WO2025002251A1 - Anticorps anti-gprc5d et son utilisation médicale - Google Patents

Anticorps anti-gprc5d et son utilisation médicale Download PDF

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Publication number
WO2025002251A1
WO2025002251A1 PCT/CN2024/101975 CN2024101975W WO2025002251A1 WO 2025002251 A1 WO2025002251 A1 WO 2025002251A1 CN 2024101975 W CN2024101975 W CN 2024101975W WO 2025002251 A1 WO2025002251 A1 WO 2025002251A1
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Prior art keywords
seq
gprc5d
amino acid
acid sequence
variable region
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Ceased
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PCT/CN2024/101975
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English (en)
Chinese (zh)
Inventor
柳颖
周雅琼
聂磊
李娜
王琪
吴振华
高章照
朱珊珊
齐健
许静雅
徐统
武兵元
王海彬
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Bioray Pharmaceutical Co Ltd
Bioray Pharmaceutical Hangzhou Co Ltd
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Bioray Pharmaceutical Co Ltd
Bioray Pharmaceutical Hangzhou Co Ltd
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Priority to CN202480031437.3A priority Critical patent/CN121311505A/zh
Publication of WO2025002251A1 publication Critical patent/WO2025002251A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure relates to biological, medical and clinical fields. Specifically, the present disclosure relates to anti-GPRC5D antibodies, bispecific antibodies based on the anti-GPRC5D antibodies, and medical uses thereof.
  • MM Multiple myeloma
  • the annual incidence of MM ranks second among hematological tumors, and with the aging of the population, the incidence of MM has an upward trend, and it is still incurable.
  • the targeted treatment methods for MM in clinical practice mainly include immunomodulatory drugs (thalidomide, lenalidomide, pomalidomide), proteasome inhibitors (bortezomib, carfilzomib, ixazomib) and anti-CD38 monoclonal antibodies (daratumumab).
  • immunomodulatory drugs thalidomide, lenalidomide, pomalidomide
  • proteasome inhibitors bortezomib, carfilzomib, ixazomib
  • anti-CD38 monoclonal antibodies daratumumab.
  • Monoclonal antibodies and CAR-T cell therapies targeting BCMA have also shown encouraging results, but for relapse cases related to low or negative BCMA expression, the efficacy of such drugs is not optimistic, so there is an urgent need for new treatment options.
  • G protein coupled receptor C5 family subtype D is an orphan receptor first identified in 2001. It is a seven-transmembrane protein.
  • the GPRC5D encoding gene is mapped to chromosome 12p13.3. The gene contains three exons and spans about 9.6 kb (H. Brauner-Osborne et al., Biochimica et Biophysica Actal. 2001, 1518(3): p.237-248).
  • GPRC5D is highly expressed on the surface of primary MM cells, while its expression in normal tissues is limited to the hair follicle area (Shinichi Inoue et al., Journal of Investigative Dermatology. 2004, 122(3): 565-573; Gao Y et al., PLoS ONE. 2016, 11(3): e0151118).
  • GPRC5D has an expression threshold of more than 50% in 65% of MM patients, and it is independent of BCMA expression (Smith et al., SCIENCE TRANSLATIONAL MEDICINE. 2019, eaau7746), suggesting that GPRC5D can still be used as a target for treatment of BCMA-negative cells.
  • the strong selective expression of GPRC5D in MM cells makes this gene and its encoded surface protein a promising marker for monitoring tumor burden and is expected to become a target for anti-myeloma antibodies (Yossi Cohen et al., Hematology. 2013, 18(6): 348-351).
  • Autoimmune diseases are characterized by autoantibodies, which are secreted by plasma cells. Autoantibodies provide diagnostic and prognostic criteria, play an essential role in disease pathogenesis, and serve as surrogate markers of disease activity (Martin and Chan, Immunity, 2004, 20(5)). Therefore, effective depletion of autoreactive plasma cells may be the key to curing these diseases.
  • an anti-GPRC5D antibody or an antigen-binding fragment thereof which comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein the GPRC5D-VH and the GPRC5D-VL are selected from any one of the following groups:
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 31;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 36;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 91;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 96;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 21;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 26;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 61;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 66;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 81;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 86;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 1;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 6;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 11;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 16;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 51;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 56;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 71;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 76;
  • the GPRC5D-VH comprises HCDR1, HCDR2, HCDR3;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 106;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 111;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 116;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 121;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 126;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 131;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 136;
  • the HCDRs and the LCDRs are identified according to the Kabat, Chothia or IMTG numbering systems.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein the GPRC5D-VH and the GPRC5D-VL are selected from any one of the following groups:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 45;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 49, and LCDR3 shown in SEQ ID NO: 50;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 33, HCDR2 shown in SEQ ID NO: 34, and HCDR3 shown in SEQ ID NO: 35;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 93, HCDR2 shown in SEQ ID NO: 94, and HCDR3 shown in SEQ ID NO: 95;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 98, LCDR2 shown in SEQ ID NO: 99, and LCDR3 shown in SEQ ID NO: 100;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, and HCDR3 shown in SEQ ID NO: 25;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 28, LCDR2 shown in SEQ ID NO: 29, and LCDR3 shown in SEQ ID NO: 30;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 83, SEQ ID NO: 84
  • the GPRC5D-VL comprises the HCDR2 shown in SEQ ID NO: 84, the HCDR3 shown in SEQ ID NO: 85;
  • the GPRC5D-VL comprises the LCDR1 shown in SEQ ID NO: 88, the LCDR2 shown in SEQ ID NO: 89, and the LCDR3 shown in SEQ ID NO: 90;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 8, LCDR2 shown in SEQ ID NO: 9, and LCDR3 shown in SEQ ID NO: 10;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 15;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 20;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 73, HCDR2 shown in SEQ ID NO: 74, and HCDR3 shown in SEQ ID NO: 75;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 78, LCDR2 shown in SEQ ID NO: 79, and LCDR3 shown in SEQ ID NO: 80;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 103, HCDR2 shown in SEQ ID NO: 104, and HCDR3 shown in SEQ ID NO: 105;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 108, LCDR2 shown in SEQ ID NO: 109, and LCDR3 shown in SEQ ID NO: 110;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 113, HCDR2 shown in SEQ ID NO: 114, and HCDR3 shown in SEQ ID NO: 115;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 118, LCDR2 shown in SEQ ID NO: 119, and LCDR3 shown in SEQ ID NO: 120;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 123, HCDR2 shown in SEQ ID NO: 124, and HCDR3 shown in SEQ ID NO: 125;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 128, LCDR2 shown in SEQ ID NO: 129, and LCDR3 shown in SEQ ID NO: 130;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 133, HCDR2 shown in SEQ ID NO: 134, and HCDR3 shown in SEQ ID NO: 135;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 138, LCDR2 shown in SEQ ID NO: 139, and LCDR3 shown in SEQ ID NO: 140.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 45;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 49, and LCDR3 shown in SEQ ID NO: 50.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 33, HCDR2 shown in SEQ ID NO: 34, and HCDR3 shown in SEQ ID NO: 35;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof as described above comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 93, HCDR2 shown in SEQ ID NO: 94, and HCDR3 shown in SEQ ID NO: 95;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 98, LCDR2 shown in SEQ ID NO: 99, and LCDR3 shown in SEQ ID NO: 100.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof as described in any of the preceding items comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein the GPRC5D-VH and the GPRC5D-VL are selected from any of the following groups:
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 41 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 41
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 46 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 46;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 31 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 31
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 36 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 36;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 91 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 91
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 96 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 96;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 21 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 21
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 26 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 26;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO:61 or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:61
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO:66 or comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:66;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 81 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 81
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 86 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 86;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 1 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 6 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 6;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 11 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 16 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 16;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 51 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 51
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 56 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 56;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 71 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 71
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 76 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 76;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 101 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 101
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 106 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 106;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 111 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 111
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 116 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 116;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 121 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 121
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 126 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 126;
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 131 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 131
  • GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 136 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 136.
  • the anti-GPRC5D antibody or its antigen binding A fragment where:
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 41 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 41
  • GPRC5D-VL comprises the amino acid sequence shown in SEQ ID NO: 46 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 46.
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 31 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 31
  • GPRC5D-VL comprises the amino acid sequence shown in SEQ ID NO: 36 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 36.
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 91 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 91
  • GPRC5D-VL comprises the amino acid sequence shown in SEQ ID NO: 96 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 96.
  • At least 85% means at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or a range between any two of the foregoing values, which can be an integer or a decimal.
  • the anti-GPRC5D antibody or antigen-binding fragment thereof as described in any of the preceding items is a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen-binding fragment thereof, or a chimeric antibody or antigen-binding fragment thereof.
  • the heavy chain variable region GPRC5D-VH and/or the light chain variable region GPRC5D-VL are humanized.
