WO2024088342A1 - Anticorps dirigés contre cd24 et leurs utilisations - Google Patents

Anticorps dirigés contre cd24 et leurs utilisations Download PDF

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Publication number
WO2024088342A1
WO2024088342A1 PCT/CN2023/126795 CN2023126795W WO2024088342A1 WO 2024088342 A1 WO2024088342 A1 WO 2024088342A1 CN 2023126795 W CN2023126795 W CN 2023126795W WO 2024088342 A1 WO2024088342 A1 WO 2024088342A1
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seq
set forth
amino acid
acid sequence
antibody
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PCT/CN2023/126795
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English (en)
Inventor
Junli Liu
Puwei YUAN
Ying Lei
Mingchen CHEN
Bingshi GUO
Fan Liu
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Beijing Neox Biotech Limited
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Publication of WO2024088342A1 publication Critical patent/WO2024088342A1/fr

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Definitions

  • CD24 is a highly glycosylated protein with a small protein core that is linked to the plasma membrane via a glycosyl-phosphatidylinositol anchor. CD24 is primarily expressed by immune cells but is often overexpressed in human tumors. In cancer, CD24 is a regulator of cell migration, invasion and proliferation. Thus, CD24 makes an attractive target for monocolonal antibodies as a cancer therapy.
  • Described herein are antibodies that specifically bind to CD24.
  • the antibodies described herein possess the useful properties of not binding to either B cells or activated T cells. Such antibodies may possess an enhanced safety profile with reduced immune side effects.
  • an antibody, or antigen binding fragment thereof, that binds to CD24 wherein the antibody does not bind to T lymphocytes. In certain embodiments, the antibody does not bind to activated T lymphocytes. In certain embodiments, the antibody does not bind to B lymphocytes. In certain embodiments, the antibody binds to CD24 with a K D of less than 1x10 -8 . In certain embodiments, the antibody binds to CD24 with a K D of less than 1x10 -9 . In certain embodiments, the antibody is a humanized or chimeric antibody. In certain embodiments, the antibody is a Fab, F (ab) 2 , or a single chain variable fragment (scFv) .
  • the antibody is a heavy chain antibody or antigen binding fragment thereof.
  • the antibody is an IgG antibody.
  • the antibody is an IgG 1 isotype.
  • the antibody is an IgG 4 isotype.
  • the antibody binds to the serine at residue 18 of SEQ ID NO: 500.
  • the antibody requires the presence of a serine at residue 18 of SEQ ID NO: 500 for binding.
  • the antibody binds to an epitope consisting of the amino acid sequence of SEQ ID NO 501.
  • an antibody or antigen binding fragment thereof that binds to CD24, wherein the antibody or antigen binding fragment thereof comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 401; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 405; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 407; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 409; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 411.
  • LCDR1 light chain complementarity determining region
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 401; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 405; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 407; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 409; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 411.
  • LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 401
  • LCDR2 comprising an amino acid sequence set forth in S
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 402; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 406; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 408; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 409; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 412.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 403
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 402; (b) a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 406; (d) a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 408; (e) a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 409; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 412.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 light chain complementarity determining region 2
  • LCDR3 light chain complementarity determining region 3
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) as set forth in SEQ ID NO: 402; (b) a light chain complementarity determining region 2 (LCDR2) as set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) as set forth in SEQ ID NO: 406; (d) a heavy chain complementarity determining region 1 (HCDR1) as set forth in SEQ ID NO: 408; (e) a heavy chain complementarity determining region 2 (HCDR2) as set forth in SEQ ID NO: 409; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) as set forth in SEQ ID NO: 412; and wherein the antibody does not bind to, or does not substantially bind to either B cells or activated T cells.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 light chain complementarity determining region 2
  • LCDR3 light chain complementar
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 83, 703, 713, 723, 733, and 743; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 704, 714, 724, 734, and 744; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 25, 35, 45, 55, 65, 75, 85, 705, 715, 725, 735, and 745; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 26, 36, 46, 56, 66,
  • the antibody comprises a combination of LCDR1, LCDR2, and LCDR3 and HCDR1, HCDR2, and HCDR3, wherein the combination is selected from the group consisting of: (i) LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 14, LCDR3 comprising SEQ ID NO: 15, HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, HCDR3 comprising SEQ ID NO: 18; (ii) LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ ID NO: 24, LCDR3 comprising SEQ ID NO: 25, HCDR1 comprising SEQ ID NO: 26, HCDR2 comprising SEQ ID NO: 27, HCDR3 comprising SEQ ID NO: 28; (iii) LCDR1 comprising SEQ ID NO: 33, LCDR2 comprising SEQ ID NO: 34, LCDR3 comprising SEQ ID NO: 35, HCDR1 comprising SEQ ID NO: 36, HCDR2 comprising
  • the antibody comprises a combination of LCDR1, LCDR2, and LCDR3 and HCDR1, HCDR2, and HCDR3, wherein the combination is selected from the group consisting of: (i) LCDR1 as set forth in SEQ ID NO: 13, LCDR2 as set forth in SEQ ID NO: 14, LCDR3 as set forth in SEQ ID NO: 15, HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18; (ii) LCDR1 as set forth in SEQ ID NO: 23, LCDR2 as set forth in SEQ ID NO: 24, LCDR3 as set forth in SEQ ID NO: 25, HCDR1 as set forth in SEQ ID NO: 26, HCDR2 as set forth in SEQ ID NO: 27, HCDR3 as set forth in SEQ ID NO: 28; (iii) LCDR1 as set forth in SEQ ID NO: 33, LCDR2 as set forth in SEQ ID NO: 34, LCDR
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 13; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 14; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 15; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 16; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 17; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 18.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 14
  • LCDR3 a
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 23; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 24; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 25; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 26; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 27; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 28.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 24
  • LCDR3
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 33; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 34; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 35; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 36; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 37; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 38.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 34
  • LCDR3
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 43; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 44; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 45; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 46; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 47; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 48.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 44
  • LCDR3 compris
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 53; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 54; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 55; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 56; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 57; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 58.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 54
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 63; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 64; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 65; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 66; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 67; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 68.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 64
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 73; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 74; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 75; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 76; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 77; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 78.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 74
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 83; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 84; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 85; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 86; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 87; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 88.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 84
  • the antibody comprises an immunoglobulin light chain amino acid variable region sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.
  • the antibody comprises an immunoglobulin light chain amino acid variable region sequence identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy1 chain variable region amino acid sequence identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to any one of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined using a hybrid scheme of any two or three of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Kabat definition.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Chothia definition.
  • VL light chain variable region
  • LCDR2 LCDR2
  • LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319
  • VH heavy chain variable region
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 275; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 276.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 275; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 276.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 277; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 278.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 277; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 278.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 309; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 310.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 309; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 310.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 155; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 156.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 155; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 156.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 157; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 158.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 157; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 158.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 169; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 170.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 169; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 170.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 267; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 268.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 267; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 268.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 271; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 272.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 271; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 272.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of the immunoglobulin light chain variable region sequences of Table 1; and an immunoglobulin heavy chain amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of the immunoglobulin heavy chain variable region sequences of Table 1.
  • the antibody comprises an immunoglobulin light chain amino acid sequence identical to any one of the immunoglobulin light chain variable region sequences of Table 1; and an immunoglobulin heavy chain variable region amino acid sequences identical to any one of the immunoglobulin heavy chain variable region sequences of Table 1.
  • in the antibody comprises an arginine substitution at position number 17 of the immunoglobulin light chain according to Kabat numbering. In certain embodiments, wherein in the antibody comprises a tyrosine substitution at position number 12 of the immunoglobulin light chain according to Kabat numbering.
  • described herein is a pharmaceutical composition comprising an antibody of this disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. In certain embodiments, the antibody or pharmaceutical composition is formulated for intravenous administration. Also described is a nucleic acid encoding an antibody of this disclosure. In certain embodiments, described herein is an expression vector comprising a nucleic acid of this disclosure. Also described herein is a eukaryotic cell comprising the expression vector.
  • the eukaryotic cell is a CHO cell line. Also described herein is a method of treating a cancer or tumor comprising administering to an individual afflicted with the cancer or tumor an antibody or a pharmaceutical composition comprising an antibody of the disclosure.
  • the cancer or tumor is a solid tissue cancer.
  • the cancer or tumor comprises breast cancer, colorectal cancer, or ovarian cancer.
  • the breast cancer is triple negative breast cancer.
