the human IgG4 isotype.

[0422] Figures 1J–1R show the binding affinity of the antibodies to acute lymphoblastic leukemia cells (Nalm6), B-cell lymphoma cells (SU-DHL6 and DoHH2), human B cells, colorectal cancer cells (HT29), hepatocellular carcinoma cells (Huh7), breast cancer cells (MCF7), and lung cancer cells (NCI-H1975 and A549).

[0423] [Example 3] Antibody characterization: phagocytosis 1. Method Monocytes were isolated from PBMCs using the Easysep™ Human CD14 Positive Selection Kit II (StemCell, 17858). Monocyte-derived macrophages (MDMs) were induced by culturing monocytes for 7 days in RPMI 1640 medium supplemented with 10% FBS and 100 ng / mL rhM-CSF. Half of the medium was replaced and cytokines were added every 3–4 days. 100 ng / mL rhTGF-β and rhIL-10 were added and cultured for an additional 5 days. Half of the medium was replaced and cytokines were added every 3–4 days.

[0424] HT-29 cells (human colorectal adenocarcinoma cell line) were labeled with a fluorescence-enhancing ligand (CFSE, Invitrogen, C34554) according to the protocol (1 x 10^6 / ml cells were labeled with 2 μM CFSE and incubated for 20 minutes at 37°C in a cell incubator). 100 μL of 40,000 cells / well were seeded into a 96-well U-bottom sterile plate (Corning, cat:3894). Then, 50 μL of serially diluted antibodies listed in Table 6 were added to each well, and the plate was incubated for 2 hours at 37°C and 5% CO2. Meanwhile, MDM cells were harvested and resuspended to 1 x 10^6 / ml in RPMI 1640 medium (Gibco, Cat No. #11835-030) + 10% FBS. The above MDM cells were dissociated by trypsin and resuspended in complete 1640 medium at 10 6 The cells were resuspended at 40,000 cells / mL. 50 μL of the MDM (40,000 cells) described above was added to each well of the assay plate. After 2 hours of incubation at 37°C and 5% CO2, the cell mixture was washed, and the cells were stained with anti-CD11b-APC for 20 minutes at 4°C. The cells were then washed, and the samples were analyzed using a flow cytometer.

[0425] 2.Results 2.1 Anti-CD24 antibody could enhance the phagocytosis of HT-29 colorectal cancer cells. All of the antibodies of the present inventors enhanced the phagocytic effect on HT-29 cells. The antibodies of the present inventors showed a higher maximum phagocytic index (phagocytic index = (number of macrophages containing phagocytosed cells / total number of macrophages counted) × 100) on HT-29 cells, and four of them showed a lower EC50 compared to the benchmark antibody SN3 (Table 11, Figure 2). The phagocytosis assay was performed according to the method described above. The effector-to-target cell ratio (E:T) of macrophages and tumor cells (E / T ratio) was 1:1.

[0426] [Table 11]

[0427] Some of our chimeric antibodies also enhanced phagocytosis against hCD24-overexpressing HEK293F cells and the colorectal cancer (CRC) cell line HT-29. Some antibodies showed higher maximum phagocytic indexes against these cells, and others showed much stronger EC50 values ​​compared to the reference antibody SN3. The hIgG1 chimeric antibody induced ADCP, resulting in much better detectable phagocytic activity (Table 11, Figure 2).

[0428] 2.2 Combination of anti-CD24 and anti-CD47 antibodies Combinations of anti-CD47 and our anti-CD24 chimeric antibodies (chAb110D4G4, chAb111F3A2 and chAb81A1 F8) were tested. HT29 colorectal tumor cells were selected as target cells.