  • the antigen-binding fragment of any of the foregoing anti-GPRC5D antibodies is selected from any one of the following: Fab, scFv, Fv, Fab', F(ab') 2 , single domain antibody, scFab, linear antibody, and multispecific antibody.
  • any of the aforementioned anti-GPRC5D antibodies or antigen-binding fragments thereof further comprises a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is selected from human IgG1, human IgG2, human IgG3 and human IgG4, and the light chain constant region is selected from ⁇ and ⁇ light chain constant regions.
  • the heavy chain constant region is the heavy chain constant region of human IgG1, and the light chain constant region is the ⁇ light chain constant region.
  • the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 143, and the light chain constant region comprises the amino acid sequence of SEQ ID NO: 144.
  • any of the foregoing anti-GPRC5D antibodies or antigen-binding fragments thereof further comprises an Fc.
  • any of the aforementioned anti-GPRC5D antibodies comprises Fc of human IgG1, Fc of human IgG2, Fc of human IgG3, or Fc of human IgG4; preferably Fc of human IgG1.
  • the anti-GPRC5D antibody comprises a heavy chain set forth in SEQ ID NO: 149 or a sequence having at least 85% identity thereto, and a light chain set forth in SEQ ID NO: 150 or a sequence having at least 85% identity thereto.
  • the anti-GPRC5D antibody comprises:
  • the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 147 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 147, and
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 148 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 148.
  • the anti-GPRC5D antibody comprises:
  • the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 155 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 155, and
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 156 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 156.
  • the anti-GPRC5D antibody comprises:
  • the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 145 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 145, and
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 146 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 146.
  • the anti-GPRC5D antibody comprises:
  • the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 151 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 151, and
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 152 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 152.
  • the anti-GPRC5D antibody comprises:
  • the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 153 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 153, and
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 154 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 154.
  • the anti-GPRC5D antibody comprises:
  • the heavy chain comprises the amino acid sequence as shown in SEQ ID NO: 149 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 149, and
  • the light chain comprises the amino acid sequence shown in SEQ ID NO: 150 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 150.
  • the anti-GPRC5D antibody comprises the heavy chain shown in SEQ ID NO: 145 and the light chain shown in SEQ ID NO: 146.
  • the anti-GPRC5D antibody comprises the A heavy chain, and a light chain as shown in SEQ ID NO:148.
  • the anti-GPRC5D antibody comprises the heavy chain shown in SEQ ID NO: 149 and the light chain shown in SEQ ID NO: 150.
  • the anti-GPRC5D antibody comprises the heavy chain shown in SEQ ID NO: 151 and the light chain shown in SEQ ID NO: 152.
  • the anti-GPRC5D antibody comprises the heavy chain shown in SEQ ID NO: 153 and the light chain shown in SEQ ID NO: 154.
  • the anti-GPRC5D antibody comprises the heavy chain shown in SEQ ID NO: 155 and the light chain shown in SEQ ID NO: 156.
  • the anti-GPRC5D antibodies or antigen-binding fragments thereof of the present disclosure bind to cells expressing human GPRC5D with an EC50 of no greater than 0.4, 0.39, 0.38, 0.37, 0.36, 0.35, 0.34, 0.33, 0.32, 0.31, 0.30, 0.29, 0.28, 0.27, or 0.26 nM.
  • the present disclosure provides a bispecific antibody comprising:
  • the present disclosure provides a bispecific antibody comprising at least one first binding moiety that specifically binds to GPRC5D and at least one second binding moiety that specifically binds to CD3, wherein the first binding moiety that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein the GPRC5D-VH and the GPRC5D-VL are selected from any one of the following groups:
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 41;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 46;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 31;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 36;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 91;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 96;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 21;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 26;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 61;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 66;
  • the GPRC5D-VH comprises HCDR1, HCDR2, HCDR3;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 86;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 1;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 6;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 11;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 16;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 51;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 56;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 71;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 76;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 101;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 106;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 111;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 116;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 121;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 126;
  • the GPRC5D-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 131;
  • the GPRC5D-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 136;
  • amino acid sequences of the HCDR and the LCDR are determined according to the Kabat, Chothia or IMTG numbering system.
  • a bispecific antibody comprising at least one first binding moiety that specifically binds to GPRC5D and at least one second binding moiety that specifically binds to CD3, wherein the first binding moiety that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein the GPRC5D-VH and the GPRC5D-VL are selected from any one of the following groups:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 45;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 49, and LCDR3 shown in SEQ ID NO: 50;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 33, HCDR2 shown in SEQ ID NO: 34, and HCDR3 shown in SEQ ID NO: 35;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 93, HCDR2 shown in SEQ ID NO: 94, and HCDR3 shown in SEQ ID NO: 95;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 98, LCDR2 shown in SEQ ID NO: 99, and LCDR3 shown in SEQ ID NO: 100;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, and HCDR3 shown in SEQ ID NO: 25;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 28, LCDR2 shown in SEQ ID NO: 29, and LCDR3 shown in SEQ ID NO: 30;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 63, HCDR2 shown in SEQ ID NO: 64, and HCDR3 shown in SEQ ID NO: 65;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 68, LCDR2 shown in SEQ ID NO: 69, and LCDR3 shown in SEQ ID NO: 70;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 83, HCDR2 shown in SEQ ID NO: 84, and HCDR3 shown in SEQ ID NO: 85;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 88, LCDR2 shown in SEQ ID NO: 89, and LCDR3 shown in SEQ ID NO: 90;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 8, LCDR2 shown in SEQ ID NO: 9, and LCDR3 shown in SEQ ID NO: 10;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 15;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 20;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 53, HCDR2 shown in SEQ ID NO: 54, and HCDR3 shown in SEQ ID NO: 55;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 58, LCDR2 shown in SEQ ID NO: 59, and LCDR3 shown in SEQ ID NO: 60;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 73, HCDR2 shown in SEQ ID NO: 74, and HCDR3 shown in SEQ ID NO: 75;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 78, LCDR2 shown in SEQ ID NO: 79, and LCDR3 shown in SEQ ID NO: 80;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 103, SEQ ID NO:
  • the GPRC5D-VL comprises the HCDR1 shown in SEQ ID NO: 108, the LCDR2 shown in SEQ ID NO: 109, and the LCDR3 shown in SEQ ID NO: 110;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 113, HCDR2 shown in SEQ ID NO: 114, and HCDR3 shown in SEQ ID NO: 115;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 118, LCDR2 shown in SEQ ID NO: 119, and LCDR3 shown in SEQ ID NO: 120;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 123, HCDR2 shown in SEQ ID NO: 124, and HCDR3 shown in SEQ ID NO: 125;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 128, LCDR2 shown in SEQ ID NO: 129, and LCDR3 shown in SEQ ID NO: 130;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 133, HCDR2 shown in SEQ ID NO: 134, and HCDR3 shown in SEQ ID NO: 135;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 138, LCDR2 shown in SEQ ID NO: 139, and LCDR3 shown in SEQ ID NO: 140.
  • a bispecific antibody comprising at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 45;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 49, and LCDR3 shown in SEQ ID NO: 50.
  • a bispecific antibody comprising at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 33, HCDR2 shown in SEQ ID NO: 34, and HCDR3 shown in SEQ ID NO: 35;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
  • a bispecific antibody comprising at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 93, HCDR2 shown in SEQ ID NO: 94, and HCDR3 shown in SEQ ID NO: 95;
  • the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 98, LCDR2 shown in SEQ ID NO: 99, and LCDR3 shown in SEQ ID NO: 100.
  • a bispecific antibody comprising at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 41 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 41
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 46 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 46;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 31 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 31
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 36 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 36;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 91 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 91
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 96 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 96;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 21 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 21
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 26 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 26;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 61 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 61
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 66 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 66;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 81 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 81
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 86 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 86;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 1 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 6 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 6;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 11 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 11
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 16 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 16;
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 51 or comprises the amino acid sequence shown in SEQ ID NO: 51 has at least 85% sequence identity
  • GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 56 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 56;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 71 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 71
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 76 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 76;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 101 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 101
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 106 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 106;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 111 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 111
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 116 or comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 116;
  • GPRC5D-VH comprises the amino acid sequence set forth in SEQ ID NO: 121 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 121
  • GPRC5D-VL comprises the amino acid sequence set forth in SEQ ID NO: 126 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 126;
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 131 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 131
  • GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 136 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 136.
  • a bispecific antibody comprising at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 41 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 41
  • GPRC5D-VL comprises the amino acid sequence shown in SEQ ID NO: 46 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 46.
  • a bispecific antibody comprising at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 31 or comprises the amino acid sequence of SEQ ID NO: 31 has at least 85% sequence identity
  • GPRC5D-VL comprises the amino acid sequence shown in SEQ ID NO: 36 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 36.
