WO2023030272A1 - 抗gprc5d抗原结合蛋白及其用途 - Google Patents

抗gprc5d抗原结合蛋白及其用途 Download PDF

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WO2023030272A1
WO2023030272A1 PCT/CN2022/115590 CN2022115590W WO2023030272A1 WO 2023030272 A1 WO2023030272 A1 WO 2023030272A1 CN 2022115590 W CN2022115590 W CN 2022115590W WO 2023030272 A1 WO2023030272 A1 WO 2023030272A1
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seq
hcdr3
hcdr2
hcdr1
binding protein
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PCT/CN2022/115590
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French (fr)
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何晓文
周金财
陈思晔
杨月
史中军
李霄培
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原启生物科技(上海)有限责任公司
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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    • C07K2319/00—Fusion polypeptide
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15044—Chimeric viral vector comprising heterologous viral elements for production of another viral vector

Definitions

  • the present application relates to the field of biomedicine, in particular to an anti-GPRC5D antigen-binding protein, a chimeric antigen receptor comprising the antigen-binding protein, and their use in treating tumors.
  • Multiple myeloma is the second most common hematologic malignancy and ranks second in cancer-related mortality.
  • MM is a plasma cell malignancy often accompanied by multiple osteolytic lesions, renal impairment, bone marrow infiltration, hypercalcemia, and anemia.
  • the current main treatment for MM is systemic chemotherapy with severe side effects, and it cannot be completely cured.
  • the G protein-coupled receptor family group C 5 member D (GPRC5D) protein is an atypical surface orphan receptor.
  • GPRCD5 like other C5 family receptors, has a short amino-terminus, so it is very similar to the C4 family in conformation. Its expression in normal tissues is limited to hair follicles, but it is also highly expressed specifically in the bone marrow of MM patients and is highly correlated with plasma cell tumor burden and genetic aberrations.
  • MM With the intervention of new drugs such as protease inhibitors (PIs), immunomodulatory drugs (IMiDs) and monoclonal antibodies (mAbs), the treatment of MM has been expanded, but there is still a very high recurrence rate and drug resistance.
  • PIs protease inhibitors
  • IMDs immunomodulatory drugs
  • mAbs monoclonal antibodies
  • CAR-T chimeric antigen receptor T cell
  • MM patients with low expression of BCMA have the problem of target escape.
  • the specific high expression of GPRC5D on plasma cells makes it an ideal therapeutic target for MM, but the drug therapy products targeting it are not yet mature.
  • the present application provides an isolated antigen-binding protein, which can specifically bind to G protein-coupled receptor family C group 5 member D (GPRC5D) protein.
  • the present application also provides a chimeric antigen receptor comprising the antigen-binding protein, and cells comprising and/or expressing the chimeric antigen receptor, and the cells have one or more of the following characteristics: (1) expanding Strong ability, (2) capable of killing target cells expressing GPRC5D, (3) secreting cytokines under the stimulation of target cells, (4) having strong proliferation ability under the stimulation of target cells, and (4) inhibiting tumor growth.
  • the present application provides an isolated antigen-binding protein, which can bind G protein-coupled receptor family C group 5 member D (GPRC5D) protein with an EC50 value lower than 1.0 ⁇ g/mL.