  • the cancer or tumor is a blood cancer.
  • a method of inhibiting CD24 signaling in a cancer cell comprising contacting the cancer cell to an antibody of the disclosure or a pharmaceutical composition of the disclosure.
  • CD24 signaling is not inhibited in non-neoplastic B lymphocytes or non-neoplastic T lymphocytes.
  • the cancer cell is a cancer cell is in vivo in an individual.
  • FIG. 1A and 1B illustrate dose response curves of maturated CD24 antibodies (single mutation) against CD24 (27-56aa) -GST (NEOX-4) by ELISA.
  • FIG. 2A and 2B illustrate dose response curve of maturated CD24 antibodies (combined mutations) against CD24 (27-56aa) -GST (NEOX-4) by ELISA.
  • FIG. 3A and 3B illustrate dose response curves of CD24 antibodies on NALM6 cells.
  • FIG. 4A and 4B illustrate dose response curves of CD24 antibodies on MDA-MB-468 cells.
  • FIG. 5A, 5B, and 5C illustrate the ADCC effect induced by anti-CD24 antibody on NALM6 and MDA-MB-468 cells.
  • FIG. 6A, 6B, 6C, and 6D illustrate the ADCP effect induced by anti-CD24 antibody on NALM6 and MDA-MB-468 cells.
  • FIG. 7A, 7B, 7C and 7D illustrate dose response a binding curve with primary B cells and activated T cells.
  • FIG. 8 illustrates antibody binding by ELISA with N-Glycanase, Neuraminidase or N-Glycanase + Neuraminidase treated NEOX-4.
  • FIG. 9A, 9B, and 9C illustrate CD24 antibody binding with N-Glycanase, Neuraminidase or N-Glycanase + Neuraminidase treated NALM6 (9A) , MDA-MB-468 (9B) , and primary B cells (9C) .
  • FIG. 10 illustrates epitope binning by blocking peptides.
  • FIG. 11 illustrates epitope determination by alanine spanning mutagenesis.
  • FIG. 12 illustrates an epitope comparison from binning studies.
  • FIG. 13 illustrates CD24 antibody in vivo efficacy in the MC38-hCD24 syngeneic model.
  • FIG. 14A and 14B illustrates CD24 antibody in vivo efficacy in the NALM6 CDX model.
  • FIG. 15A and 15B illustrates CD24 antibody in vivo efficacy in the MDA-MB-468 CDX model.
  • Described herein in one aspect is an antibody, or antigen binding fragment thereof, that binds to CD24, wherein the antibody does not bind to T lymphocytes. In certain embodiments, the antibody does not bind to activated T lymphocytes. In certain embodiments, the antibody does not bind to B lymphocytes.
  • the term “individual, ” “patient, ” or “subject” refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease for which the described compositions and method are useful for treating.
  • the individual is a mammal.
  • the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak.
  • the individual is a human.
  • an antibody includes, but is not limited to, full-length and native antibodies, as well as fragments and portion thereof retaining the binding specificities thereof, such as any specific binding portion thereof including those having any number of, immunoglobulin classes and/or isotypes (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM) ; and biologically relevant (antigen-binding) fragments or specific binding portions thereof, including but not limited to Fab, F (ab’) 2 , Fv, and scFv (single chain or related entity) .
  • immunoglobulin classes and/or isotypes e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and IgM
  • biologically relevant (antigen-binding) fragments or specific binding portions thereof including but not limited to Fab, F (ab’) 2 , Fv
  • a monoclonal antibody is generally one within a composition of substantially homogeneous antibodies; thus, any individual antibodies comprised within the monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • the monoclonal antibody can comprise a human IgG1 constant region.
  • the monoclonal antibody can comprise a human IgG4 constant region.
  • the human constant region can comprise natural human immunoglobulin constant regions or variants thereof further comprising common modifications that confer altered effector functions, extended half-life or other properties. Such modifications include D356E, L358M, L234A, L235A, P329G, M252Y, S254T, and/or T256E per EU numbering.
  • antibody herein is used in the broadest sense and includes monoclonal antibodies, and includes intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F (ab') 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments (e.g., heavy chain antibodies) , including single chain variable fragments (sFv or scFv) , and single domain antibodies (e.g., sdAb, sdFv, nanobody, VHH) or fragments thereof.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antibody can comprise a human IgG1 constant region.
  • the antibody can comprise a human IgG4 constant region.
  • Suitable antibodies e.g., recombinant, monoclonal, or polyclonal antibodies
  • many techniques known in the art can be used (see, e.g., Kohler & Milstein, Nature 256: 495-497 (1975) ; Kozbor et al., Immunology Today 4: 72 (1983) ; Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985) ; Coligan, Current Protocols in Immunology (1991) ; Harlow & Lane, Antibodies, A Laboratory Manual (1988) ; and Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986) ) .
  • the genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, e.g., the genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce a recombinant monoclonal antibody.
  • Gene libraries encoding heavy and light chains of monoclonal antibodies can also be made from hybridoma or plasma cells. Random combinations of the heavy and light chain gene products generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997) ) . Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. Nos.
  • transgenic mice or other organisms such as other mammals, may be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos.
  • phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g., McCafferty et al., Nature 348: 552-554 (1990) ; Marks et al., Biotechnology 10: 779-783 (1992) ) .
  • Antibodies can also be made bispecific, i.e., able to recognize two different antigens (see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10: 3655-3659 (1991) ; and Suresh et al., Methods in Enzymology 121: 210 (1986) ) .
  • Antibodies can also be heteroconjugates, e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089) .
  • linker herein is also referred to as “linker sequence” “spacer” “tethering sequence” or grammatical equivalents thereof.
  • a “linker” as referred herein connects two distinct molecules that by themselves possess target binding, catalytic activity, or are naturally expressed and assembled as separate polypeptides, or comprise separate domains of the same polypeptide. For example, two distinct binding moieties or a heavy-chain/light-chain pair. A number of strategies may be used to covalently link molecules together.
  • Linkers described herein may be utilized to join a light chain variable region and a heavy chain variable region in an scFv molecule; or may be used to tether an scFv or other antigen binding fragment on the N-or C-terminus of an antibody heavy chain; or the N-or C-terminus of a light chain to create a bispecific or multispecific binding molecule.
  • These include but are not limited to polypeptide linkages between N-and C-termini of proteins or protein domains, linkage via disulfide bonds, and linkage via chemical cross-linking reagents.
  • the linker is a peptide bond, generated by recombinant techniques or peptide synthesis.
  • the linker peptide may predominantly include the following amino acid residues: Gly, Ser, Ala, or Thr.
  • the linker peptide should have a length that is adequate to link two molecules in such a way that they assume the correct conformation relative to one another so that they retain the desired activity.
  • the linker is from about 1 to 50 amino acids in length or about 1 to 30 amino acids in length. In one embodiment, linkers of 1 to 20 amino acids in length may be used.
  • Useful linkers include glycine-serine polymers, including for example (GS) n, (GSGGS) n, (GGGGS) n, and (GGGS) n, where n is an integer of at least one, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
  • linkers for linking antibody fragments or single chain variable fragments can include AAEPKSS, AAEPKSSDKTHTCPPCP, GGGG, or GGGGDKTHTCPPCP.
  • non-proteinaceous polymers including but not limited to polyethylene glycol (PEG) , polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, may find use as linkers use as linkers.
  • PEG polyethylene glycol
  • polypropylene glycol polypropylene glycol
  • polyoxyalkylenes polyoxyalkylenes
  • copolymers of polyethylene glycol and polypropylene glycol may find use as linkers use as linkers.
  • CDR complementarity determining region
  • CDR-H1, CDR-H2, CDR-H3 three CDRs in each heavy chain variable region
  • CDR-L1, CDR-L2, CDR-L3 three CDRs in each light chain variable region
  • Framework regions and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains.
  • FR-H1, FR-H2, FR-H3, and FR-H4 there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) , and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4) .
  • FR-H1, FR-H2, FR-H3, and FR-H4 four FRs in each full-length heavy chain variable region
  • FR-L1, FR-L2, FR-L3, and FR-L4 four FRs in each full-length light chain variable region.
  • the precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991) , “Sequences of Proteins of Immunological Interest, ” 5th Ed.
  • the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or combinations thereof.
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a, ” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ( “indels” ) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (V H and V L , respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs (See e.g., Kindt et al. Kuby Immunology, 6th ed., W. H. Freeman and Co., page 91 (2007) ) .