  • a bispecific antibody comprising at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion that specifically binds to GPRC5D comprises a heavy chain variable region GPRC5D-VH and a light chain variable region GPRC5D-VL, wherein:
  • GPRC5D-VH comprises the amino acid sequence shown in SEQ ID NO: 91 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 91
  • GPRC5D-VL comprises the amino acid sequence shown in SEQ ID NO: 96 or comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 96.
  • At least 85% means at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or a range between any two of the foregoing values, which can be an integer or a decimal.
  • any of the aforementioned bispecific antibodies, wherein the second binding moiety that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein,
  • the CD3-VH comprises HCDR1, HCDR2, and HCDR3 in the sequence shown in SEQ ID NO: 202;
  • the CD3-VL comprises LCDR1, LCDR2, and LCDR3 in the sequence shown in SEQ ID NO: 203;
  • the HCDR and the LCDR are determined according to the Kabat, Chothia or IMTG numbering system.
  • any of the aforementioned bispecific antibodies, wherein the second binding moiety that specifically binds to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein,
  • the CD3-VH comprises HCDR1 shown in SEQ ID NO: 204, HCDR2 shown in SEQ ID NO: 205, and HCDR3 shown in SEQ ID NO: 206; and the CD3-VL comprises LCDR1 shown in SEQ ID NO: 207, LCDR2 shown in SEQ ID NO: 208, and LCDR3 shown in SEQ ID NO: 209.
  • the second binding portion that specifically binds to CD3 comprises the heavy chain variable region CD3-VH shown in SEQ ID NO: 202, and the light chain variable region CD3-VL shown in SEQ ID NO: 203.
  • the heavy chain variable region GPRC5D-VH and/or the light chain variable region GPRC5D-VL are humanized.
  • the bispecific antibody comprises at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion comprises GPRC5D-VH shown in SEQ ID NO: 41 and GPRC5D-VL shown in SEQ ID NO: 46; the second binding portion comprises the heavy chain variable region CD3-VH shown in SEQ ID NO: 202, and the light chain variable region CD3-VL shown in SEQ ID NO: 203.
  • the bispecific antibody comprises at least one specific binding A first binding portion of GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion comprises GPRC5D-VH shown in SEQ ID NO: 31 and GPRC5D-VL shown in SEQ ID NO: 36; the second binding portion comprises the heavy chain variable region CD3-VH shown in SEQ ID NO: 202, and the light chain variable region CD3-VL shown in SEQ ID NO: 203.
  • the bispecific antibody comprises at least one first binding portion that specifically binds to GPRC5D and at least one second binding portion that specifically binds to CD3, wherein the first binding portion comprises GPRC5D-VH shown in SEQ ID NO: 91 and GPRC5D-VL shown in SEQ ID NO: 96; the second binding portion comprises the heavy chain variable region CD3-VH shown in SEQ ID NO: 202, and the light chain variable region CD3-VL shown in SEQ ID NO: 203.
  • the bispecific antibody as described in any of the preceding items, wherein the bispecific antibody further comprises an Fc region, wherein the Fc region comprises two subunits (a first subunit Fc1 and a second subunit Fc2).
  • the two subunits are a first subunit Fc1 and a second subunit Fc2 having identical or different amino acid sequences.
  • the first subunit Fc1 and the second subunit Fc2 each independently have one or more amino acid substitutions that reduce Fc region homodimerization.
  • the first subunit Fc1 and the second subunit Fc2 each independently have an amino acid substitution of the knob-in-hole technique.
  • the first subunit Fc1 comprises a hole structure according to the knob-in-hole KIH technology
  • the second subunit Fc2 comprises a knob structure according to the knob-in-hole KIH technology.
  • the second subunit Fc2 comprises a hole structure according to the knob-in-hole KIH technology; and the first subunit Fc1 comprises a knob structure according to the knob-in-hole KIH technology.
  • the amino acid at position 366 of the first subunit Fc1 is S
  • the amino acid at position 368 is A
  • the amino acid at position 407 is V
  • the amino acid at position 366 of the second subunit Fc2 is W; numbering is based on the EU index.
  • the Fc region is an IgG Fc region, particularly an IgG1 Fc region.
  • the Fc region is a human IgG1 Fc region and the amino acids at positions 234 and 235 are A, numbering according to the EU index.
  • the Fc1 has the amino acid sequence shown in SEQ ID NO: 199
  • the Fc2 has the amino acid sequence shown in SEQ ID NO: 197 or 198.
  • the bispecific antibody as described in any of the preceding items comprises at least one first binding moiety that specifically binds to GPRC5D, at least one second binding moiety that specifically binds to CD3, and an Fc region.
  • the first binding moiety that specifically binds GPRC5D is a Fab; and
  • the second binding moiety that specifically binds to CD3 is a scFv.
  • the first binding moiety that specifically binds GPRC5D is a scFv; and the second binding moiety that specifically binds CD3 is a Fab.
  • the first binding moiety that specifically binds GPRC5D is a Fab
  • the second binding moiety that specifically binds CD3 is a Fab
  • the first binding moiety that specifically binds GPRC5D, the second binding moiety that specifically binds CD3, and one subunit of the Fc region are connected directly or through a linker in the order from N-terminus to C-terminus.
  • the second binding moiety that specifically binds CD3, the first binding moiety that specifically binds GPRC5D, and one subunit of the Fc region are connected directly or through a linker in the order from N-terminus to C-terminus.
  • the first binding moiety that specifically binds to GPRC5D, a subunit of the Fc region, and the second binding moiety that specifically binds to CD3 are connected directly or through a linker in the order from N-terminus to C-terminus.
  • the second binding moiety that specifically binds to CD3, a subunit of the Fc region, and the first binding moiety that specifically binds to GPRC5D are connected directly or through a linker in the order from N-terminus to C-terminus.
  • the bispecific antibody as described in any of the preceding items, wherein the bispecific antibody comprises: a first chain shown in formula a, a second chain shown in formula b, and a third chain shown in formula c;
  • the first subunit and the second subunit are interchangeable;
  • the bispecific antibody as described in any of the preceding items, wherein the bispecific antibody comprises a first chain shown in formula a, a second chain shown in formula b, and a third chain shown in formula c;
  • the bispecific antibody as described in any of the preceding items, wherein the bispecific antibody comprises: a first chain shown in formula a, a second chain shown in formula b, a third chain shown in formula d, and a fourth chain shown in formula e;
  • the first subunit and the second subunit are interchangeable;
  • the bispecific antibody as described in any of the preceding items wherein the bispecific antibody
  • the body comprises a first chain represented by formula a, a second chain represented by formula b, a third chain represented by formula d, and a fourth chain represented by formula e.
  • the bispecific antibody as described in any of the preceding items wherein the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 41, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 46; and
  • the CD3-VH contains the amino acid sequence of SEQ ID NO: 202
  • the CD3-VL contains the amino acid sequence of SEQ ID NO: 203.
  • the bispecific antibody as described in any of the preceding items wherein the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 31, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 36; and
  • the CD3-VH contains the amino acid sequence of SEQ ID NO: 202
  • the CD3-VL contains the amino acid sequence of SEQ ID NO: 203.
  • the bispecific antibody as described in any of the preceding items wherein the GPRC5D-VH comprises the amino acid sequence of SEQ ID NO: 91, and the GPRC5D-VL comprises the amino acid sequence of SEQ ID NO: 96; and
  • the CD3-VH comprises the amino acid sequence of SEQ ID NO: 202
  • the CD3-VL comprises the amino acid sequence of SEQ ID NO: 203.
  • the linker is a peptide linker, preferably a flexible peptide linker.
  • the length of the peptide linker is 3-15 amino acid residues.
  • the peptide linker has a structure of (GGGGS)n(SEQ ID NO: 217), wherein n is an integer from 1 to 8. In some embodiments, n is 2, 3, 4, 5 or 6.
  • the peptide linker is as shown in SEQ ID NO: 200.
  • the bispecific antibody as described in any of the preceding items wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 201.
  • the antigen binding molecule as described in any of the preceding items wherein the CL is the light chain constant region of an antibody. In some embodiments, the antigen binding molecule as described in any of the preceding items, wherein the CL is the light chain constant region of kappa or lamada. In some embodiments, wherein the CL comprises the amino acid sequence of SEQ ID NO: 144.
  • the bispecific antibody comprises: the first chain shown in SEQ ID NO: 161, the second chain shown in SEQ ID NO: 163, and the third chain shown in SEQ ID NO: 159.
  • the bispecific antibody comprises: the first chain shown in SEQ ID NO: 165, the second chain shown in SEQ ID NO: 167, and the third chain shown in SEQ ID NO: 159.
  • the bispecific antibody comprises: the first chain shown in SEQ ID NO: 169, the second chain shown in SEQ ID NO: 171, and the third chain shown in SEQ ID NO: 159.