  • GPRC5D G protein-coupled receptor family C group 5 member D
  • the antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antigen-binding fragment is VHH.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the antigen-binding protein can compete with a reference antibody for binding to the GPRC5D protein, wherein the reference antibody comprises an antibody heavy chain variable region VH, and the VH of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the reference antibody comprises any set of amino acid sequences selected from the following:
  • HCDR1 SEQ ID NO: 102
  • HCDR2 SEQ ID NO: 103
  • HCDR3 SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 105
  • HCDR2 SEQ ID NO: 106
  • HCDR3 SEQ ID NO: 107;
  • HCDR1 SEQ ID NO: 109
  • HCDR2 SEQ ID NO: 110
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 105
  • HCDR2 SEQ ID NO: 114
  • HCDR3 SEQ ID NO: 115;
  • HCDR1 SEQ ID NO: 116
  • HCDR2 SEQ ID NO: 117
  • HCDR3 SEQ ID NO: 118;
  • HCDR1 SEQ ID NO: 119
  • HCDR2 SEQ ID NO: 120
  • HCDR3 SEQ ID NO: 121;
  • HCDR1 SEQ ID NO: 123
  • HCDR2 SEQ ID NO: 124
  • HCDR3 SEQ ID NO: 125;
  • HCDR1 SEQ ID NO: 127
  • HCDR2 SEQ ID NO: 128, and HCDR3: SEQ ID NO: 129;
  • HCDR1 SEQ ID NO: 130
  • HCDR2 SEQ ID NO: 131
  • HCDR3 SEQ ID NO: 132
  • HCDR1 SEQ ID NO:95
  • HCDR2 SEQ ID NO:96
  • HCDR3 SEQ ID NO:97;
  • HCDR1 SEQ ID NO: 133
  • HCDR2 SEQ ID NO: 134
  • HCDR3 SEQ ID NO: 135;
  • HCDR1 SEQ ID NO: 136
  • HCDR2 SEQ ID NO: 134
  • HCDR3 SEQ ID NO: 137;
  • HCDR1 SEQ ID NO: 138
  • HCDR2 SEQ ID NO: 139
  • HCDR3 SEQ ID NO: 140;
  • HCDR1 SEQ ID NO: 142
  • HCDR2 SEQ ID NO: 143
  • HCDR3 SEQ ID NO: 144;
  • HCDR1 SEQ ID NO:145
  • HCDR2 SEQ ID NO:146
  • HCDR3 SEQ ID NO:147;
  • HCDR1 SEQ ID NO: 148
  • HCDR2 SEQ ID NO: 149
  • HCDR3 SEQ ID NO: 150;
  • HCDR1 SEQ ID NO: 152
  • HCDR2 SEQ ID NO: 153
  • HCDR3 SEQ ID NO: 154;
  • HCDR1 SEQ ID NO: 123
  • HCDR2 SEQ ID NO: 155
  • HCDR3 SEQ ID NO: 156;
  • HCDR1 SEQ ID NO:157
  • HCDR2 SEQ ID NO:153
  • HCDR3 SEQ ID NO:158;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:160
  • HCDR3 SEQ ID NO:161;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:162
  • HCDR3 SEQ ID NO:163;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:164
  • HCDR3 SEQ ID NO:165;
  • HCDR1 SEQ ID NO: 166
  • HCDR2 SEQ ID NO: 167
  • HCDR3 SEQ ID NO: 168;
  • HCDR1 SEQ ID NO:169
  • HCDR2 SEQ ID NO:153
  • HCDR3 SEQ ID NO:170
  • HCDR1 SEQ ID NO:157
  • HCDR2 SEQ ID NO:171
  • HCDR3 SEQ ID NO:172
  • HCDR1 SEQ ID NO:174
  • HCDR2 SEQ ID NO:175
  • HCDR3 SEQ ID NO:176;
  • HCDR1 SEQ ID NO: 177
  • HCDR2 SEQ ID NO: 178
  • HCDR3 SEQ ID NO: 179;
  • HCDR1 SEQ ID NO: 180
  • HCDR2 SEQ ID NO: 181
  • HCDR3 SEQ ID NO: 182;
  • HCDR1 SEQ ID NO: 184
  • HCDR2 SEQ ID NO: 106
  • HCDR3 SEQ ID NO: 185;
  • HCDR1 SEQ ID NO: 186
  • HCDR2 SEQ ID NO: 187
  • HCDR3 SEQ ID NO: 188;
  • HCDR1 SEQ ID NO: 189
  • HCDR2 SEQ ID NO: 190
  • HCDR3 SEQ ID NO: 191;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:192
  • HCDR3 SEQ ID NO:179;
  • HCDR1 SEQ ID NO: 193
  • HCDR2 SEQ ID NO: 194, and HCDR3: SEQ ID NO: 195;
  • HCDR1 SEQ ID NO: 196
  • HCDR2 SEQ ID NO: 197
  • HCDR3 SEQ ID NO: 198.