  • a single V H or V L domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a V H or V L domain from an antibody that binds the antigen to screen a library of complementary V L or V H domains, respectively (See e.g., Portolano et al., J. Immunol. 150: 880-887 (1993) ; Clarkson et al., Nature 352: 624-628 (1991) ) .
  • Specific binding or binding of antibody molecules described herein refers to binding mediated by one or more CDR portions of the antibody. Not all CDRs may be required for specific binding. Specific binding can be demonstrated for example by an ELISA against a specific recited target or antigen that shows significant increase in binding compared to an isotype control antibody.
  • an “epitope” refers to the binding determinant of an antibody or fragment described herein minimally necessary for specific binding of the antibody or fragment thereof to a target antigen.
  • the target antigen is a polypeptide the epitope will be a continuous or discontinuous epitope.
  • a continuous epitope is formed by one region of the target antigen, while a discontinuous epitope may be formed from two or more separate regions.
  • a discontinuous epitope for example, may form when a target antigen adopts a tertiary structure that brings two amino acid sequences together and forms a three-dimensional structure bound by the antibody.
  • the target antigen is a polypeptide the epitope will generally be a plurality of amino acids linked into a polypeptide chain.
  • a continuous epitope may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids. While an epitope may comprise a contiguous polymer of amino acids, not every amino acid of the polymer may be contacted by an amino acid residue of the antibody. Such non-contacted amino acids will still comprise part of the epitope as they may be important for the structure and linkage of the contacted amino acids.
  • the skilled artisan may determine if any given antibody binds an epitope of a reference antibody, for example, by cross-blocking experiments with a reference antibody.
  • described herein are antibodies that bind the same epitope of the described antibodies.
  • described herein are antibodies that are competitively blocked by the described antibodies.
  • antibody fragments refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F (ab’) 2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv or sFv) ; and multispecific antibodies formed from antibody fragments.
  • the antibodies are single-chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., polypeptide linkers, and/or those that are not produced by enzyme digestion of a naturally-occurring intact antibody.
  • the antibody fragments are scFvs.
  • a “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs.
  • a humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) , e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the CDR residues are derived
  • human antibodies are human antibodies.
  • a “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries.
  • the term excludes humanized forms of non-human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human.
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. Human antibodies also may be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody-encoding sequences derived from a human repertoire.
  • CD24 refers to the sialoglycoprotein expressed at the surface of most B lymphocytes and activated T lymphocytes.
  • the amino acid sequence of human CD24 can found at www. uniprot. org/uniprotkb/P25063/entry, with accession number P2506.
  • the anti-CD24 binding antibodies described herein bind to mammalian CD24. In certain embodiments, the anti-CD24 binding antibodies described herein bind to human CD24.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.
  • Polypeptides including the provided antibodies and antibody chains and other peptides, e.g., linkers and binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity.
  • amino acid sequence variants of the antibodies provided herein are contemplated.
  • a variant typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions.
  • Such variants can be naturally occurring or can be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of known techniques.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.
  • Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • %amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • the %amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B.
  • the antibodies are altered to increase or decrease their glycosylation (e.g., by altering the amino acid sequence such that one or more glycosylation sites are created or removed) .
  • a carbohydrate attached to an Fc region of an antibody may be altered.
  • Native antibodies from mammalian cells typically comprise a branched, biantennary oligosaccharide attached by an N-linkage to Asn 297 of the CH2 domain of the Fc region (See e.g., Wright et al. TIBTECH 15: 26-32 (1997) ) .
  • the oligosaccharide can be various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc) , galactose, sialic acid, fucose attached to a GlcNAc in the stem of the biantennar oligosaccharide structure. Modifications of the oligosaccharide in an antibody can be made, for example, to create antibody variants with certain improved properties.
  • Antibody glycosylation variants can have improved ADCC and/or CDC function.
  • antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn 297 , relative to the sum of all glycostructures attached to Asn297 (See e.g., WO 08/077546) .
  • Asn 297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues; See e.g., Edelman et al. Proc Natl Acad Sci U S A. 1969 May; 63 (1) : 78–85) .
  • Asn 297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies.
  • Such fucosylation variants can have improved ADCC function (See e.g., Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004) ; and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ) .
  • Cell lines e.g., knockout cell lines and methods of their use can be used to produce defucosylated antibodies, e.g., Lec13 CHO cells deficient in protein fucosylation and alpha-1, 6-fucosyltransferase gene (FUT8) knockout CHO cells (See e.g., Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986) ; Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004) ; Kanda, Y. et al., Biotechnol. Bioeng., 94 (4) : 680-688 (2006) ) .
  • Other antibody glycosylation variants are also included (See e.g., U.S. Pat. No. 6,602,684) .
  • an antibody provided herein has a dissociation constant (K D ) of about 1 ⁇ M, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or less (e.g., 10 -8 M or less, e.g., from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M) for the antibody target.
  • K D dissociation constant
  • an antibody provided herein has a dissociation constant (K D ) of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or greater (e.g., 10 -8 M or less, e.g., from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M) for the antibody target.
  • the antibody target can be CD24.
  • K D can be measured by any suitable assay. In certain embodiments, K D can be measured using surface plasmon resonance assays (e.g., using a a or Octet) .
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • An Fc region herein is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • An Fc region includes native sequence Fc regions and variant Fc regions.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant.
  • An Fc region herein is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • An Fc region includes native sequence Fc regions and variant Fc regions.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the Fc region of an immunoglobulin is important for many important antibody functions (e.g. effector functions) , such as antigen-dependent cellular cytotoxicity (ADCC) , complement dependent cytotoxicity (CDC) , and antibody-dependent cell-mediated phagocytosis (ADCP) , result in killing of target cells, albeit by different mechanisms.
  • ADCC antigen-dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • the antibodies described herein comprise the variable domains of the invention combined with constant domains comprising different Fc regions, selected based on the biological activities of the antibody for the intended use.
  • Human IgGs for example, can be classified into four subclasses, IgG1, IgG2, IgG3, and IgG4, and each these of these comprises an Fc region having a unique profile for binding to one or more of Fc ⁇ receptors (activating receptors Fc ⁇ RI (CD64) , Fc ⁇ RIIA, Fc ⁇ RIIC (CD32) ; Fc ⁇ RIIIA and Fc ⁇ RIIIB (CD16) and inhibiting receptor Fc ⁇ RIIB) , and for the first component of complement (C1q) .
  • Fc ⁇ receptors activating receptors Fc ⁇ RI (CD64) , Fc ⁇ RIIA, Fc ⁇ RIIC (CD32) ; Fc ⁇ RIIIA and Fc ⁇ RIIIB (CD16) and inhibiting receptor Fc ⁇ RIIB
  • C1q first component of complement
  • Human IgG1 and IgG3 bind to all Fc ⁇ receptors; IgG2 binds to Fc ⁇ RIIA H131 , and with lower affinity to Fc ⁇ RIIA R131 Fc ⁇ RIIIA V158 ; IgG4 binds to Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIB, Fc ⁇ RIIC, and Fc ⁇ RIIIA V158 ; and the inhibitory receptor Fc ⁇ RIIB has a lower affinity for IgG1, IgG2 and IgG3 than all other Fc ⁇ receptors. Studies have shown that Fc ⁇ RI does not bind to IgG2, and Fc ⁇ RIIIB does not bind to IgG2 or IgG4. Id. In general, with regard to ADCC activity, human IgG1 ⁇ IgG3>>IgG4 ⁇ IgG2.
  • the antibodies of this disclosure are variants that possess reduced effector functions, which make it a desirable candidate for applications in which certain effector functions (such as complement fixation and ADCC) are unnecessary or deleterious.
  • Such antibodies can have decreased complement-dependent cytotoxicity (CDC) , antibody-dependent cell cytotoxicity (ADCC) , or antibody dependent cellular phagocytosis (ADCP) .
  • the antibodies of this disclosure are variants that possess increased effector functions for applications in which increased immunogenicity would be beneficial.
  • Such antibodies can have increased CDC, ADCC, or ADCP, or a combination thereof.
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No.
  • non-radioactive assays methods may be employed (e.g., ACTI TM and CytoTox non-radioactive cytotoxicity assays) .
  • useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) , monocytes, macrophages, and Natural Killer (NK) cells.
  • Antibodies can have increased half-lives and improved binding to the neonatal Fc receptor (FcRn) (See e.g., US 2005/0014934) .