  • the bispecific antibody comprises: a first chain as shown in SEQ ID NO: 173, a second chain as shown in SEQ ID NO: 175, a third chain as shown in SEQ ID NO: 177, The fourth chain is shown in SEQ ID NO:179.
  • the bispecific antibody comprises: a first chain shown in SEQ ID NO: 161, a second chain shown in SEQ ID NO: 163, and a third chain shown in SEQ ID NO: 159.
  • the bispecific antibody comprises: a first chain shown in SEQ ID NO: 165, a second chain shown in SEQ ID NO: 167, and a third chain shown in SEQ ID NO: 159.
  • the bispecific antibody comprises: a first chain shown in SEQ ID NO: 169, a second chain shown in SEQ ID NO: 171, and a third chain shown in SEQ ID NO: 159.
  • the bispecific antibody comprises: a first chain shown in SEQ ID NO: 173, a second chain shown in SEQ ID NO: 175, a third chain shown in SEQ ID NO: 177, and a fourth chain shown in SEQ ID NO: 179.
  • the bispecific antibodies of the present disclosure bind to cells expressing human GPRC5D; preferably HEK293T cells expressing hGPRC5D, with an EC50 of no higher than 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.49, 2.48, 2.47, 2.46, 2.45, or 2.44 nM.
  • the present disclosure provides a pharmaceutical composition comprising:
  • the present disclosure also provides an isolated nucleic acid encoding any of the aforementioned bispecific antibodies or anti-GPRC5D antibodies or antigen-binding fragments thereof.
  • the nucleic acid is selected from any of the following groups:
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 42 and SEQ ID NO: 47;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 32 and SEQ ID NO: 37;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 92 and SEQ ID NO: 97;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 22 and SEQ ID NO: 27;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 62 and SEQ ID NO: 67;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 82 and SEQ ID NO: 87;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 7;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 17;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 52 and SEQ ID NO: 57;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 72 and SEQ ID NO: 77;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 102 and SEQ ID NO: 107;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 112 and SEQ ID NO: 117;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 122 and SEQ ID NO: 127;
  • the nucleic acid contains the sequences shown in SEQ ID NO: 132 and SEQ ID NO: 137.
  • the nucleic acid is selected from any of the following groups:
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 166 and SEQ ID NO: 168;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 162 and SEQ ID NO: 164;
  • the nucleic acid comprises the sequences shown in SEQ ID NO: 160, SEQ ID NO: 170 and SEQ ID NO: 172;
  • the nucleic acid contains the sequences shown in SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178 and SEQ ID NO: 180.
  • nucleotide sequences capable of "encoding the bispecific antibodies or anti-GPRC5D antibodies or antigen-binding fragments thereof of the present disclosure are not limited to these specific sequences, because equivalent nucleotide sequences can be used to encode the same amino acid sequence depending on the codon preference and codon degeneracy of the expression host.
  • the present disclosure further provides a host cell, which comprises the aforementioned nucleic acid or the aforementioned vector or is used to express the bispecific antibody or anti-GPRC5D antibody or antigen-binding fragment thereof of the present disclosure.
  • the present disclosure also provides a method for treating a disease, comprising administering to a subject a preventively or therapeutically effective amount of any of the aforementioned bispecific antibodies, anti-GPRC5D antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions.
  • the disease is cancer or an autoimmune disease.
  • the disease is selected from the group consisting of: multiple myeloma, Waldenstrom's macroglobulinemia, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma, and malignant melanoma.
  • the disease is associated with abnormal expression of GPRC5D.
  • cancer cells of the cancer express GPRC5D protein.
  • the cancer is a hematological cancer.
  • the cancer is a solid tumor.
  • the present disclosure also provides a method for preventing or treating multiple myeloma, comprising administering to a subject a preventively or therapeutically effective amount of any of the aforementioned bispecific antibodies, anti-GPRC5D antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions.
  • the present disclosure also provides any of the aforementioned bispecific antibodies, anti-GPRC5D antibodies Use of a nucleic acid or an antigen-binding fragment thereof, a nucleic acid or a pharmaceutical composition in the preparation of a drug for treating or preventing a disease.
  • the disease is cancer or an autoimmune disease.
  • the disease is selected from the group consisting of: multiple myeloma, Waldenstrom's macroglobulinemia, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma, and malignant melanoma.
  • the disease is associated with abnormal expression of GPRC5D protein.
  • cancer cells of the cancer express GPRC5D protein.
  • the cancer is a hematological cancer.
  • the cancer is a solid tumor.
  • the disease is multiple myeloma.
  • the present disclosure also provides any of the aforementioned bispecific antibodies, anti-GPRC5D antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions for use as a medicament.
  • the medicament is for treating a disease.
  • the disease is cancer or an autoimmune disease.
  • the disease is selected from the group consisting of: multiple myeloma, Waldenstrom's macroglobulinemia, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, liver cancer, pancreatic cancer, colorectal cancer, esophageal cancer, bladder cancer, cervical cancer, blood cancer, lymphoma and malignant melanoma.
  • the disease is associated with abnormal expression of GPRC5D protein.
  • cancer cells of the cancer express GPRC5D protein.
  • the cancer is a hematological cancer.
  • the cancer is a solid tumor.
  • the medicament is for treating multiple myeloma.
  • the bispecific antibodies, anti-GPRC5D antibodies or antigen-binding fragments thereof disclosed herein are used for immunoassays.
  • the immunoassays include contacting the biological samples with the bispecific antibodies, anti-GPRC5D antibodies or antigen-binding fragments disclosed herein to detect cancer cells in the biological samples from the patients, or the expression of GPRC5D.
  • the specific antibodies, anti-GPRC5D antibodies or antigen-binding fragments disclosed herein can be coupled with fluorescent labels or other labels.
  • the present disclosure further provides a medicine kit, comprising at least one container, wherein each container independently comprises: any one of the aforementioned bispecific antibodies, anti-GPRC5D antibodies or antigen-binding fragments thereof, nucleic acids or pharmaceutical compositions of the present disclosure.
  • kits contain one or more of the following components: assay reagents, buffers, and antibodies of the disclosure.
  • these reagents/kits may include instructions.
  • Figure 1 shows the results of the binding activity test between the anti-GPRC5D antibody produced by hybridoma and GPRC5D of different species, wherein WT represents wild-type HEK293T cells.
  • FIG2 shows the results of the binding activity test of the anti-GPRC5D antibody produced by the hybridoma to human GPRC5A, GPRC5B and GPRC5C.
  • FIG3 shows the results of binding activity detection between anti-GPRC5D ⁇ CD3 bispecific antibodies Bs71D8, Bs80A8, Bs155C2 and positive control BM03 and HEK293T_hGPRC5D cells overexpressing human_GPRC5D.
  • FIG4 shows the results of binding activity detection of anti-GPRC5D ⁇ CD3 bispecific antibodies Bs80A8, 80A8KIH and positive control DuobodyBM with HEK293T_hGPRC5D cells overexpressing human_GPRC5D.
  • FIG. 5 shows the specific activation of human T cells by anti-GPRC5D ⁇ CD3 bispecific antibodies Bs71D8, Bs80A8, Bs155C2 and the positive control BM03 in the presence of MM.1R cells.
  • FIG6 shows the specific activation of human T cells by the anti-GPRC5D ⁇ CD3 bispecific antibody Bs80A8 and the positive control DuobodyBM in the presence of MM.1R cells.
  • FIG. 7 shows the results of detecting T cell killing activity induced by anti-GPRC5D ⁇ CD3 bispecific antibodies Bs71D8, Bs80A8, Bs155C2 and the positive control BM03 in the presence of MM.1R cells.
  • FIG8 shows the results of detecting T cell killing activity induced by the anti-GPRC5D ⁇ CD3 bispecific antibody Bs80A8 and the positive control DuobodyBM in the presence of MM.1R cells.
  • FIG. 9 shows the results of detecting T cell killing activity induced by anti-GPRC5D ⁇ CD3 bispecific antibodies Bs71D8, Bs80A8, Bs155C2 and the positive control BM03 in the presence of MM.1S cells.
  • FIG. 10 shows the results of detecting T cell killing activity induced by the anti-GPRC5D ⁇ CD3 bispecific antibody Bs80A8 and the positive control DuobodyBM in the presence of MM.1S cells.
  • Figure 11 shows the results of T cell killing activity detection induced by anti-GPRC5D ⁇ CD3 bispecific antibodies Bs71D8, Bs80A8, Bs155C2 and positive control BM03 in the presence of RPMI 8226 cells.
  • Figure 12 shows the results of T cell killing activity detection induced by anti-GPRC5D ⁇ CD3 bispecific antibody Bs80A8 and positive control DuobodyBM in the presence of RPMI 8226 cells.
  • FIG. 13 shows the in vivo inhibitory effect of the anti-GPRC5D ⁇ CD3 bispecific antibody on multiple myeloma.