[0429] The chimeric antibodies chAb110D4G4, chAb111F3A2, and chA81A1F8 were applied in combination with different concentrations of anti-CD47 antibodies (hu5F9, hu5F9-hIgG4, or hu5F9-G4 are used interchangeably in Figure 3) to detect the overall phagocytic effect. The antibody concentration of the anti-CD24 chimeric antibody was set at 0.1 nM, and the hu5F9 concentrations were set at 0.0033 nM, 0.013 nM, 0.052 nM, and 0.208 nM. The combination of anti-CD24 and anti-CD47 improved the phagocytic index. Figure 3 shows that the combination of anti-CD24 and anti-CD47 antibodies induced significantly better phagocytic effects than anti-CD47 antibodies alone.

[0430] 2.3 Bispecific antibodies (IgG+scFv format) were investigated for phagocytosis assays. A bispecific antibody in IgG-scFv format was constructed using chAb81A1F8-IgG4 and Hu5F9-derived scFvs, each containing two heavy chains in the VH-CH1-hinge-CH2-CH3-spacer-scFv format, linked to a light chain in the VL-CL format. The affinity of this bispecific antibody was tested, and then a phagocytosis assay was also performed (see Figure 4). All antibody concentrations were set at 100 nM, sufficient for maximal phagocytosis. The E / T ratios were set at 1:5 and 1:1. The bispecific antibody showed improved phagocytosis compared to hu5F9, and at an E / T ratio of 1:5, the combination group showed nearly 100% phagocytosis. As shown in Figure 4, the bispecific antibody and the combination of an anti-CD24 antibody and an anti-CD47 antibody were tested by phagocytosis assay. The maximum index reached 85% when the E / T ratio was 1:1, as in the previous assay, and 98% when the E / T ratio was 1:5. The bispecific antibody showed excellent phagocytic ability.

[0431] [Example 4] Antibody humanization Humanization of 81A1F8 and 101H9G9A2 was performed using the following steps: 1) modeling the mouse antibody VH and VL domains, 2) alignment with a set of preferred human germline sequences, 3) evaluation of competition between the nonhuman CDRs and human FRs and designing back mutations to prevent loss of affinity in the final product, and 4) CDR grafting onto the preferred germline framework. Five different humanized sequences were generated, followed by cloning and small-scale production of all humanized variants and chimeras in mammalian expression systems.

[0432] [Example 5] Characterization of humanized antibodies: affinity Humanized variants 81A1F8-VH-11 / VL-21 (81A1F8-1121), 81A1F8-VH-21 / VL-21 (81A1F8-2121), 81A1F8-VH-11 / VL-41 (81A1F8-1141), and 81A1F8-VH-21 / VL-41 (81A1F8-2141) were tested in a cell-based binding assay using CD24-positive cells HT-29 and Nalm 6. Humanized variants of 101H9G9A2 were tested in a cell-based binding assay using CD24-positive cells HT-29.

[0433] Briefly, HT-29 or Nalm6 cells were washed three times with PBS and first incubated with the humanized variants. After three washes, the secondary antibody was incubated with the cells. The MFI of the samples was analyzed by flow cytometry, and cell-based affinity was calculated.

[0434] The binding affinities of the above-described humanized antibody of 81A1F8 to HT29 and Nalm6 were comparable to those of chimeric 81A1F8, as shown in Figures 5A and 5B, respectively.

[0435] The binding affinity of the above-mentioned humanized antibody 101H9G9A2 to HT29 is shown in FIG. 5C.

[0436] The protein-based KD of the 81A1F8 humanized antibody was tested using the Gatorprime system (hCD24-Fc protein as the antigen), and the KD data are shown in Figure 5D.

[0437] [Example 6] Glycosylated / sialylated epitopes of CD24 The epitope patterns recognized by the antibodies provided herein were examined, and some of the antibodies provided herein were found to recognize glycosylation- or sialylation-modified epitopes of CD24.