  • Such antibodies can comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn, and include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 according to the EU numbering system (See e.g., U.S. Pat. No. 7,371,826) .
  • Fc region variants are also contemplated (See e.g., Duncan & Winter, Nature 322: 738-40 (1988) ; U.S. Pat. Nos. 5,648,260 and5,624,821; and WO94/29351) .
  • cysteine engineered antibodies e.g., “thioMAbs, ” in which one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • Reactive thiol groups can be positioned at sites for conjugation to other moieties, such as drug moieties or linker drug moieties, to create an immunoconjugate.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known and available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG) , copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers) , and dextran or poly (n vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylen oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol) ,
  • PEG poly
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • 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 if two or more polymers are attached, they can be the same or different molecules.
  • the antibodies described herein can be encoded by a nucleic acid.
  • a nucleic acid is a type of polynucleotide comprising two or more nucleotide bases.
  • the nucleic acid is a component of a vector that can be used to transfer the polypeptide encoding polynucleotide into a cell.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of vector is a genomic integrated vector, or “integrated vector, ” which can become integrated into the chromosomal DNA of the host cell.
  • vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors. ”
  • Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors and the like.
  • regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral or insect genes. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated.
  • Vectors derived from viruses may be employed. Plasmid vectors can be linearized for integration into a genomic region.
  • the expression vector is a plasmid.
  • the expression vector is a lentivirus, adenovirus, or adeno-associated virus.
  • the expression vector is an adenovirus.
  • the expression vector is an adeno-associated virus.
  • the expression vector is a lentivirus.
  • homology when used herein to describe to an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993) . Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990) . Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.
  • BLAST basic local alignment search tool
  • the nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise render a suitable cell transgenic for the nucleic acid, thus enabling the production of antibodies for commercial or therapeutic uses.
  • Standard cell lines and methods for the production of antibodies from a large scale cell culture are known in the art. See e.g., Li et al., “Cell culture processes for monoclonal antibody production. ” Mabs. 2010 Sep-Oct; 2 (5) : 466–477.
  • the cell is a Eukaryotic cell.
  • the Eukaryotic cell is a mammalian cell.
  • the mammalian cell is a cell line useful for producing antibodies is a Chines Hamster Ovary cell (CHO) cell, an NS0 murine myeloma cell, or a PER. cell.
  • the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing antibodies.
  • described herein is a method of making an antibody comprising culturing a cell comprising a nucleic acid encoding an antibody under conditions in vitro sufficient to allow production and secretion of said antibody.
  • a master cell bank comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody described herein integrated at a genomic location; and (b) a cryoprotectant.
  • the cryoprotectant comprises glycerol or DMSO.
  • the master cell bank is contained in a suitable vial or container able to withstand freezing by liquid nitrogen.
  • Such methods comprise incubating a cell or cell-line comprising a nucleic acid encoding the antibody in a cell culture medium under conditions sufficient to allow for expression and secretion of the antibody, and further harvesting the antibody from the cell culture medium.
  • the harvesting can further comprise one or more purification steps to remove live cells, cellular debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components.
  • the additional purification step (s) include centrifugation, ultracentrifugation, protein A, protein G, protein A/G, or protein L purification, and/or ion exchange chromatography.
  • Treatment refers to, e.g., a deliberate intervention to a physiological disease state resulting in the reduction in severity of a disease or condition; the reduction in the duration of a condition; the amelioration or elimination of one or more symptoms associated with a disease or condition; or the provision of beneficial effects to a subject with a disease or condition. Treatment does not require curing the underlying disease or condition.
  • a “therapeutically effective amount, ” “effective dose, ” “effective amount, ” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • the ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • “pharmaceutically acceptable” with reference to a carrier” “excipient” or “diluent” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion) .
  • the active compound i.e., antibody
  • the active compound i.e., antibody
  • the active compound can be coated in a material to protect the compound from the action of acids and other natural conditions that can inactivate the compound.
  • the pharmaceutical compounds described herein can include one or more pharmaceutically acceptable salts.
  • a “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19) .
  • Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as ⁇ , ⁇ '-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • Described herein are antibodies that specifically bind to CD24.
  • the antibodies described herein possess the useful properties of not binding to either B cells or activated T cells. Such antibodies may possess an enhanced safety profile with reduced immune side effects.
  • the antibody does not bind to T lymphocytes. In certain embodiments, the antibody has weak binding to T lymphocytes. In certain embodiments, antibody does not bind to activated T lymphocytes. In certain embodiments, the antibody has weak binding to activated T lymphocytes. In certain embodiments, said activated T lymphocytes are activated human T lymphocytes. In certain embodiments, the antibody does not bind to B lymphocytes. In certain embodiments, the antibody has weak binding to B lymphocytes. In certain embodiments, said B lymphocytes are human B lymphocytes. In certain embodiments, the antibody binds to CD24 with a KD of less than 1x10 -8 .
  • the antibody binds to CD24 with a KD of less than 1x10 -9 .
  • the antibody is a humanized or chimeric antibody.
  • the antibody is a Fab, F (ab) 2, or a single chain variable fragment (scFv) .
  • the antibody is a heavy chain antibody or antigen binding fragment thereof.
  • the antibody is an IgG antibody.
  • the antibody is an IgG1 isotype.
  • the antibody is an IgG4 isotype.
  • the antibody binds to the serine at residue 18 of SEQ ID NO: 500.
  • the antibody requires the presence of a serine at residue 18 of SEQ ID NO: 500 for binding. In certain embodiments, the antibody binds to the serine at residue 18 of SEQ ID NO: 500 but does not bind to the asparagine at residue 17 of SEQ ID NO: 500. In certain embodiments, the antibody binds to an epitope consisting of the amino acids sequence of SEQ ID NO 501.
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 401; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 405; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 407; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 409; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 411.
  • LCDR1 comprising an amino acid sequence set forth in SEQ ID NO: 401
  • LCDR2 comprising an amino acid sequence set forth in S
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 402; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 403; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 406; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 408; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 409; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 412.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 403
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 83, 703, 713, 723, 733, and 743; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 84, 704, 714, 724, 734, and 744; (c) a light chain complementarity determining region 3 (LCDR3) comprising having an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 25, 35, 45, 55, 65, 75, 85, 705, 715, 725, 735, and 745; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16, 26, 36, 46, 56, 66
  • the antibody comprises a combination of LCDRs 1-3 and HCDRs 1-3, wherein the combination is selected from the group consisting of: (i) LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 14, LCDR3 comprising SEQ ID NO: 15, HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, HCDR3 comprising SEQ ID NO: 18; (ii) LCDR1 comprising SEQ ID NO: 23, LCDR2 comprising SEQ ID NO: 24, LCDR3 comprising SEQ ID NO: 25, HCDR1 comprising SEQ ID NO: 26, HCDR2 comprising SEQ ID NO: 27, HCDR3 comprising SEQ ID NO: 28; (iii) LCDR1 comprising SEQ ID NO: 33, LCDR2 comprising SEQ ID NO: 34, LCDR3 comprising SEQ ID NO: 35, HCDR1 comprising SEQ ID NO: 36, HCDR2 comprising SEQ ID NO: 37, HCDR
  • the antibody comprises a combination of LCDRs 1-3 and HCDRs 1-3, wherein the combination is selected from the group consisting of: (i) LCDR1 as set forth in SEQ ID NO: 13, LCDR2 as set forth in SEQ ID NO: 14, LCDR3 as set forth in SEQ ID NO: 15, HCDR1 as set forth in SEQ ID NO: 16, HCDR2 as set forth in SEQ ID NO: 17, HCDR3 as set forth in SEQ ID NO: 18; (ii) LCDR1 as set forth in SEQ ID NO: 23, LCDR2 as set forth in SEQ ID NO: 24, LCDR3 as set forth in SEQ ID NO: 25, HCDR1 as set forth in SEQ ID NO: 26, HCDR2 as set forth in SEQ ID NO: 27, HCDR3 as set forth in SEQ ID NO: 28; (iii) LCDR1 as set forth in SEQ ID NO: 33, LCDR2 as set forth in SEQ ID NO: 34, LCDR3 as set forth in SEQ ID NO: 35
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 13; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 14; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 15; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 16; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 17; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 18.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 14
  • LCDR3 a
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 23; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 24; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 25; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 26; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 27; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 28.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 24
  • LCDR3
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 33; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 34; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 35; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 36; € a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 37; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 38.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 34
  • LCDR3 light chain complement
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 43; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 44; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 45; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 46; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 47; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 48.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 44
  • LCDR3 compris
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 53; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 54; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 55; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 56; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 57; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 58.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 54
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 63; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 64; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 65; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 66; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 67; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 68.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 64
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 73; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 74; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 75; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 76; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 77; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 78.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 74
  • the antibody comprises: (a) a light chain complementarity determining region 1 (LCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 83; (b) a light chain complementarity determining region 2 (LCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 84; (c) a light chain complementarity determining region 3 (LCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 85; (d) a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence set forth in SEQ ID NO: 86; (e) a heavy chain complementarity determining region 2 (HCDR2) comprising an amino acid sequence set forth in SEQ ID NO: 87; and/or (f) a heavy chain complementarity determining region 3 (HCDR3) comprising an amino acid sequence set forth in SEQ ID NO: 88.