  • the target refers to the object to which the therapeutic active ingredient (bispecific antibody, anti-GPRC5D antibody or its antigen-binding fragment) of the present disclosure is directed.
  • the target can be a nucleic acid (gene, mRNA, etc.) or a protein (precursor, mature protein, isoform, modified form, free, surface expressed).
  • the target refers in particular to a protein.
  • the bispecific antibody, anti-GPRC5D antibody or its antigen-binding fragment of the present disclosure targets GPRC5D.
  • the bispecific antibody of the present disclosure targets GPRC5D and CD3.
  • nucleotide or amino acid information of GPRC5D and CD3 as targets is well known in the art, and can be obtained, for example but not limited to, from literature or databases.
  • GPRC5D should be understood broadly, including various forms of molecules of GPRC5D in various stages, such as, but not limited to, molecules produced by the GPRC5D gene during amplification, replication, transcription, splicing, processing, translation, and modification, such as cDNA, mRNA, precursor protein, mature protein, and fragments thereof.
  • GPRC5D refers to human GPRC5D.
  • human GPRC5D refers to mature GPRC5D protein, especially GPRC5D expressed on the cell surface.
  • CD3 should be understood broadly, including various forms of CD3 molecules at various stages, such as, but not limited to, molecules generated during amplification, replication, transcription, splicing, processing, translation, and modification of CD3 genes, such as cDNA, mRNA, precursor protein, mature protein, and fragments thereof.
  • CD3 refers to human CD3.
  • human CD3 refers to mature CD3 protein, especially CD3 expressed on the cell surface.
  • Antigen refers to a molecule or part thereof that can be selectively recognized or bound by an antigen binding molecule (e.g., a bispecific antibody, an anti-GPRC5D antibody, or an antigen binding fragment thereof of the present disclosure).
  • An antigen may have one or more epitopes.
  • Epitope refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment.
  • An epitope can be formed by continuous amino acids (linear epitope); or comprise non-continuous amino acids (conformational epitope).
  • An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids. Routine methods in the art can be used to screen for antibodies or their antigen-binding fragments that bind to a specific epitope, such as, but not limited to, alanine scanning, peptide cleavage analysis, epitope extraction, and chemical modification of the antigen.
  • Antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and full-length antibodies, as long as they exhibit the desired antigen-binding activity.
  • natural IgG antibodies are heterotetrameric proteins consisting of two light chains and two heavy chains bound by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH) and three constant domains (CH1, CH2, and CH3). From N to C-terminus, each light chain has a variable region (VL) and a constant light domain (CL).
  • VH variable region
  • CH1, CH2, and CH3 constant domains
  • CL constant light domain
  • a technician can determine the specific meaning of "antibody”.
  • the "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. Antibodies are divided into five main classes: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes). For example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as ⁇ , ⁇ , ⁇ , ⁇ and ⁇ , respectively.
  • Specific binding means that the bispecific antibody, anti-GPRC5D antibody or its antigen-binding fragment of the present disclosure binds to the targeted antigen or its epitope with higher affinity than other antigens or their epitopes.
  • KD equilibrium dissociation constant
  • KD can be measured using known methods, such as FACS or surface plasmon resonance.
  • Affinity is used to describe the strength of non-covalent interactions between bispecific antibodies, anti-GPRC5D antibodies or their antigen-binding fragments and their antigens or their epitopes. Affinity is generally represented by an equilibrium dissociation constant (KD).
  • kassoc or “ka” refers to the association rate of a specific antibody-antigen interaction
  • kdis or “kd” is intended to refer to the dissociation rate of a specific antibody-antigen interaction.
  • KD is obtained from the ratio of kd to ka (ie, kd/ka), expressed as a molar concentration.
  • “Specific binding” does not exclude cross-reactivity to homologous antigens in other species (e.g. crab-eating macaque (Macaca fascicularis), chimpanzee (Pan troglodytes) or marmoset (Callithrix jacchus).
  • species e.g. crab-eating macaque (Macaca fascicularis), chimpanzee (Pan troglodytes) or marmoset (Callithrix jacchus).
  • anti-GPRC5D "specifically binds to GPRC5D” means that the bispecific antibody, anti-GPRC5D antibody or its antigen-binding fragment can specifically recognize or bind to GPRC5D (or its epitope).
  • anti-GPRC5D "specifically binds to GPRC5D” means that the bispecific antibody, anti-GPRC5D antibody or its antigen-binding fragment can specifically recognize or bind to GPRC5D (or its epitope).
  • Specifically binds to CD3 means that the bispecific antibody can specifically recognize or bind to CD3 (or its epitope).
  • Binding moiety refers to a module that specifically binds to an antigen or an epitope thereof.
  • exemplary binding moieties include antigen binding domains of antibodies, such as but not limited to heavy chain variable regions, light chain variable regions.
  • a binding moiety that specifically binds to GPRC5D refers to a module that can bind to GPRC5D or its epitope with sufficient affinity.
  • the binding moiety comprises an antigen binding fragment as defined in the present disclosure.
  • the binding moiety that specifically binds to GPRC5D is a Fab or scFv.
  • a binding moiety that specifically binds to CD3 refers to a module that can bind to CD3 or an epitope thereof with sufficient affinity.
  • the binding moiety comprises an antigen binding fragment as defined in the present disclosure.
  • the binding moiety that specifically binds to CD3 is a Fab or scFv.
  • Fully human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • a “humanized antibody” refers to an antibody produced by grafting non-human CDR sequences into the framework of a human antibody variable region.
  • a “chimeric antibody” is an antibody formed by fusing the variable region of an antibody from a first species (such as mouse) with the constant region of an antibody from another species (such as human).
  • Antibody fragments or "antigen-binding fragments” are molecules other than intact antibodies, which contain a portion of an intact antibody that retains the antigen-binding ability of the intact antibody.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibodies (e.g., scFv); and multispecific antibodies formed from antibody fragments.
  • Fab consists of a light chain and the CH1 and variable region of a heavy chain.
  • Fab' fragment which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain.
  • F(ab') 2 fragment which is a bivalent fragment formed by two Fab' fragments connected by a disulfide bridge in the hinge region part.
  • Fab'-SH refers to Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • Single-chain Fab fragment is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following orders from N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein the linker is a peptide linker.
  • the peptide linker is a polypeptide of at least 30 amino acids, preferably a polypeptide of 32-50 amino acids.
  • “Diabodies” have two antigen-binding sites and include a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to each other in the same polypeptide chain.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • a “linear antibody” comprises a pair of tandem Fd segments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding regions.
  • a "Fv fragment” has the VL and VH domains of one arm of an antibody.
  • a “single domain antibody” is an antibody fragment that comprises all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody.
  • Single-chain antibody is an antibody composed of the heavy chain variable region and the light chain variable region connected by a linker.
  • Fc contains two heavy chain fragments including CH2 and CH3 domains. The two heavy chain fragments are held together by disulfide bonds and by the hydrophobic interaction of the CH3 domain.
  • Variable region refers to the domain of an antibody or antigen-binding fragment that binds to an antigen.
  • CDR complementarity determining region
  • VH and VL each contain four framework regions (FRs) and three complementarity determining regions (CDRs).
  • VH contains three CDR regions: HCDR1, HCDR2, and HCDR3;
  • VL contains three CDR regions: LCDR1, LCDR2, and LCDR3.
  • Each VH and VL has the following sequence from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • FR Framework or "FR” refers to the variable domain residues outside the hypervariable region (CDR) residues.
  • the FR of the variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Therefore, HVR and FR sequences usually appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.
  • the heavy chain variable region in the binding portion that specifically binds to GPRC5D is denoted as GPRC5D-VH, and the light chain variable region is denoted as GPRC5D-VL.
  • CD3-VH The heavy chain variable region in the binding portion that specifically binds to CD3
  • CD3-VL The light chain variable region
  • the three CDR regions in GPRC5D-VH are represented as GPRC5D-HCDR1, GPRC5D-HCDR2 and GPRC5D-HCDR3; the three CDR regions in GPRC5D-VL are represented as GPRC5D-LCDR1, GPRC5D-LCDR2 and GPRC5D-LCDR3.
  • CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 The three CDR regions in CD3-VH are denoted as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3; the three CDR regions in CD3-VL are represented as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 respectively.
  • the amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: the Kabat numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the Chothia numbering system, and the ImMunoGenTics (IMGT) numbering system (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278).
  • the Kabat numbering system see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD
  • the Chothia numbering system the Chothia numbering system
  • IMGT ImMunoGenTics
  • Linker refers to a connecting unit that connects two polypeptide fragments.
  • the linker may be a peptide linker, which comprises one or more amino acids, a typical example being about 1-40 amino acids.
  • the linker disclosed herein may be any suitable peptide chain, as long as the antibody or antigen-binding fragment can exhibit the desired antigen-binding activity.