[0438] HT-29, Nalm6, HEK293T-hCD24, and MCF7-hCD24 cells were digested with neuraminidase or deglycosylation mix kits. Compared to the PBS control, 81A1F8 and 110D4G4 maintained their affinity for target cells and their MFI signals did not decrease. However, the MFIs of 81A7A10, 111F3A2, 100F2E3, 101H9G9A2, and 107D10D11 were significantly reduced after treatment of target cells with neuraminidase or deglycosylation mix, as shown in Figures 6A and 6B. The benchmark antibody SN3 also recognized a previously reported glycosylated epitope. As shown in Figures 6C and 6D, 101H9G9A2 bound to HT-29 cells with high affinity but failed to bind to 293T-hCD24 cells at all. 107D10D11 bound to 293T-hCD24 with low affinity, whereas SN3 bound normally to 293T-hCD24 and HT-29.

[0439] [Example 7] Affinity testing for anti-CD24 mAb and humanized antibody by SPR The affinity of antibodies for mFc-tagged human CD24 (hCD24-mFc-biotin) was determined using a BIAcore 8K (GE Healthcare). 0.8 or 2.5 μg / ml of hCD24-mFc-biotin was immobilized on a Series S Sensor Chip SA for 120 seconds at a flow rate of 10 μl / min, achieving an immobilization level of approximately 1200 RU. Antibodies were injected at room temperature in a concentration gradient (1.56–50 nM) at a flow rate of 30 μl / min. The contact time was set to 180 seconds, and the dissociation time was 400 seconds. At the end of each cycle, 10 mM glycine pH 1.5 was injected to remove the tested antibodies from the surface. Finally, binding kinetics were calculated using BIAcore Insight Evaluation Software, and a 1:1 binding model was used for curve fitting. The results of SPR analysis for several anti-CD24 mAbs provided herein are shown in Figure 7A. The results of SPR analysis of the humanized 81A1F8 candidate are shown in Figure 7B.

[0440] [Example 8] ELISA-based receptor binding assay for 81A1F8 1. Method Human CD24-mFc protein was coated onto the plate. Biotinylated human SigLec10 protein (SIG-HM510B, Kactus) was mixed with different concentrations of 81A1F8 and then added to the plate. The binding buffer we used was 1% Triton-X, 0.3 mM MnCl2, 1 mM CaCl2, 25 mM Tris, 150 mM NaCl, 1 mM MgCl2, pH 7.6. The samples were washed three times with 1x PBS buffer. The sample signals were detected and normalized to the original SigLec-10 readings.

[0441] 2.Results The results of the ELISA-based receptor binding assay for different concentrations of 81A1F8 are shown in FIG.

[0442] [Example 9] In vitro efficacy assays for 81A1F8 and its humanized variants 1. Phagocytosis Assay Human monocytes were isolated and derived as described in Example 3. A and B: Phagocytosis of target tumor cells (HT-29 and Nalm6) was performed using anti-CD24 mAb (hIgG4 Fc) and benchmark antibody SWA11 and anti-CD47 mAb 5F9-IgG4. The results are shown in Figure 9A and Figure 9B.

[0443] C: We investigated whether 81A1F8-LALAPG, which has rare Fc-dependent functions, could enhance the ADCP effect of rituximab by combining it with rituximab. We found that an additive effect was detectable (see Figure 9C).

[0444] D: The 81A1F8-hIgG4 chimeric antibody also enhanced the phagocytic effect of the anti-CD47 antibody hu5F9 when administered in combination (see Figure 9D).

[0445] 2. ADCC and CDC Assays A: The ADCC effect of 81A1F8 and its humanized variants was tested against target cells HT-29. HT-29 cells were incubated with PBMC. Different concentrations of 81A1F8 and humanized variants 81A1F8-1141 and 81A1F8-2141 were added to the samples. Target cell lysis was tested via LDH kit. As shown in Figure 10A, the ADCC effect can only be induced by hIgG1 isotype antibodies.

[0446] B: The CDC effect of 81A1F8 and its humanized variants was tested on target cells hCD24-MCF7. Different concentrations of the antibody were incubated with the target cells and the complement system. As shown in Figure 10B, more than 90% lysis of target cells could be detected at high concentrations of the 81A1F8 antibody variant.