  • LCDR1 light chain complementarity determining region 1
  • LCDR2 comprising an amino acid sequence set forth in SEQ ID NO: 84
  • the antibody comprises an immunoglobulin light chain amino acid variable region sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.
  • the antibody comprises an immunoglobulin light chain amino acid variable region sequence identical to any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, or 319; and an immunoglobulin heavy1 chain variable region amino acid sequence identical to any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, or 320.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to any one of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined using a hybrid scheme of any two or three of the Kabat definition, Chothia definition, Aho definition, Abm definition, IMGT definition, Contact definition and North definition.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Kabat definition.
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to the Chothia definition.
  • VL light chain variable region
  • LCDR2 LCDR2
  • LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319
  • VH heavy chain variable region
  • the antibody comprises: (i) a light chain variable region (VL) having the identical LCDR1, LCDR2, and LCDR3 with any one of SEQ ID NOs: 155, 157, 169, 267, 271, 275, 277, 309, 311, 313, 315, 317, and 319; and (ii) a heavy chain variable region (VH) having the identical HCDR1, HCDR2, and HCDR3 with any one of SEQ ID NOs: 156, 158, 170, 268, 272, 276, 278, 310, 312, 314, 316, 318, and 320; wherein the CDRs are defined according to Kabat combined with Chothia definitions.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 275; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 276.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 275; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 276.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 277; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 278.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 277; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 278.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 309; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 310.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 309; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 310.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 155; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 156.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 155; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 156.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 157; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 158.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 157; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 158.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 169; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 170.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 169; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 170.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 267; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 268.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 267; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 268.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 271; and an immunoglobulin heavy chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 272.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence identical to SEQ ID NOs: 271; and an immunoglobulin heavy chain variable region amino acid sequence identical to SEQ ID NO: 272.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to a immunoglobulin light chain variable region sequence of Table 1; and an immunoglobulin heavy chain amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%identical to a heavy chain immunoglobulin sequence of Table 1.
  • the antibody comprises an arginine substitution at position number 17 of the immunoglobulin light chain according to Kabat numbering.
  • the antibody comprises a tyrosine substitution at position number 12 of the immunoglobulin light chain according to Kabat numbering.
  • the antibody comprises an immunoglobulin light chain variable region amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%or 100%identical to any one of the immunoglobulin light chain variable region sequences of Table 1; and an immunoglobulin heavy chain amino acid sequence at least about 90%, 95%, 96%, 97%, 98%, or 99%or 100%identical to any one of the immunoglobulin heavy chain variable region sequences of Table 1.
  • the antibody comprises an immunoglobulin light chain amino acid sequence identical to any one of the immunoglobulin light chain variable region sequences of Table 1; and an immunoglobulin heavy chain variable region amino acid sequence identical to any one of the immunoglobulin heavy chain variable region sequences of Table 1.
  • *CDRs are defined by Kabat Definition in Table 1 and in the hereinafter Examples.
  • Described herein is a method of inhibiting CD24 signaling in a cancer cell comprising contacting the cancer cell with an antibody of the present disclosure.
  • CD24 signaling is not inhibited in non-neoplastic B lymphocytes or non-neoplastic T lymphocytes.
  • the cancer cell is a cancer cell is in vivo in an individual.
  • signaling though CD24 is reduced by about 10%, 20%, 35%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more.
  • signaling through CD24 is reduced to levels not detectable above background levels.
  • signaling through SHP2 is reduced.
  • signaling through SHP2 is reduced by about 10%, 20%, 35%, 30%, 40%, 50%, 60%, 70%, 80%, 90%or more. Signaling through SHP2 can be evidence by levels of phosphorylation of SHP2.
  • the cancer or tumor is a solid cancer or tumor.
  • the cancer or tumor is a blood cancer or tumor.
  • the cancer or tumor comprises breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovarian, prostate, brain, pancreatic, skin, bone, bone marrow, blood, thymus, uterine, testicular, and liver tumors.
  • tumors which can be treated with the antibodies of the invention comprise adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and/or teratoma.
  • the tumor/cancer is selected from the group of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenomas, adenosarcoma, adenosquamous carcinoma, astrocytic tumors, Bartholin gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing's sarcoma, focal nodular hyperplasia, gastronoma, germ line tumors, glioblastoma, glucagonoma, hemangioblastom
  • the tumor/cancer to be treated with one or more antibodies of the invention comprise brain cancer, head and neck cancer, colorectal carcinoma, acute myeloid leukemia, pre-B-cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell cancer, and non-small cell cancer, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and breast cancer, preferably breast ductal cancer, and/or breast carcinoma.
  • the cancer treated with the antibodies of this disclosure comprises glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises lung cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises prostate cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises colon cancer. In certain embodiments, the cancer treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In a certain embodiment, the cancer is refractory to other treatment. In a certain embodiment, the cancer treated is relapsed.
  • the antibodies can be administered to a subject in need thereof by any route suitable for the administration of antibody-containing pharmaceutical compositions, such as, for example, subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral, etc.
  • the antibodies are administered intravenously.
  • the antibodies are administered subcutaneously.
  • the antibodies are administered intratumoral.
  • the antibodies are administered on a suitable dosage schedule, for example, weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once a month etc.
  • the antibodies are administered once every three weeks.
  • the antibodies can be administered in any therapeutically effective amount.
  • the therapeutically acceptable amount is between about 0.1 mg/kg and about 50 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg/kg and about 40 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg/kg and about 20 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg/kg and about 10 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg/kg and about 30 mg/kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg/kg and about 20 mg/kg. Therapeutically effective amounts include amounts sufficient to ameliorate one or more symptoms associated with the disease or affliction to be treated.
  • the anti-CD24 antibodies of the current disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents.
  • Pharmaceutically acceptable excipients, carriers and diluents can be included to increase shelf-life, stability, or the administrability of the antibody.
  • Such compounds include salts, pH buffers, detergents, anti-coagulants, and preservatives.
  • the antibodies of the current disclosure are administered suspended in a sterile solution.
  • the solution comprises about 0.9%NaCl.
  • the solution comprises about 5%dextrose.
  • the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris) ; surfactants, for example, polysorbate 80 (Tween 80) , polysorbate 20 (Tween 20) , and poloxamer 188; polyol/disaccharide/polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA or EGTA.
  • buffers for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris)
  • surfactants for example, polysorbate 80 (Tween 80)
  • the antibodies of the current disclosure can be shipped/stored lyophilized and reconstituted before administration.
  • lyophilized antibody formulations comprise a bulking agent such as, mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof.
  • the lyophilized formulation can be contained in a vial comprised of glass or other suitable non-reactive material.
  • the antibodies when formulated, whether reconstituted or not, can be buffered at a certain pH, generally less than 7.0. In certain embodiments, the pH can be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.
  • kits comprising one or more of the antibodies described herein in a suitable container and one or more additional components selected from: instructions for use; a diluent, an excipient, a carrier, and a device for administration (e.g., a needle and/or syringe) .
  • described herein is a method of preparing a cancer treatment comprising admixing one or more pharmaceutically acceptable excipients, carriers, or diluents and an antibody of the current disclosure. In certain embodiments, described herein is a method of preparing a cancer treatment for storage or shipping comprising lyophilizing one or more antibodies of the current disclosure.
  • the amino acid sequences of the antigen and proteins for use in raising the antibodies of the present disclosure were obtained from UniProt (human CD24, Uniprot number: P25063) .
  • a SUMO-his tag and GST tag were fused to the CD24 protein.