  • the peptide linker may be a flexible peptide of 1-40 amino acid residues.
  • the peptide linkers each independently have a structure of (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8.
  • the peptide linker is shown in SEQ ID NO: 200.
  • identity refers to the degree (percentage) to which the amino acids/nucleic acids of the two sequences are identical at equivalent positions when the two sequences are optimally aligned. During the alignment process, gaps are introduced if necessary to obtain maximum sequence identity. Sequence identity is determined by techniques known in the art, such as computer software (BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign).
  • the heavy chain variable region comprises an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 41
  • the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 45, and amino acid mutations are allowed to be introduced in regions outside the CDRs, thereby having at least 85% sequence identity with SEQ ID NO: 41.
  • amino acid mutation includes amino acid substitution, deletion, insertion, modification or any combination thereof. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, as long as the final construct has the desired characteristics, such as amino acid substitutions that reduce homodimerization in the Fc region. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for the desired activity, such as retained/improved antigen binding, reduced immunogenicity. Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain.
  • the amino acid mutation is a non-conservative amino acid substitution, that is, an amino acid is replaced with another amino acid having different structural and/or chemical properties.
  • Amino acid mutations can be generated using genetic or chemical methods known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc.
  • the Fc region is substituted so that it has a hole structure or a protrusion structure according to the knob-into-hole technique (KIH).
  • the hole structure (hole) is introduced in the first subunit and the protrusion structure (knob) is introduced in the second subunit.
  • the protrusion structure is positioned in the hole structure, promoting the formation of heterodimers and inhibiting the generation of homodimers.
  • the protrusion structure is constructed by replacing smaller amino acid side chains with larger amino acid side chains (e.g., tyrosine or tryptophan).
  • the hole structure is constructed by replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine).
  • Optional amino acid substitutions are shown in the following table:
  • the amino acid at position 366 is substituted with S
  • the amino acid at position 368 is substituted with A
  • the amino acid at position 407 is substituted with V
  • the amino acid at position 366 in the second subunit is substituted with W (numbering is according to the EU index).
  • other techniques for modifying the CH3 domain of the heavy chain to achieve heterodimerization are also known in the art, such as WO1996027011A1, WO1998050431, EP1870459, WO2007110205, WO2009089004, WO2010129304, WO201190754, WO2011143545, WO2012058768, WO2013157954 and WO2013096291.
  • amino acid residue at a specific site can be indicated by position + amino acid residue, for example, 366S, 368A means that the amino acid residue at site 366 is S and the amino acid residue at site 368 is A.
  • the C-terminus of the Fc region is a complete C-terminus ending with PGK, it can still be a truncated C-terminus.
  • one or two C-terminal residues are removed from the truncated C-terminus (e.g., a truncated C-terminus ending with PG).
  • antibodies with complete C-termini and antibodies with truncated C-termini may be included.
  • Bispecific antibody refers to an antibody that can specifically bind to two different antigens (or two different epitopes of the same antigen).
  • the prior art has disclosed bispecific antibodies of various structures.
  • Exemplary bispecific antibody structural models include KIH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMA
  • the bispecific antibodies disclosed herein are not limited to a specific molecular structure, as long as they have the desired
  • the antigen binding portion can be any antibody fragment with antigen binding activity, which is fused via a peptide linker.
  • the bispecific antibody of the present disclosure comprises a first chain having a structure shown in Formula a, a second chain having a structure shown in Formula b, and a third chain having a structure shown in Formula c:
  • Fc1 and Fc2 are interchangeable.
  • the bispecific antibody of the present disclosure comprises a first chain having a structure shown in Formula a, a second chain having a structure shown in Formula b, a third chain having a structure shown in Formula d, and a fourth chain having a structure shown in Formula e:
  • Fc1 and Fc2 are interchangeable.
  • anti-GPRC5D antibodies or antigen-binding fragments thereof are provided.
  • the anti-GPRC5D antibodies or antigen-binding fragments thereof of the present disclosure comprise a GPRC5D-VH and a GPRC5D-VL selected from any one of the following groups:
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 45; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 48, LCDR2 shown in SEQ ID NO: 49, and LCDR3 shown in SEQ ID NO: 50;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 33, HCDR2 shown in SEQ ID NO: 34, and HCDR3 shown in SEQ ID NO: 35; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 93, HCDR2 shown in SEQ ID NO: 94, and HCDR3 shown in SEQ ID NO: 95; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 98, LCDR2 shown in SEQ ID NO: 99, and LCDR3 shown in SEQ ID NO: 100;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, and HCDR3 shown in SEQ ID NO: 25; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 28, LCDR2 shown in SEQ ID NO: 29, and LCDR3 shown in SEQ ID NO: 30;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 63, SEQ ID NO: 64
  • the GPRC5D-VL comprises the HCDR1 of SEQ ID NO: 68, the LCDR2 of SEQ ID NO: 69, and the LCDR3 of SEQ ID NO: 70;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 8, LCDR2 shown in SEQ ID NO: 9, and LCDR3 shown in SEQ ID NO: 10;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 13, HCDR2 shown in SEQ ID NO: 14, and HCDR3 shown in SEQ ID NO: 15; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 18, LCDR2 shown in SEQ ID NO: 19, and LCDR3 shown in SEQ ID NO: 20;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 53, HCDR2 shown in SEQ ID NO: 54, and HCDR3 shown in SEQ ID NO: 55; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 58, LCDR2 shown in SEQ ID NO: 59, and LCDR3 shown in SEQ ID NO: 60;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 73, HCDR2 shown in SEQ ID NO: 74, and HCDR3 shown in SEQ ID NO: 75; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 78, LCDR2 shown in SEQ ID NO: 79, and LCDR3 shown in SEQ ID NO: 80;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 103, HCDR2 shown in SEQ ID NO: 104, and HCDR3 shown in SEQ ID NO: 105; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 108, LCDR2 shown in SEQ ID NO: 109, and LCDR3 shown in SEQ ID NO: 110;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 113, HCDR2 shown in SEQ ID NO: 114, and HCDR3 shown in SEQ ID NO: 115; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 118, LCDR2 shown in SEQ ID NO: 119, and LCDR3 shown in SEQ ID NO: 120;
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 123, HCDR2 shown in SEQ ID NO: 124, and HCDR3 shown in SEQ ID NO: 125; and the GPRC5D-VL comprises LCDR1 shown in SEQ ID NO: 128, LCDR2 shown in SEQ ID NO: 129, and LCDR3 shown in SEQ ID NO: 130; and
  • the GPRC5D-VH comprises HCDR1 shown in SEQ ID NO: 133, HCDR2 shown in SEQ ID NO: 134, and HCDR3 shown in SEQ ID NO: 135;
  • the GPRC5D-VL comprises SEQ LCDR1 shown in SEQ ID NO: 138, LCDR2 shown in SEQ ID NO: 139, LCDR3 shown in SEQ ID NO: 140;
  • CDR is determined according to Kabat, Chothia or IMTG numbering system.
  • the anti-GPRC5D antibodies or antigen-binding fragments thereof of the present disclosure comprise a GPRC5D-VH and a GPRC5D-VL selected from any one of the following groups:
  • the bispecific antibody of the present disclosure comprises a group selected from:
  • Nucleic acid is used interchangeably with “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form.
  • the nucleic acids are synthetic, naturally occurring, and non-naturally occurring. Unless otherwise indicated, a nucleic acid sequence also encompasses conservative variants (e.g., degenerate substitutions) and complementary sequences thereof as well as explicitly specified sequences.
  • the term "nucleic acid molecule” includes a plurality of nucleic acid molecules.
  • Vector means a polynucleotide molecule capable of transporting another polynucleotide to which it is attached.
  • plasmid refers to a circular double-stranded DNA loop into which an exogenous DNA segment is attached.
  • viral vector such as an adeno-associated viral vector (AAV), in which an exogenous DNA segment is attached to the viral genome.
  • AAV adeno-associated viral vector
  • “Host cell” and “cell line” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced and their progeny, regardless of the number of generations of the progeny.
  • the progeny are not allowed to be completely identical to the parent cell in terms of nucleic acid, but may contain mutations, as long as the progeny with the mutation have the same desired function or activity as the original cell.
  • the host cell cannot develop into a complete animal or plant individual.
  • Host cells include prokaryotic and eukaryotic host cells.
  • Eukaryotic host cells include, but are not limited to, mammalian cells, insect cells, plant cells, and fungal cells.
  • Mammalian cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells.
  • Exemplary host cells include, but are not limited to, CHO, NSO, COS, SP2 cells, HeLa cells, BHK cells, human hepatocellular carcinoma cells, A549 cells, 3T3 cells, and HEK-293 cells.
  • Fungal cells include yeast, such as, but not limited to, Pichia, Saccharomyces, Hansenula polymorpha, and Kluyveromyces.