[0447] [Example 10] In vivo efficacy assay of anti-CD24 antibodies The in vivo efficacy of 81A1F8 was tested in a C57BL / 6J syngeneic mouse model. Briefly, one million MC38-hCD24 cells were implanted subcutaneously into 6-8 week old female C57BL / 6J mice. The mean tumor volume was approximately 60 mm. 3 When tumor size reached 100 mg / kg / day, mice were randomly grouped and treated as follows: hIgG4 isotype antibodies were tested as single agents (10 mpk, Q3D) or in combination with oxaliplatin (6 mpk, QW). Tumor volume and mouse weights were measured twice weekly. The in vivo efficacy of 81A1F8 is shown in Figures 11A-11D.

[0448] All treatment groups showed tumor growth inhibition (see Figure 11A). When combined with oxaliplatin, 81A1F8-hIgG4 showed much better efficacy, and a synergistic effect could be observed. All mice did not lose weight (see Figure 11B). An overview of the in vivo efficacy study is shown in Figure 11C. Tumor-infiltrating lymphocytes were isolated from tumor tissues and analyzed by flow cytometry. Figure 11D showed that treatment with the combination of 81A1F8-hIgG4 and oxaliplatin significantly increased the CD8 / CD4 ratio and the M1 / M2 macrophage ratio compared to the control (CTRL).

[0449] The in vivo efficacy of 81A1F8-mIgG2a (a homolog of human IgG1) was also evaluated in the MC38-hCD24 syngeneic model. When performing a dosage-efficacy study, we found that a dosage of 6mpk showed the best efficacy, as four out of six mice were tumor-free after administration of 81A1F8-mIgG2a (see Figure 12A).

[0450] When rechallenge assays were performed by re-inoculating MC38-Hcd24 cells, tumors were able to grow, but tumors did not recur in the treated group, indicating the formation of immune memory after 81A1F8 treatment (see Figure 12B).

[0451] FIG. 12C shows the tumor growth curves for each mouse treated with vehicle control or 81A1F8-mIgG2a.

[0452] Figure 12D provides an overview of the in vivo efficacy of 81A1F8-mIgG2a (also referred to as "ATG031-mAb011-mIgG2a").

[0453] The in vivo efficacy of humanized 81A1F8 (81A1F8-2141-mIgG2a) was also tested. Briefly, MC38-hCD24 tumor cells were inoculated. The mean tumor volume was approximately 120 mm. 3Once the tumor size reached 100 μg / mL, mice were divided into groups and treated with single agents (81A1F8-2141-mIgG2a, oxaliplatin, or atezolizumab) or combination strategies. The tumor-inhibitory effects of the tested humanized 81A1F8 (81A1F8-2141-mIgG2a) are shown in Figures 13A-13C. Figure 13A shows the tumor growth curves after drug administration, Figure 13B shows the tumor growth curves for each mouse in the treatment group, and Figure 13C shows an overview of the in vivo efficacy of 81A1F8-2141-mIgG2a.

[0454] An in vivo efficacy assay for 101H9G9A2-mIgG2a was also tested. Briefly, MC38-hCD24 tumor cells were inoculated as described above. The mean tumor volume was approximately 120 mm. 3 Once the tumor size reached 101H9G9A2-mIgG2a, 81A1F8-2141-mIgG2a, oxaliplatin, and atezolizumab, or a combination thereof, were administered to the mice. The tumor-inhibitory effects of the tested 101H9G9A2-mIgG2a and humanized 81A1F8 (81A1F8-2141-mIgG2a) are shown in Figures 14A-14C. Figure 14A shows the tumor volume curves of mice grouped as indicated, Figure 14B shows the body weights of the mice, and Figure 14C shows a summary of the efficacy of the different treatments.