  • the antigen binding protein comprises at least one CDR derived from an antibody heavy chain variable region VH comprising SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO :42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58 , SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and the amino
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:215 or SEQ ID NO:218.
  • the antigen binding protein comprises HCDR3, and the HCDR3 comprises SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:115 , SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO : 165, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 176, SEQ ID NO: 179, SEQ ID NO: 182, SEQ ID NO: 185, SEQ ID NO: 188 , the amino acid sequence shown in
  • the antigen binding protein comprises HCDR2, and the HCDR2 comprises an amino acid sequence represented by X 1 X 2 X 3 X 4 X 5 X 6 X 7 T, wherein X 1 is I or T , X2 is N, S or T, X3 is A, P, R, S or W, X4 is G, R, S, T or does not exist, X5 is D or G, X6 is G or S , and X 7 is D, G, I, N, R, S, T or V.
  • the antigen binding protein comprises HCDR2, and the HCDR2 comprises SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:114 , SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 134, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO:146, SEQ ID NO:149, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO :171, SEQ ID NO:175, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:187, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194 and SEQ ID NO:197
  • the antigen-binding protein includes HCDR1, and the HCDR1 includes the amino acid sequence shown as GX 2 X 3 X 4 SX 6 X 7 X 8 , wherein X 2 is F, G, I, L, N, R, S or Y, X 3 is I or T, X 4 is F or L, X 6 is I, L, N, R, S, T or Y, X 7 is D, N or Y, And X8 is A, D, G, I, N, R, S, T, V or Y.
  • the antigen binding protein comprises HCDR1, and the HCDR1 comprises SEQ ID NO:95, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:116 , SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:166, SEQ ID NO:169, SEQ ID NO:174, SEQ ID NO :177, SEQ ID NO:180, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:189, SEQ ID NO:193 and the amino acid sequence shown in any one of SEQ ID NO:196.
  • the antigen binding protein comprises HCDR1, HCDR2, HCDR3, and the HCDR1, HCDR2, HCDR3 comprises any set of amino acid sequences selected from the group consisting of:
  • HCDR1 SEQ ID NO: 102
  • HCDR2 SEQ ID NO: 103
  • HCDR3 SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 105
  • HCDR2 SEQ ID NO: 106
  • HCDR3 SEQ ID NO: 107;
  • HCDR1 SEQ ID NO: 109
  • HCDR2 SEQ ID NO: 110
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 105
  • HCDR2 SEQ ID NO: 114
  • HCDR3 SEQ ID NO: 115;
  • HCDR1 SEQ ID NO: 116
  • HCDR2 SEQ ID NO: 117
  • HCDR3 SEQ ID NO: 118;
  • HCDR1 SEQ ID NO: 119
  • HCDR2 SEQ ID NO: 120
  • HCDR3 SEQ ID NO: 121;
  • HCDR1 SEQ ID NO: 123
  • HCDR2 SEQ ID NO: 124
  • HCDR3 SEQ ID NO: 125;
  • HCDR1 SEQ ID NO: 127
  • HCDR2 SEQ ID NO: 128, and HCDR3: SEQ ID NO: 129;
  • HCDR1 SEQ ID NO: 130
  • HCDR2 SEQ ID NO: 131
  • HCDR3 SEQ ID NO: 132
  • HCDR1 SEQ ID NO:95
  • HCDR2 SEQ ID NO:96
  • HCDR3 SEQ ID NO:97;
  • HCDR1 SEQ ID NO: 133
  • HCDR2 SEQ ID NO: 134
  • HCDR3 SEQ ID NO: 135;
  • HCDR1 SEQ ID NO: 136
  • HCDR2 SEQ ID NO: 134
  • HCDR3 SEQ ID NO: 137;
  • HCDR1 SEQ ID NO: 138
  • HCDR2 SEQ ID NO: 139