  • bispecific antibodies, anti-GPRC5D antibodies, or antigen-binding fragments thereof of the present disclosure can be produced using recombinant methods.
  • the bispecific antibody is a heterodimer
  • three encoding nucleic acids are required, one for encoding the first heavy chain comprising the first subunit of the Fc region, one for encoding the first light chain; and one for encoding the scFv comprising the second subunit of the Fc region.
  • four encoding nucleic acids may be used, one for encoding the first light chain, one for encoding the first heavy chain comprising the first subunit of the Fc region; one for encoding the second light chain, and one for encoding the second heavy chain comprising the second subunit of the Fc region.
  • Three or four nucleic acids are expressed in one or more expression vectors.
  • a host cell comprising the expression vector is also provided.
  • a host cell comprising the expression vector is cultured under conditions suitable for expression of the antibody, and the antibody is recovered (optionally purified) from the host cell (or its culture).
  • a “pharmaceutical composition” refers to a composition containing one or more bispecific antibodies, anti-GPRC5D antibodies, Or a mixture of its antigen-binding fragment and other chemical components, wherein the "other chemical components” are, for example, pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • “Pharmaceutically acceptable carrier, diluent, buffer or excipient” refers to an ingredient in a pharmaceutical preparation that is different from the active ingredient and is non-toxic to the subject.
  • Pharmaceutically acceptable carriers, diluents, buffers or excipients include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • Subject refers to an animal, preferably a mammal.
  • the subject is a mammal, including, for example, camels, horses, cows, pigs, sheep, cats, dogs, rabbits, rats, guinea pigs, mice, primates (e.g., humans).
  • the subject is a human.
  • the subject is an individual susceptible to, suspected of having, or already having a disease.
  • Treatment means providing a subject with a bispecific antibody, anti-GPRC5D antibody, or antigen-binding fragment thereof, or a pharmaceutical composition thereof.
  • the subject has one or more symptoms of a disease.
  • the bispecific antibody, anti-GPRC5D antibody, or antigen-binding fragment thereof is administered in an amount effective to alleviate the symptoms of the disease in the treated subject or subject population.
  • the selection of an effective amount can be determined by those skilled in the art based on consideration of a variety of factors (e.g., through clinical trials), including the disease to be treated, the symptoms involved, the route of administration, the severity of the disease, the subject's weight, the subject's immune status, and other factors known to those skilled in the art.
  • the effective amount in the specific embodiment can be obtained from the dose-response curve derived from the animal model test system, and it is allowed to be determined according to the doctor's judgment and the situation of each subject.
  • the amount of the drug required for the subject's one-time administration can be conveniently obtained by calculating the product of the subject's body weight and the unit weight dose required for the subject's one-time medication.
  • the dosage can be initially determined by the equivalent dose conversion relationship between the unit weight dose of the experimental animal and the human.
  • the dosage of humans and mice can be converted using the body surface area conversion coefficient of 0.0026 for humans and mice.
  • the active ingredient e.g., bispecific antibody, anti-GPRC5D antibody or antigen-binding fragment thereof
  • the active ingredient can be administered once, or can be divided into many smaller unit doses and administered at certain time intervals. It should be understood that the dosage, duration, and interval of treatment are functions of the disease being treated, and can be determined by inference using animal or clinical trial data.
  • the administration may include a single administration, or two or more administrations separated by appropriate time intervals.
  • Two adjacent administrations may be spaced 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months apart.
  • Example 1 Obtaining anti-human GPRC5D human monoclonal antibodies based on humanized mouse platform and hybridoma technology
  • the humanized anti-human GPRC5D monoclonal antibody was prepared using the fully humanized antibody mouse platform (AceMouse TM ) of Shanghai Taijin Biotechnology Co., Ltd. (referred to as "Taijin Bio", English name AceMab) using hybridoma technology.
  • mice Two groups of mice were immunized using DNA and cell-based multiple rounds of immunization, respectively.
  • DNA immunogen The complete DNA coding sequence of human GPRC5D (hGPRC5D) (CDS, SEQ ID NO: 200) was inserted into the expression vector of Taijin Bio to construct the GPRC5D expression vector for immunization.
  • CHO-K1 cells expressing hGPRC5D (CHO-K1_hGPRC5D, Kangyuan Bochuang Biotechnology Co., Ltd.) were used for immunization.
  • Immunization scheme Group A used DNA immunization (8 mice), and Group B used cell immunization (8 mice). A total of six immunizations were performed, including the initial immunization, four booster immunizations, and the final booster immunization.
  • the titer of anti-hGPRC5D antibodies in mouse serum was evaluated 4 days after each immunization. According to the titer of antibodies in the serum after three booster immunizations, 10 mice with high antibody titers were selected for hybridoma fusion 3 days after the final booster immunization to obtain hybridoma cells. A positive control monoclonal antibody BM01 was set in the experiment.
  • variable region was derived from the GCDB72 antibody disclosed in Table 23 of WO 2018017786 A2, containing the GPRC5D binding portion of GCDB72, and the constant region was mouse IgG1 (antibody sequence see SEQ ID NO: 141, SEQ ID NO: 142).
  • the antibodies produced by the hybridomas were tested by binding to HEK293T cells stably expressing hGPRC5D (HEK293T_hGPRC5D), HEK293T cells stably expressing cynomolgus monkey GPRC5D (CDS, SEQ ID NO: 212) (HEK293T_cynoGPRC5D), HEK293T cells stably expressing mouse GPRC5D (CDS, SEQ ID NO: 213) (HEK293T_mGPRC5D), HEK293T cells stably expressing human GPRC5A (CDS, SEQ ID NO: 214) (HEK293T_GPRC5A), HEK293T cells stably expressing human GPRC5B (CDS, SEQ ID NO: 215) (HEK293T_GPRC5B), and HEK293T cells stably expressing human GPRC5C (CDS, SEQ ID NO: 216) (HEK
  • the antibody was diluted into 8 gradients starting from 60 ⁇ g/mL and then incubated with the above different HEK293T cells at 4°C for 30 min. The cells were washed twice by centrifugation at 1200 rpm for 5 min, and then incubated with PE goat anti-mouse IgG (Invitrogen, #12-4010-87) secondary antibody for PE signal value detection by flow cytometry. At the same time, 5 ⁇ g/mL of antibody BM01 was used as a positive control. The screening results are shown in Figures 1 and 2.
  • clones were selected, including monoclonal clones 53C10, 47G4, 71D8, 78G1, 80A8, 91A1, 56A7, 53E2, 92C5, 155C2, 165B7, 199B11, 154D12 and 192E2, which were IgG subtypes and bound to human, monkey or human, mouse and monkey GPRC5D but not to human GPRC5A, GPRC5B and GPRC5C (see Tables 2-3 for sequence numbers).
  • variable region of the antibody was fused with the human constant region (such as the constant region shown in SEQ ID NO: 143 and 144) to obtain the final fully human anti-GPRC5D antibody.
  • the variable region and CDR sequences of the 14 anti-GPRC5D antibodies obtained in the present disclosure are shown in Tables 5 and 6, among which the heavy chain and light chain sequences of 47G4, 71D8, 80A8, 78G1, 53E2 and 155C2 are shown in Table 7.
  • Flow cytometry was used to analyze the binding of fully human anti-GPRC5D antibodies to HEK293T_hGPRC5D cells.
  • a positive control monoclonal antibody BM02 was set in the experiment, whose variable region was derived from the GCDB72 antibody disclosed in Table 23 of WO2018017786A2, whose heavy chain sequence is shown in SEQ ID NO: 157, and whose light chain sequence is shown in SEQ ID NO: 158.
  • HEK293T_hGPRC5D cells were incubated with different concentrations of anti-GPRC5D antibodies (the highest concentration was 10 ⁇ g/ml, 3-fold dilution, and a total of 7 concentration points were obtained) at 4°C for 30 minutes.
  • the CD3 arm of the GPRC5D ⁇ CD3 bispecific antibody disclosed herein can be derived from any suitable anti-CD3 antibody, and the structure of the bispecific antibody can be any structure known in the art.
  • the bispecific antibody disclosed herein has the following structure:
  • the fourth chain formula e.[CD3-VL]-[CL].
  • bispecific antibodies constructed are shown in Tables 8, 9 and 10.
  • IsotypeCD3 is a non-targeted T cell bispecific antibody that only binds to CD3 but not to GPRC5D. The relevant sequences are shown in Tables 9 and 10.
  • Bispecific antibodies can be purified using Protein A pre-packed columns by standard affinity column chromatography. After equilibration of the column with 1 ⁇ PBS at 1 mL/min, load the sample at a flow rate of 1 mL/min; After washing with ⁇ PBS, collect in separate tubes, about 500 ⁇ L per tube. Read the absorbance at 280 nm using a NanoDrop instrument. Store in separate containers.