[0455] [Example 11] Discovery of cynomolgus monkey (Macaca fascicularis) CD24 (cmCD24) antibody Balb / c and SJL mice were immunized with 293F cells overexpressing cmCD24-Flag, and two antibodies that recognized cynomolgus CD24 were identified. Cell-based binding data are shown in Figure 15.

[0456] In vitro efficacy studies of anti-cmCD24 antibodies were conducted, and the results are shown in Figures 16A-16C, where Figure 16A shows results from a phagocytosis assay of anti-cmCD24 antibodies provided herein, Figure 16B shows the ADCC effect of anti-cmCD24 antibodies provided herein, and Figure 16C shows the CDC effect of anti-cmCD24 antibodies provided herein.

Claims

1. a) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; b) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO:11, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:8, SEQ ID NO:10 and SEQ ID NO:12; c) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:7, SEQ ID NO:14 and SEQ ID NO:11, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:13, SEQ ID NO:10 and SEQ ID NO:15; d) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 10 and SEQ ID NO: 15; e) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 20, SEQ ID NO: 22 and SEQ ID NO: 24; f) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:28, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:26, SEQ ID NO:22 and SEQ ID NO:24; g) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:25, SEQ ID NO:30 and SEQ ID NO:28, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:29, SEQ ID NO:22 and SEQ ID NO:31; h) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36, and a light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 37; i) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:38, SEQ ID NO:40 and SEQ ID NO:42, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:43; j) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 48, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 49; k) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:95, SEQ ID NO:96 and SEQ ID NO:97, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO:100; l) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 37; m) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 13, SEQ ID NO: 116 and SEQ ID NO: 117; n) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 126, SEQ ID NO: 127 and SEQ ID NO: 128, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 129, SEQ ID NO: 130 and SEQ ID NO: 131; o) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:25, SEQ ID NO:136 and SEQ ID NO:137, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:138, SEQ ID NO:139 and SEQ ID NO:140; p) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:25, SEQ ID NO:30 and SEQ ID NO:28, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:29, SEQ ID NO:145 and SEQ ID NO:24; q) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 150, SEQ ID NO: 151 and SEQ ID NO: 152, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 153, SEQ ID NO: 154 and SEQ ID NO: 155; r) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 162, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 163, SEQ ID NO: 154 and SEQ ID NO: 155; s) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 168, SEQ ID NO: 169 and SEQ ID NO: 170, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 171, SEQ ID NO: 172 and SEQ ID NO: 173; t) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 184, SEQ ID NO: 108 and SEQ ID NO: 37; u) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 185, SEQ ID NO: 108 and SEQ ID NO: 37; v) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 186, SEQ ID NO: 108 and SEQ ID NO: 37; w) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 187, SEQ ID NO: 108 and SEQ ID NO: 37; x) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106 and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO: 188, SEQ ID NO: 108 and SEQ ID NO: 37; or y) a heavy chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:222, SEQ ID NO:223 and SEQ ID NO:224, and a kappa light chain variable region comprising one, two or three CDR sequences selected from SEQ ID NO:225, SEQ ID NO:226 and SEQ ID NO:

227.