  • HCDR3 SEQ ID NO: 140;
  • HCDR1 SEQ ID NO: 142
  • HCDR2 SEQ ID NO: 143
  • HCDR3 SEQ ID NO: 144;
  • HCDR1 SEQ ID NO:145
  • HCDR2 SEQ ID NO:146
  • HCDR3 SEQ ID NO:147;
  • HCDR1 SEQ ID NO: 148
  • HCDR2 SEQ ID NO: 149
  • HCDR3 SEQ ID NO: 150;
  • HCDR1 SEQ ID NO: 152
  • HCDR2 SEQ ID NO: 153
  • HCDR3 SEQ ID NO: 154;
  • HCDR1 SEQ ID NO: 123
  • HCDR2 SEQ ID NO: 155
  • HCDR3 SEQ ID NO: 156;
  • HCDR1 SEQ ID NO:157
  • HCDR2 SEQ ID NO:153
  • HCDR3 SEQ ID NO:158;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:160
  • HCDR3 SEQ ID NO:161;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:162
  • HCDR3 SEQ ID NO:163;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:164
  • HCDR3 SEQ ID NO:165;
  • HCDR1 SEQ ID NO: 166
  • HCDR2 SEQ ID NO: 167
  • HCDR3 SEQ ID NO: 168;
  • HCDR1 SEQ ID NO:169
  • HCDR2 SEQ ID NO:153
  • HCDR3 SEQ ID NO:170
  • HCDR1 SEQ ID NO:157
  • HCDR2 SEQ ID NO:171
  • HCDR3 SEQ ID NO:172
  • HCDR1 SEQ ID NO:174
  • HCDR2 SEQ ID NO:175
  • HCDR3 SEQ ID NO:176;
  • HCDR1 SEQ ID NO:177
  • HCDR2 SEQ ID NO:178
  • HCDR3 SEQ ID NO:179;
  • HCDR1 SEQ ID NO: 180
  • HCDR2 SEQ ID NO: 181
  • HCDR3 SEQ ID NO: 182;
  • HCDR1 SEQ ID NO: 184
  • HCDR2 SEQ ID NO: 106
  • HCDR3 SEQ ID NO: 185;
  • HCDR1 SEQ ID NO: 186
  • HCDR2 SEQ ID NO: 187
  • HCDR3 SEQ ID NO: 188;
  • HCDR1 SEQ ID NO: 189
  • HCDR2 SEQ ID NO: 190
  • HCDR3 SEQ ID NO: 191;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:192
  • HCDR3 SEQ ID NO:179;
  • HCDR1 SEQ ID NO: 193
  • HCDR2 SEQ ID NO: 194, and HCDR3: SEQ ID NO: 195;
  • HCDR1 SEQ ID NO: 196
  • HCDR2 SEQ ID NO: 197
  • HCDR3 SEQ ID NO: 198.
  • the antigen-binding protein comprises an antibody heavy chain variable region VH, wherein the VH includes a framework region H-FR1, and the C-terminus of the H-FR1 is directly or directly connected to the N-terminus of the HCDR1. Indirectly connected, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:98.
  • the VH in the antigen binding protein comprises a framework region H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO : the amino acid sequence shown in 216.
  • the H-FR2 in the antigen binding protein comprises SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:122, SEQ ID NO:151 and The amino acid sequence shown in any one of SEQ ID NO:173.
  • the VH in the antigen binding protein comprises a framework region H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises SEQ ID NO : the amino acid sequence shown in 217.
  • the H-FR3 in the antigen binding protein comprises SEQ ID NO:100, SEQ ID NO:113, SEQ ID NO:126, SEQ ID NO:141 and SEQ ID NO:183 The amino acid sequence shown in any one.
  • the VH in the antigen binding protein comprises a framework region H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID The amino acid sequence shown in NO:101.
  • the VH in the antigen binding protein comprises SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO: 30.
  • the antigen binding protein is VHH
  • the VHH comprises SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO :46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62 , the amino acid sequence shown in any one of SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:
  • the antigen binding protein comprises an antibody heavy chain constant region derived from IgG.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from human IgG.