  • Example 4 Binding activity detection of anti-GPRC5D ⁇ CD3 bispecific antibody and HEK293T_hGPRC5D cells
  • Flow cytometry was used to analyze the expression of dual antibodies Bs71D8, Bs80A8, Bs155C2, 80A8KIH and HEK293T_ Binding of hGPRC5D cells.
  • the viability of HEK293T_hGPRC5D cells was measured and counted, and the cells were divided according to the number of samples.
  • the staining buffer (2% FBS-PBS) was prepared, the cells were washed by centrifugation, and the culture medium was removed; then, the HEK293T_hGPRC5D cells were incubated with different concentrations of bispecific antibodies (initial concentration was 160nM, and a total of 8 concentration points were diluted) at 4°C for 30 minutes.
  • Example 5 Detection of the activation activity of anti-GPRC5D ⁇ CD3 bispecific antibody on T cells
  • MM.1R cells MM cell line, from Shanghai Cell Bank, Chinese Academy of Sciences
  • Jurkat/NFAT-Luc reporter cells 4E2 fluorescein reporter gene effector cells, homemade in the laboratory
  • T cell activation medium 1640 + 10% FBS; take MM.1R cells (target cells) and 4E2 cells (effector cells) in the logarithmic growth phase, centrifuge at 1200rpm for 5min, resuspend and count, adjust the cell density, and inoculate them in the plate at 2 ⁇ 10 4 cells/well; the control group is only inoculated with the same number of 4E2 cells and blank medium.
  • Bs80A8, Bs71D8, Bs155C2, BM03, DuobodyBM, IsotypeCD3 were diluted to a starting concentration of 80nM, 10-fold gradient dilution for 5 points, 100-fold dilution for 3 points, and a total of 8 concentration points were obtained.
  • the 96-well plate was placed in a 37°C, 5% CO 2 incubator for 6h and detected with the Bio-Lite luciferase detection kit.
  • the intensity of the luminescent signal detected by the microplate reader (obtained by adding luciferase substrate) is proportional to the intensity of CD3 activation and signal transduction, and the results are shown in Figures 5 and 6.
  • FIG. 5 shows that Bs80A8, Bs71D8, Bs155C and the positive control BM03 all showed a dose-dependent The activation of Jurkat/NFAT-Luc cells was induced by the method, while no obvious activation signal was obtained in the presence of the negative control molecule IsotypeCD3; Bs80A8 molecule had the strongest specific activation potency, followed by Bs155C2 and Bs71D8, and the activation potency was better than BM03.
  • Figure 6 shows that compared with DuobodyBM, Bs80A8 has a stronger specific activation potency.
  • Example 6 Detection of T cell killing activity induced by anti-GPRC5D ⁇ CD3 bispecific antibody
  • TDCC medium was prepared: RPMI1640+Glutamax medium+2% FBS+1nM sodium pyruvate+1 ⁇ NEAA (all the above reagents were from Gibco), MM cells MM.1R, MM.1S and RPMI8226 in the logarithmic growth phase (all the above MM cell lines were from the Cell Bank of the Chinese Academy of Sciences), centrifuged at 1200rpm for 5min, washed once with TDCC medium, resuspended and counted to adjust the density to 250 cells/ ⁇ L for use; a PBMC cell was taken, resuscitated in a 37°C water bath, centrifuged at 1200rpm for 5min, the supernatant was discarded, and the TDCC medium was resuspended at a density of 2500 cells/ ⁇ L for use
  • the 96-well culture plate was placed in a 37°C, 5% CO 2 incubator for 24 hours.
  • the killing rate was determined using an LDH kit (Roche).
  • group iii maximal release well
  • 5 ⁇ L/well lysis buffer was added, gently tapped to mix, and the cells were lysed at 37°C for 5 minutes.
  • 1 mL of ultrapure water was added to the Catalyst to dissolve it, diluted 1:45 with the staining solution, 100 ⁇ L/well was added to the culture plate, and incubated at room temperature in the dark for 20 minutes. 50 ⁇ L/well stop solution was used to terminate the reaction.
  • the OD value was read at a wavelength of 492 nm, and all OD values were subtracted from the value of the blank well for data analysis.
  • the percentage of cells lysed by TDCC was calculated according to the following formula:
  • Target cell lysis (%) (OD value of the experimental group - OD value of the TDCC background well) / (OD value of the maximum release well - OD value of the minimum release well) ⁇ 100%.
  • Example 7 Anti-tumor efficacy of bispecific antibodies in the human myeloma NCI-H929 subcutaneously transplanted female NPG mouse MiXeno model
  • NCI-H929 cells from ATCC were cultured in RPMI1640 medium containing 10% FBS and 0.05mM ⁇ -ME. Cells in the exponential growth phase were collected and resuspended to a suitable concentration for subcutaneous tumor inoculation in female MiXeno model NPG mice (from Sino-US Crown Science). Each mouse was subcutaneously inoculated with 5 ⁇ 10 6 NCI-H929 cells on the right back, and the cells were resuspended in 0.1mL of 1:1 PBS and Matrigel.
  • mice 5 ⁇ 10 6 PBMC/0.1mL PBS was injected intraperitoneally (ip) into each mouse on the day of NCI-H929 cell inoculation (donor PBMC was from Miaoshun). Tumor growth was observed regularly, and the mice were randomly divided into 4 groups when the tumor grew to an average volume of approximately 115mm 3 . The day of grouping was defined as day 0. On the day of grouping, mice were administered dual antibodies BM03, Bs80A8, and Bs155C2 by intravenous infusion (iv). The vehicle control was treated with an equal amount of PBS solution. The specific dosing schedule is shown in Table 14. The dosing cycle of this experiment was from day 0 to day 1. On day 18, some experimental groups extended the observation period to day 32. The evaluation indicators of this experiment are: absolute tumor proliferation rate (The T/C value, T/C) and absolute tumor growth inhibition rate (Tumor growth inhibition, TGI) and other mouse health information.
  • T/C% TmTV / CmTV ⁇ 100%; TmTV : average tumor weight of the treatment group; CmTV: average tumor weight of the control group;
  • TGI% (1-T/C) ⁇ 100%; T/C% is the absolute tumor proliferation rate, which is the percentage value of the relative tumor volume (or body weight) of the treatment group and the control group at a certain time point.
  • mice The administration was stopped on the 18th day of the experiment. No mice died during the experiment. The lowest weight change rate was -2.26% (BM03 group). The mice generally had good tolerance. The results are shown in FIG13 and Table 14.
  • the average tumor volume of the vehicle control group was 3303.18 mm 3
  • the average tumor volumes of the drug-treated groups BM03, Bs80A8 and Bs155C2 were 2429.01 mm 3 , 85.62 mm 3 and 370.06 mm 3 , respectively.

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Abstract

La présente invention concerne un anticorps anti-GPRC5D et son utilisation médicale. Spécifiquement, la présente invention concerne un anticorps anti-GPRC5D, un anticorps bispécifique basé sur l'anticorps anti-GPRC5D, et son utilisation dans le traitement de cancers.
PCT/CN2024/101975 2023-06-28 2024-06-27 Anticorps anti-gprc5d et son utilisation médicale Ceased WO2025002251A1 (fr)

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Citations (5)

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WO2018017786A2 (fr) * 2016-07-20 2018-01-25 Janssen Pharmaceutica Nv Anticorps anti-gprc5d, molécules bispécifiques de liaison à l'antigène qui se lient à gprc5d et cd3 et leurs utilisations
CN110462038A (zh) * 2017-02-07 2019-11-15 第一三共株式会社 抗gprc5d抗体和包含所述抗体的分子
CN111788231A (zh) * 2018-02-09 2020-10-16 豪夫迈·罗氏有限公司 结合gprc5d的抗体
CN115232209A (zh) * 2021-04-22 2022-10-25 南京北恒生物科技有限公司 靶向gprc5d的抗体及其用途
CN115386006A (zh) * 2021-05-23 2022-11-25 上海祥耀生物科技有限责任公司 抗gprc5d抗体、其制备方法与用途

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WO2018017786A2 (fr) * 2016-07-20 2018-01-25 Janssen Pharmaceutica Nv Anticorps anti-gprc5d, molécules bispécifiques de liaison à l'antigène qui se lient à gprc5d et cd3 et leurs utilisations
CN109715667A (zh) * 2016-07-20 2019-05-03 詹森药业有限公司 抗-gprc5d抗体、结合gprc5d和cd3的双特异性抗原结合分子及其用途
CN110462038A (zh) * 2017-02-07 2019-11-15 第一三共株式会社 抗gprc5d抗体和包含所述抗体的分子
CN111788231A (zh) * 2018-02-09 2020-10-16 豪夫迈·罗氏有限公司 结合gprc5d的抗体
CN115232209A (zh) * 2021-04-22 2022-10-25 南京北恒生物科技有限公司 靶向gprc5d的抗体及其用途
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