2. An isolated antibody or antigen-binding fragment thereof comprising:

2. a) a heavy chain variable region comprising SEQ ID NO:50 and a light chain variable region comprising SEQ ID NO:52; b) a heavy chain variable region comprising SEQ ID NO:54 and a light chain variable region comprising SEQ ID NO:56; c) a heavy chain variable region comprising SEQ ID NO:58 and a light chain variable region comprising SEQ ID NO:64; d) a heavy chain variable region comprising SEQ ID NO:62 and a light chain variable region comprising SEQ ID NO:64; e) a heavy chain variable region comprising SEQ ID NO:66 and a light chain variable region comprising SEQ ID NO:68; f) a heavy chain variable region comprising SEQ ID NO: 70 and a light chain variable region comprising SEQ ID NO: 72; g) a heavy chain variable region comprising SEQ ID NO: 74 and a light chain variable region comprising SEQ ID NO: 76; h) a heavy chain variable region comprising SEQ ID NO: 78 and a light chain variable region comprising SEQ ID NO: 80; i) a heavy chain variable region comprising SEQ ID NO: 82 and a light chain variable region comprising SEQ ID NO: 84; j) a heavy chain variable region comprising SEQ ID NO: 86 and a light chain variable region comprising SEQ ID NO: 88; k) a heavy chain variable region comprising SEQ ID NO: 101 and a light chain variable region comprising SEQ ID NO: 103; l) a heavy chain variable region comprising SEQ ID NO: 109 and a light chain variable region comprising SEQ ID NO: 111; m) a heavy chain variable region comprising SEQ ID NO: 118 and a light chain variable region comprising SEQ ID NO: 120 or SEQ ID NO: 221; n) a heavy chain variable region comprising SEQ ID NO: 122 and a light chain variable region comprising SEQ ID NO: 124; o) a heavy chain variable region comprising SEQ ID NO: 132 and a light chain variable region comprising SEQ ID NO: 134; p) a heavy chain variable region comprising SEQ ID NO: 141 and a light chain variable region comprising SEQ ID NO: 143; q) a heavy chain variable region comprising SEQ ID NO: 146 and a light chain variable region comprising SEQ ID NO: 148; r) a heavy chain variable region comprising SEQ ID NO: 156 and a light chain variable region comprising SEQ ID NO: 158; s) a heavy chain variable region comprising SEQ ID NO: 164 and a light chain variable region comprising SEQ ID NO: 166; t) a heavy chain variable region comprising SEQ ID NO: 174 and a light chain variable region comprising SEQ ID NO: 176; u) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 180; v) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 180; w) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 180; x) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 181; y) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 181; z) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 181; aa) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 182; bb) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 182; cc) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 182; dd) a heavy chain variable region comprising SEQ ID NO: 177 and a light chain variable region comprising SEQ ID NO: 183; ee) a heavy chain variable region comprising SEQ ID NO: 178 and a light chain variable region comprising SEQ ID NO: 183; ff) a heavy chain variable region comprising SEQ ID NO: 179 and a light chain variable region comprising SEQ ID NO: 183; gg) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 195; hh) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 196; ii) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 197; jj) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 198; kk) a heavy chain variable region comprising SEQ ID NO: 189 and a light chain variable region comprising SEQ ID NO: 199; ll) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 200; mm) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 201; nn) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 202; oo) a heavy chain variable region comprising SEQ ID NO: 190 and a light chain variable region comprising SEQ ID NO: 203; pp) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 200; qq) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 201; rr) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 202; ss) a heavy chain variable region comprising SEQ ID NO: 191 and a light chain variable region comprising SEQ ID NO: 203; tt) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 200; uu) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 201; vv) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 202; w) a heavy chain variable region comprising SEQ ID NO: 192 and a light chain variable region comprising SEQ ID NO: 203; xx) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 200; yy) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 201; zz) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 202; aaa) a heavy chain variable region comprising SEQ ID NO: 193 and a light chain variable region comprising SEQ ID NO: 203; bbb) a heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 200; ccc) a heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 201; ddd) a heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 202; or eee) A heavy chain variable region comprising SEQ ID NO: 194 and a light chain variable region comprising SEQ ID NO: 203 The antibody or antigen-binding fragment thereof of claim 1 .

3. The antibody or antigen-binding fragment thereof of claim 1 which is bispecific.

4. The antibody or antigen-binding fragment thereof according to claim 3, which is capable of specifically binding to a first and a second epitope of CD24, or is capable of specifically binding to CD24 and a second antigen.

5. The antibody or antigen-binding fragment thereof according to claim 4, which binds to CD24 via the Fab and binds to a second antigen via the scFv.