  • the gene encoding SUMO-his tag and GST tag fusion protein was cloned into a pET21b vector (Novagen, Cat No. 69741) , expressed by an E. coli expression system and purified.
  • the gene encoding the GST-tag fusion protein was cloned into a pTT5 vector, and was transiently expressed with Expi293F cells (Thermo, Cat No. A14527) and purified.
  • CD24 antigens referred to in this section refer to human CD24 protein, unless otherwise specified.
  • CD24 (27-56aa) extracellular domain and GST i.e., CD24 (27-56aa) -GST (NEOX-2, SEQ ID NO: 503) , for mouse immunization and detection:
  • underlined portion denotes CD24 (27-56aa)
  • italic portion denotes GST tag.
  • CD24 (27-56aa) extracellular domain and SUMO-his i.e., CD24 (27-56aa) -SUMO (NEOX-27, SEQ ID NO: 504) , for detection
  • underlined portion denotes CD24 (27-56aa)
  • italic portion denotes SUMO-his tag.
  • CD24 (27-56aa) extracellular domain and GST i.e., CD24 (27- 56aa) -GST (NEOX-4, SEQ ID NO: 505) , for detection
  • the column was rinsed with Buffer B (Buffer A containing 20 mM imidazole) and Buffer C (Buffer A containing 50 mM imidazole) sequentially, and then eluted with Buffer E (Buffer A containing 250 mM imidazole) and the target CD24 (27-56aa) -SUMO his protein was collected. Finally, the CD24 (27-56aa) -SUMO his protein was changed from Buffer E to PBS buffer by dialysis. The presence and purity of the CD24 (27-56aa) -SUMO his protein was confirmed by SDS-PAGE and HPLC.
  • the column was eluted with Buffer F (Buffer A containing 10 mM Glutathione, RSH) and the target CD24 (27-56aa) -GST protein was collected. Finally, the CD24 (27-56aa) -GST protein was changed from Buffer F to PBS buffer by dialysis. The presence and purity of the CD24 (27-56aa) -GST protein was confirmed by SDS-PAGE and HPLC.
  • Buffer F Buffer A containing 10 mM Glutathione, RSH
  • Anti-human CD24 monoclonal antibodies were produced by immunizing mice.
  • SJL white mice, female, 6-8 weeks old (Shanghai Slac Laboratory Animal Co. Ltd., Animal production license number: SCXK (Shanghai) 2017-0005) were used in the experiment.
  • the breeding environment was SPF grade. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12/12 hours light/dark cycle adjustment, at a temperature of 20-25 °C and a humidity of 40-60%.
  • the mice having adapted to the environment were immunized in accordance with the following protocol.
  • the immunizing antigen was a GST-tagged human CD24 extracellular domain fusion protein (CD24 (27-56aa) -GST (NEOX-2) ) .
  • mice were immunized with CFA/IFA/Alum/CpG.
  • the ratio of antigen to adjuvant was 1: 1, and the antigen and adjuvant were inoculated after being mixed quickly and thoroughly.
  • First immunization 50 ⁇ g antigen/mice/time; booster immunization: 25 ⁇ g antigen/mouse/time.
  • the inoculation interval was 7 days, and a total of 5 immunizations were performed.
  • blood was collected 7 days after each immunization, and the antibody titer in the mouse serum was determined by ELISA method. Mice with high antibody titers in the serum and titers tended to plateau were subjected to splenocyte fusion.
  • the booster immunization was performed by intraperitoneal (IP) injection of antigen solution prepared in normal saline at a dose of 50 ⁇ g/mouse.
  • IP intraperitoneal
  • Mouse spleen lymphocytes were mixed with Sp2/0 myeloma cells (CRL-8287TM) at a ratio of 1: 1-10: 1 using a TBX electroporator, and the hybridoma cells were obtained by a round of electric shock (60V, 30s; pulse 40ms, 1700V) .
  • the fused hybridoma cells were resuspended in complete medium (containing 20%FBS, 1 ⁇ HAT, 1 ⁇ DMEM medium) at a density of 4-5E5/ml, and were seeded at 200 ⁇ l/well in a 96-well plate and cultured at 37°C, 5%CO 2 for 7 days.
  • the culture medium was switched to HT complete medium (containing 20%FBS, 1 ⁇ HT and 1 ⁇ DMEM medium) at 200ul/well, and the cells were cultured at 37°C, 5%CO 2 for 2 days, then subjected to ELISA detection.
  • HT complete medium containing 20%FBS, 1 ⁇ HT and 1 ⁇ DMEM medium
  • ELISA and FACS (antigen being detected: CD24 (27-56aa) -SUMO his (NEOX-27) ) were used to screen for positive hybridomas. Clones positive by ELISA and FACS were expanded, and subjected to 2 to 3 rounds of subclone screening until single cell clones were obtained. The hybridoma monoclonal G14 was obtained through the above experimental screening.
  • Hybridoma cells in logarithmic growth phase were collected, and RNA was extracted with Trizol (Invitrogen, Cat No. 15596-018) according to the kit instructions and reverse transcribed with PrimeScript TM Reverse Transcriptase Kit (Takara, Cat No. 2680A) .
  • the cDNA obtained by reverse-transcription was PCR-amplified with a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) to amplify the variable region sequences of the antibody heavy and light chains, cloned into a vector, and then subjected to sequencing.
  • the amino acid sequences corresponding to the DNA sequences of the heavy and light chain variable regions of murine monoclonal antibody G14 were obtained as:
  • G14 heavy chain variable region (G14VH, SEQ ID NO: 2)
  • G14 light chain variable region (G14VL, SEQ ID NO: 1)
  • the germline genes of heavy chain and light chain variable regions with high homology to the murine monoclonal antibody G14 antibody were selected as templates respectively, so as to grating the CDRs of the murine antibodies into the corresponding humanization templates to form humanized variable region sequences with the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the heavy chain variable region sequence of the murine antibody G14 was humanized using germline IGHV3-15*01 IGHJ6*019 as template.
  • the humanized variable region sequence combination huG14-1_graft was as follows:
  • the light chain variable region was humanized using the germline IGKV2-28*01 IGKJ2*01 as template, and the heavy chain variable region was humanized using the germline IGHV3-15*01 IGHJ6*019 as template.
  • the grafted variable region sequence combination huG14-2_graft was as obtained as follows:
  • huG14VL1-1 (SEQ ID NO: 209) :
  • huG14VL1-3 (SEQ ID NO: 213) :
  • huG14VL1-15 (SEQ ID NO: 279) :
  • huG14VH (SEQ ID NO: 280) :
  • huG14VL1-16 (SEQ ID NO: 281) :
  • huG14VH (SEQ ID NO: 282) :
  • the light chain variable region was humanized using the germline GKV2-30*01 IGKJ2*01 as template, and the heavy chain variable region was humanized using the germline IGHV3-15*01 IGHJ6*019 as template.
  • the humanized variable region sequence combination huG14-3_graft was obtained as follows:
  • G14VL_Graft2 sequence to obtain the humanized light chain variable region sequence huG14VL2.
  • Three mutations (G49A, N79S, L81V) were introduced into the FR region of the G14VH_Graft sequence to obtain the humanized heavy chain variable region sequence huG14VH.
  • the humanized antibody huG14-3 was obtained, and its VH/VL is expressed as follows:
  • huG14VL2 (SEQ ID NO: 301) :
  • huG14VH (SEQ ID NO: 302) :
  • Affinity maturation based on humanization design 1 (huG14-1) : single mutation site design and multiple mutation combination design for CDR
  • VH and VL sequences were paired to obtain multiple heavy chain/light chain variable region sequence combinations (i.e., affinity matured antibodies) , expressed as follows:
  • VH and VL sequences were paired to obtain multiple heavy chain/light chain variable region sequence combinations (i.e., affinity matured antibodies) , expressed as follows:
  • VH and VL sequences were combined to obtain multiple heavy chain/light chain variable region sequence combinations (i.e., affinity matured antibodies) , expressed as follows:
  • Deamidation is a common chemical modification in antibodies that may potentially affect stability in solution. Deamidation, oxidation, or isomerization of some amino acids in the CDR region of the described antibodies were reduced by introducing mutations that would prevent such undesirable reactions. An amino acid sequence of “NG” is prone to deamidation, and the above antibodies possess an NG at positions 33-34.
  • mutations to the CDR1 of the CD24 antibody light chain KSSQSLLYSX1X2ETYLN wherein X1 and X2 are the amino acid residues at positions 33-34 in the variable region of the CD24 antibody light chain, respectively; X1 can be selected from Ser, Tyr or Gln, X2 can be selected from His, Lys, Arg, Trp or Ala, were constructed.