  • the antigen binding protein comprises an antibody heavy chain constant region derived from human IgG1.
  • the present application provides a chimeric antigen receptor comprising a targeting moiety, and the targeting moiety comprises the antigen-binding protein.
  • the chimeric antigen receptor comprises a co-stimulatory signal domain, wherein the cellular co-stimulatory signal domain comprises an intracellular co-stimulatory signal derived from one or more proteins selected from the group Regions: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, Ligands of DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83, CD40 and MyD88.
  • the group Regions CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI
  • the co-stimulatory signal domain in the chimeric antigen receptor is an intracellular co-stimulatory signal domain derived from 4-1BB.
  • the co-stimulatory signal region in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:78.
  • the chimeric antigen receptor comprises an intracellular signaling region comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpesvirus (HSKV), DAP10, DAP- 12 and a domain containing at least one ITAM.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpesvirus
  • the intracellular signaling domain in the chimeric antigen receptor is a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling region in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:80.
  • the chimeric antigen receptor comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4 -1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM , DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane region in the chimeric antigen receptor is a transmembrane region derived from CD8.
  • the transmembrane region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:76.
  • the chimeric antigen receptor includes a hinge region between the targeting moiety and the transmembrane region, the hinge region comprising a hinge derived from one or more proteins selected from the group consisting of Regions: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • Regions CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region in the chimeric antigen receptor is a hinge region derived from CD8.
  • the hinge region of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:74.
  • the chimeric antigen receptor further comprises a low-density lipoprotein receptor-associated protein or a fragment thereof, and the low-density lipoprotein receptor-associated protein or a fragment thereof is located in the intracellular signaling region C-terminal.
  • the low-density lipoprotein receptor-associated protein or fragment thereof in the chimeric antigen receptor comprises one or more selected from the group consisting of: low-density lipoprotein receptor-associated protein 1 -12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or fragment thereof in the chimeric antigen receptor is low-density lipoprotein receptor-related protein 6 or a fragment thereof.
  • the low-density lipoprotein receptor-associated protein or a fragment thereof in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:84.
  • the chimeric antigen receptor further comprises a signal peptide.
  • the signal peptide in the chimeric antigen receptor is derived from the signal peptide of CD8 protein.
  • the signal peptide in the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:72.
  • the chimeric antigen receptor comprises SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:200, SEQ ID NO: 201, the amino acid sequence shown in any one of SEQ ID NO:202, SEQ ID NO:203 and SEQ ID NO:204.
  • the chimeric antigen receptor comprises the nucleotide sequence shown in any one of SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:91 .
  • the present application also provides a polypeptide comprising said antigen-binding protein.
  • the present application also provides one or more isolated nucleic acid molecules encoding the antigen binding protein and/or the chimeric antigen receptor.
  • the nucleic acid molecule further comprises a promoter.
  • the promoter in the nucleic acid molecule is a constitutive promoter.
  • the promoter in the nucleic acid molecule is the EF1 ⁇ promoter.
  • the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:91.
  • the present application also provides a vector comprising the nucleic acid molecule.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • the present application also provides a cell comprising the antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, and/or the carrier.
  • the cells are immune effector cells.
  • the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, Peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • NK cells natural killer cells
  • NKT cells monocytes, dendritic cells
  • monocytes granulocytes
  • lymphocytes granulocytes
  • leukocytes granulocytes
  • Peripheral blood mononuclear cells embryonic stem cells
  • lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells are T cells.
  • the present application also provides a method for preparing the antigen-binding protein and/or the chimeric antigen receptor, the method comprising making the antigen-binding protein and/or the chimeric antigen receptor
  • the cells were cultured as described in the root conditions for chimeric antigen receptor expression.
  • the present application also provides a method for preparing modified immune effector cells, which includes introducing the carrier into the immune effector cells.
  • the present application also provides a pharmaceutical composition, which comprises the antigen-binding protein, the chimeric antigen receptor, the polypeptide, any one of the nucleic acid molecules, any of the One of the above-mentioned carrier, and/or the above-mentioned cell, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides the antigen-binding protein, the antigen chimeric receptor, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition , the use in the preparation of medicines, the medicines are used for preventing, treating and/or alleviating diseases or diseases related to abnormal expression of GPRC5D.