6. The antibody or antigen-binding fragment thereof of claim 5 , wherein the second antigen-binding scFv is operably linked to the C-terminus of the heavy chain constant region following the CD24-binding Fab.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the bispecific antibody molecule comprises a heavy chain in the format VH(anti-CD24)-CH1-hinge-CH2-CH3-spacer-second antigen-binding scFv associated with a light chain in the format VL(anti-CD24)-CL.

8. The second antigen is optionally PD-L1, PD-L2, PD-1, CLTA-4, TIM-3, LAG3, CD160, 2B4, TGF 5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody or antigen-binding fragment thereof is an immune related target selected from the group consisting of: β, VISTA, BTLA, TIGIT, LAIR1, OX40, CD2, CD27, ICAM-1, NKG2C, SLAMF7, NKp80, CD160, B7-H3, LFA-1, ICOS, 4-1BB, GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, IL-2, IL-15, CD3, CD16, SIRPα, Siglec10, LILRB2, Clever-1, Macro, LILRB4, Siglec15, CSF1R, PSGL-1, VSIG-4, B2M and CD83.

9. The second antigen is CA-125, ganglioside G(D2), G(M2) and G(D3), CD19, CD20, CD33, CD47, CD52, Ep-CAM, CEA, CLDN18.2, bombesin-like peptide, PSA, HER2 / neu, epidermal growth factor receptor (EGFR), erbB2, erbB3 / HER3, erbB4, CD44v6, CD44v9, Ki-67, cancer-associated mucin, VEGF, VEGFR (e.g., VEGFR3 6. The antibody or antigen-binding fragment thereof of claim 5, comprising an estrogen receptor, Lewis-Y antigen, TGFβ1, IGF-1 receptor, EGFα, c-Kit receptor, transferrin receptor, IL-2R, CDH6, CEA, FOLR1, TROP2, PTK7, SLITRK6, CD142, NECTIN-4, ROR1, ROR2, CD142, CD123, CD22, CD79b, DLL3, SLC family, or CO17-1A.

10. 10. The antibody or antigen-binding fragment thereof of claim 9, comprising a heavy chain in the format VH(anti-CD24)-CH1-hinge-CH2-CH3-spacer-scFv(anti-CD47) associated with a light chain in the format VL(anti-CD24)-CL.

11. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment is linked to one or more conjugates, and optionally the conjugates are covalently bonded either directly or via a linker.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in claim 1 and a pharma- ceutically acceptable carrier.

13. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1.

14. A vector comprising the isolated polynucleotide of claim 13.

15. A host cell comprising the vector of claim 14.

16. The pharmaceutical composition of claim 12, used for a method of treating a disease or condition in a subject that would benefit from modulation of CD24 activity, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in claim 1.

17. A method for detecting the presence or amount of CD24 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in claim 1 and determining the presence or amount of CD24 in the sample.

18. 2. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, a costimulatory signaling region and a TCR signaling domain, wherein the antigen-binding domain specifically binds to CD24, and comprises the antigen-binding fragment of claim 1.

19. 1. A pharmaceutical composition for use in a method of treating cancer in a subject in need thereof, comprising: an anti-CD24 antibody or antigen-binding fragment thereof that binds only to glycosylated or sialylated CD24, preferably only to the sialic acid of CD24, and a pharma- ceutical acceptable carrier; The method comprises administering to the subject a therapeutically effective amount of the anti-CD24 antibody or antigen-binding fragment thereof.

20. 20. The pharmaceutical composition of claim 19, wherein the anti-CD24 antibody or antigen-binding fragment thereof competes for the same epitope with an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, respectively, and wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 107, 184, 185, 186, 187 and 188, LCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 37, or competes for the same epitope with an antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, respectively.

21. 21. The pharmaceutical composition of claim 20, wherein the anti-CD24 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 107, 184, 185, 186, 187 and 188, LCDR2 comprises the amino acid sequence of SEQ ID NO: 108, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 37, or comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 95, SEQ ID NO: 96 and SEQ ID NO: 97, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, respectively.

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