  • the above heavy chain variable regions can be recombinantly expressed with the human heavy chain IgG1 constant region shown in SEQ ID NO: 607 to obtain the full-length heavy chain sequence.
  • the above light chain variable regions can be recombinantly expressed with the kappa light chain constant region sequence shown in SEQ ID NO: 608 to obtain the full-length light chain sequences.
  • the above heavy and light chain variable regions can also be recombined with other heavy and light chain constant regions of the IgG family or constant regions of mutated IgG family commonly known in the art, forming the complete heavy and light chain sequences of the antibodies.
  • Exemplary heavy and light chain constant regions employed in the Examples of the present invention are as follows:
  • the positive antibody molecules obtained by hybridoma screening were sequenced to obtain the gene sequence encoding the variable region.
  • the head and tail primers were designed based on the sequences obtained by the sequencing, and the sequenced genes were used as the templates to construct the VH/VK gene fragments of the G14 antibody by PCR.
  • the VH/VK gene fragments were then subjected to homologous recombination with the expression vector pcDNA3.4 (Thermo Fisher, Cat No.
  • V001453 (with a signal peptide and hIgG1/kappa constant region gene (CH1-Fc/CL) fragment) to construct a recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pcDNA3.4/VL-CL-pcDNA3.4, forming a G14 chimeric antibody expression construct.
  • CH1-Fc/CL constant region gene
  • the full-length amino acid sequence of the chimeric antibody (G14) is as follows:
  • variable region sequences SEQ ID NO: 1 and 2, respectively
  • the rest are constant region sequences.
  • the antibody sequences of humanized antibodies and affinity matured antibodies were codon-optimized to generate human codon-preferred coding gene sequences.
  • Primers were designed to construct VH/VK gene fragments of each of the antibodies (such as huG14-1 ⁇ 3, and huG14-101 ⁇ 139, 201 ⁇ 235, 301 ⁇ 306, etc. ) via PCR, and then subjected to homologous recombination with the expression vector pcDNA3.4 (Thermo Fisher, Cat No.
  • V001453 (with signal peptide and hIgG1/kappa constant region gene (CH1-Fc/CL) fragment) to construct the recombinant humanized antibody full-length expression plasmid VH-CH1-Fc-pcDNA3.4/VL-CL-pcDNA3.4.
  • huG14-1 full-length Heavy Chain (SEQ ID NO: 610) :
  • variable region sequences SEQ ID NO: 101 and 102, respectively
  • the rest are constant region sequences.
  • the plasmid expressing antibody light/heavy chains was transfected into CHO cells at a ratio of 1.5: 1. After 4-6 days, the expression supernatant was collected, centrifuged at high speed (13,000rpm, 4°C, 30 min) to remove impurities, and purified with a Protein A column. The target antibody protein was eluted with a glycine eluent at pH3.0-pH3.5, neutralized with 1M Tris-HCl at pH8.0-9.0. The target antibody protein was replaced with PBS buffer by dialysis, aliquoted, and frozen for later use. If the antibody purity was less than 85%, the antibody was further purified by gel chromatography Superdex200 (GE) equilibrated with PBS to remove aggregates, and monomer peaks would be collected, aliquoted, and frozen for later use.
  • GE gel chromatography Superdex200
  • Example 7 Determination of the binding of chimeric antibodies, humanized antibodies and affinity matured antibodies to CD24 protein
  • An ELISA method was used to determine the binding capacity between CD24 protein and chimeric antibody (G14) , humanized antibody (huG14-1, whose VH/VL sequences are shown in SEQ ID NO: 102 and SEQ ID NO: 101, respectively) and affinity matured antibodies (respectively labeled as huG14-101 to 139, huG14-204 to 233, and their VH/VL combinations are elaborated in the table in Example 4, and the constant regions are the same as above.
  • Th4e ELISA was performed as follows: 1) coating of CD24 protein: CD24 (27-56aa) -GST (NEOX-2) or CD24 (27-56aa) -GST (NEOX-4) was diluted with PBS to 1 ⁇ g/ml, added to the ELISA plate with 100 ⁇ l per well, and coated overnight at 4 °C. 2) blocking: the plate was washed with 0.05%PBST solution for 3 times, and 200 ⁇ l of a 5%nonfat dry milk PBS solution which was pre-prepared in advance was added to each well of ELISA detection plate, and incubated for 2 hours at room temperature; 3) dilution and sample loading of primary antibody: the plate was washed 3 times with PBS.
  • the CD24 antibody was diluted to 200 ⁇ g/ml, then subjected to 4-fold gradient dilution, added to the ELISA detection plate at 100 ⁇ l/well, and incubated at 37 °C for 1 hour. 4) Addition of secondary antibody: the plate was washed with PBS for 3 times. A HRP-labeled anti-human IgG Fc antibody (Jackson, Cat No. 109-035-098) was diluted for 5000 times, added to each well at 100 ⁇ l, and incubated at 37°C for 1h. 5) color development: the plate was washed with PBS for 6 times. 50 ⁇ l of TMB (Beijing Zhizhou Biotechnology Co., Ltd., Cat No.
  • NALM6 and MDA-MB-486 are human tumor cell lines that express high levels of CD24.
  • the CD24 antibodies were co-incubated with NALM6 and MDA-MB-486 cells to determine whether the CD24 antibodies can specifically bind to NALM6 and MDA-MB-486 cells.
  • the control positive antibody was P6373, the sequence of which is shown in SEQ ID NO 603 and 604 (see WO 2019/222082 A1) .
  • the process for cell binding experiment was as follows: adding 5x10E4 cells to each well of a 96-well plate, and removing the supernatant after centrifugation; diluting the CD24 antibody with PBS: starting from the initial concentration of 30 ⁇ g/mL, with a 4-fold gradient dilution, with a total of 7 dilution gradients; adding 100 ⁇ l of the diluted CD24 antibodies to each well of the 96-well plate, resuspending the cells, and then incubating at 4°C for 1 hour; removing the supernatant by centrifugation, washing the cells with 200 ⁇ l staining buffer (1xPBS + 1%BSA) for 3 times, and then adding 50 ⁇ l of a 1: 200 diluted fluorescent secondary antibody goat-anti-human-IgG (Fc) -PE (Jackson, Cat No.
  • both the chimeric antibody G14 and the affinity matured CD24 antibodies could specifically bind to the CD24 protein on the surface of NALM6 cells, and the binding activities of the affinity matured CD24 antibodies were further significantly higher than that of the chimeric antibody G14.
  • both the chimeric antibody G14 and the affinity matured CD24 antibodies could specifically bind to the CD24 protein on the surface of MDA-MB-468 cells, and the binding activities of the affinity matured CD24 antibodies were further significantly higher than that of the chimeric antibody G14.
  • the above results show that the antibodies of the present invention can effectively bind to CD24 on the surface of tumor cells.
  • a biotinylated CD24 protein (ACRO, Cat No. CD4-H82E9) was coupled to a CM5 biosensor chip (Cytiva, BR100530) , and then 2-fold dilutions of CD24 antibodies at different concentrations were passed on the surface of the chip.
  • the reaction signals were detected in real time using Biacore (Cytiva, Biacore 8K) to obtain the association (ka) and dissociation (kd) curves, and then the affinity (K D ) values were obtained by fitting, as shown in the table X below.
  • the results show that the binding affinities of affinity-matured antibodies huG14-234, huG14-235 and huG14-301 against CD24 protein (ACRO, Cat No. CD4-H82E9) were significantly higher than that of the chimeric antibody G14.
  • Examples 10-17 of the present invention involve three CD24 antibodies for comparison, and their full-length sequences are respectively described as follows: SWA11 (light chain: SEQ ID NO: 601, heavy chain: SEQ ID NO: 602) , P6373 (light chain: SEQ ID NO: 603, heavy chain: SEQ ID NO: 604) and IMM47H (light chain: SEQ ID NO: 605, heavy chain: SEQ ID NO: 606) .
  • the Jurkat cell line containing the Fc ⁇ RIIIA luciferase reporter gene and the target cells NALM6 or MDA-MB-468 were inoculated into 96-well plates at an effect-target ratio of 6: 1, and then 3-fold gradient dilutions of antibodies to be tested at a starting concentration of 20 ⁇ g/ml and Isotype (anti-HEL IgG1 isotype control antibody) were added. After incubation at 37°C for 5 h, luciferase substrate was added and the reaction was allowed to proceed for 5 min, and the fluorescence intensity was detected by a microplate reader.
  • FIG. 5A, 5B, 5C, and Table 6 show that the antibodies huG14-124, huG14-125, huG14-131, huG14-216, huG14-217, huG14-230, huG14-232, huG14-234, huG14-235, huG14-301 all possess ADCC activity against NALM6 and MDA-MB-468.
  • Monocytes were isolated from PBMCs and were induced to differentiate into M2 macrophages with M-CSF, TGF ⁇ , and IL10 for later use.
  • the tumor cells NALM6 and MDA-MB-468 were labeled with CFSE and were mixed with M2 macrophages at a ratio of 1: 1.
  • FIG. 6A, 6B, 6C, 6D and Table 7 show that all the tested antibodies showed tumor phagocytic activity against NALM6 and MDA-MB-468.
  • Example 12-CD24 antibodies of the description show reduced binding to B cells and activated T cells
  • CD24 protein is expressed in both normal B cells and activated T cells.
  • Flow cytometry was used to study the binding profile of CD24 antibodies to B cells and activated T cells.
  • Serial dilutions of CD24 antibodies were incubated with normal primary B cells or with T cells that were activated with CD3, CD28 for three days at 4°C for 1 h, respectively, and then washed with PBS for three times.
  • a 1: 200 diluted fluorescent secondary antibody Goat-anti-human-IgG (Fc) -PE was added to each well, and incubated at 4°C for 30 min. After washing with PBS for three times, the cells were resuspended and subjected to flow cytometer detection by CytoFLEX.
  • FIG. 7A, 7B, 7C, 7D and Table 8 show that huG14-124, huG14-125, huG14-131, huG14-216, huG14-217, huG14-230, huG14-232, huG14-234, huG14-235, and huG14-301 did not bind to primary B cells and activated T cells.
  • IMM47H and SWA11 showed significant binding to B cells and P6373 showed significant binding to activated T cells. This is potentially important as the antibodies of the present invention may possess significant safety advantages to previously described anti-CD34 antibodies.
  • NALM6 cells 1 ⁇ 10 5 CFSE labeled NALM6 cells were mixed with human PBMCs at a ratio of 1: 7. Then anti-CD24 antibody G14, SWA11, IMM47H, P6373 were added into the NALM6/PMBCs mixture at a final concentration of 10 ⁇ g/ml. After 4h incubation at 37°C, 5%CO 2 , cell mixtures were stained with PE labeled anti-human CD19 antibody at 4°C in dark for 30 min.
  • NALM6 elimination rate (%) 1- (CFSE+CD19+ cells in anti-CD24 treated group /CFSE+CD19+ cell in isotype control group) ⁇ 100%
  • B cells elimination rate (%) 1- (anti-CD24 treatment group CD19+CFSE-B cell percentage in PBMCs /isotype control group CD19+CFSE-B cell percentage in PBMCs) ⁇ 100%.
  • huG14-301 and P6373 can selectively eliminate tumor cells (NALM6) and spare the normal B cells in PBMCs.
  • the reference antibodies SWA11, IMM47H kill both NALM6 and normal B cells.
  • G14 may have better safety profile over previously described anti-CD24 antibodies.
  • CD24 is a highly glycosylated protein, and the affinity of antibodies against CD24 proteins with different degrees of glycosylation will vary. Studies and literatures have shown that the glycosylation of CD24 in tumor cells is different from that in normal cells. Usually, the glycosylation of CD24 in tumor cells is reduced to a certain extent, thereby exposing the amino acid residues of its protein (for such literature, see, for example, WO2019222082A1, etc. ) .
  • the binding abilities of antibodies to CD24 with different glycosylation levels were determined by ELISA.
  • the method was as follows: 1) digesting CD24 (27-56aa) -GST (NEOX-4) with N-Glycanase, Neuraminidase or N-Glycanase + Neuraminidase, respectively; 2) diluting the CD24 before and after digestion with PBS to 1 ug/ml and then coating the 96-well plate with the CD24 at 4°C overnight; 3) after blocking with BSA, adding 0.78, 0.19, 0.05 ug/ml of G14 antibodies and incubating at 37°C for 1h; 4) adding secondary antibody: washing the plate with PBS for 3 times, diluting the HRP-labeled anti-human IgG Fc antibody (Jackson, Cat No.
  • FIG. 8 shows that the affinity of G14 against NEOX-4 protein digested by N-Glycanase, Neuraminidase or N-Glycanase + Neuraminidase was higher than that before digestion.
  • Cell preparation the cells (NALM6, MDA-MB-468, primary B cells) were counted (cell viability 98%) , centrifuged and resuspended in buffer, and aliquoted into a 96-well conical bottom plate at 5 ⁇ 10 5 , 200 ul per well. After centrifugation, the supernatant was removed and the cells were resuspended in blank medium.
  • N-Glycanase, Neuraminidase, N-Glycanase +Neuraminidase were added, respectively, and then placed into an incubator for 3h at 37°C.
  • the cells were washed with PBS for three times and then resuspended with PBS containing 2ug/ml of huG14-235 or huG14-301, and then incubated at room temperature for 1 h. After washing 3 times with PBS, fluorescent secondary antibody staining was performed and fluorescence intensities were detected by CytoFLEX.
  • FIG. 9A, 9B, and 9C show that huG14-235 or huG14-301 had strong binding to NALM6 and MDA-MB-468, and when tumor cells were treated with N-Glycanase, Neuraminidase or N-Glycanase + Neuraminidase, the binding of the antibodies to NALM6, MDA-MB-468 was significantly enhanced.
  • the huG14-235 or huG14-301 did not bind to primary B cells, but could bind to primary B cells after de-glycosylation by enzymatic digestion.
  • G14 binded to CD24 at 44S, 45G, 46L, 47A, 48P, while P6373 binded at 43N, 45G, 46L, 47A, 48P (the numbering of residues is based on the full-length CD24 sequence with the signal peptide, as shown in SEQ ID NO: 502) .
  • FIG. 12 Boding Epitope Comparison
  • the hSiglec10 transgenic mice were subcutaneously inoculated with 1 ⁇ 10 6 MC38-hCD24 tumor cells, and when the tumor volumes reached to 150 mm 3 , they were administered with CD24 antibodies intraperitoneally for treatment.
  • the dose was 20 mg/kg twice a week for two consecutive weeks.
  • the tumor volumes were measured twice a week and the tumor growth curve was plotted.
  • the results in FIG. 13 show that the G14 antibody showed significant tumor-suppressive activity with a tumor suppression rate of 90%.
  • mice aged 6-8 weeks were subcutaneously inoculated with 5 ⁇ 10 6 NALM6 tumor cells, and when the tumor volumes reached to 150 mm 3 , the mice were administered with different doses of CD24 antibodies intraperitoneally for treatment. The mice were administered twice a week for three consecutive weeks. The tumor volumes were measured twice a week and the tumor growth curve was plotted.
  • the results in FIGs. 14A and 14B show that both low-dose and high-dose of G14, huG14-235 and huG14-301 antibodies showed significant tumor-suppressive activity with both tumor inhibition rates greater than 90%.
  • mice aged 6-8 weeks were subcutaneously inoculated with 1 ⁇ 10 7 triple-negative breast cancer MDA-MB-468 cells, and when the tumor volume reached to 150 mm 3 , the mice were administered with CD24 antibodies intraperitoneally for treatment.
  • the mice were administered twice a week for four consecutive weeks (FIG. 15A) or three consecutive weeks (FIG. 15B) .
  • the tumor volumes were measured twice a week and the tumor growth curve was plotted.
  • the results in FIGs. 15A and 15B show that the G14, huG14-235 and huG14-301 antibodies antibody showed significant tumor-suppressive activity (G14 with a tumor suppression rate of 66%) .

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Abstract

L'invention concerne des anticorps de liaison à CD24 et des fragments de ceux-ci.
PCT/CN2023/126795 2022-10-27 2023-10-26 Anticorps dirigés contre cd24 et leurs utilisations WO2024088342A1 (fr)

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CNPCT/CN2022/128003 2022-10-27
CN2022128003 2022-10-27
CNPCT/CN2022/128297 2022-10-28
CN2022128297 2022-10-28
CN2023121689 2023-09-26
CNPCT/CN2023/121689 2023-09-26

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