  • the diseases or conditions associated with abnormal expression of GPRC5D in the use include tumors.
  • the tumor in the use comprises a solid tumor.
  • the tumor in the use comprises a non-solid tumor.
  • the tumor in the use includes hematoma and/or lymphoma.
  • the tumor in said use comprises myeloma.
  • the present application also provides a method for preventing, treating and/or alleviating diseases or disorders related to abnormal expression of GPRC5D, the method comprising administering the antigen-binding protein to a subject in need, wherein The antigen chimeric receptor, the nucleic acid molecule, the carrier, the cell, and/or the pharmaceutical composition.
  • the disease or disorder associated with aberrant expression of GPRC5D in the method includes a tumor.
  • said method wherein said tumor comprises a solid tumor.
  • said method wherein said tumor comprises a non-solid tumor.
  • said method comprises said tumor comprising hematoma and/or lymphoma.
  • said method includes said tumor comprising myeloma.
  • Figures 1A to 1J show the flow cytometric analysis of the binding activity of the anti-CPRC5D antibody described in the present application to GPRC5D on the cell surface.
  • Figure 2 shows the elements and connection sequences of each part of CAR in the chimeric antigen receptor lentiviral expression vector and GPRC5D core plasmid described in this application.
  • Figure 3A to Figure 3C show the expansion multiple of activated GPRC5D CAR-T cells and the CAR-positive rate of T cells after infection.
  • Figure 4A to Figure 4D show the killing ability of GPRC5D CAR-T cells on target cells (CHO cells/MM1S cells).
  • Figure 5A to Figure 5B show the secretion of cytokines (IL2 ⁇ IFN- ⁇ ) after killing GPRC5D CAR-T cells in vitro.
  • Figure 6 shows the targeted proliferation of GPRC5D CAR-T cells.
  • Figure 7A to Figure 7D show the cytokine secretion, tumor elimination ability and safety of GPRC5D CAR-T cells in mice.
  • Figure 8A to Figure 8D and Figure 9A to Figure 9D show that anti-GPRC5D antibody does not bind to cells that do not express GPRC5D antigen, A: A549 cells, B: KERA cells, C: MCF-7 cells, D: MSC cells.
  • FIGS 10A to 10D show the results of the endocytosis effect of anti-GPRC5D.
  • isolated antigen-binding protein generally refers to a protein having antigen-binding ability that is removed from its naturally occurring state.
  • Said “isolated antigen binding protein” may comprise an antigen-binding moiety and optionally a framework or framework portion which permits the antigen-binding moiety to adopt a conformation which facilitates its binding to antigen.
  • Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative or artificial framework regions with grafted variable regions (CDR) or CDR derivatives.
  • the antigen binding protein may comprise an antibody or an antigen binding fragment thereof.
  • the antigen binding protein can bind GPRC5D protein.
  • the antigen binding protein can compete with a reference antibody for binding to the GPRC5D protein.
  • the antigen binding protein may comprise an antibody heavy chain variable region VH.
  • the antigen binding protein may comprise at least one CDR derived from an antibody heavy chain variable region VH.
  • the VH may comprise HCDR3, HCDR2 and/or HCDR1.
  • the VH may include a framework region H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1.
  • the VH may comprise a framework region H-FR2 located between the HCDR1 and the HCDR2.
  • the VH may comprise a framework region H-FR3 located between the HCDR2 and the HCDR3.
  • the VH may include a framework region H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3.
  • the antigen binding protein can be VHH.
  • the antigen binding protein may comprise an antibody heavy chain constant region, which may be derived from IgG.
  • the antibody heavy chain constant region may be derived from human IgG.
  • the antibody heavy chain constant region may be derived from human IgG1.
  • antibody as used includes whole antibodies and binding fragments thereof. Typically, a fragment competes with the intact antibody from which it is derived for specific binding to the antigen.
  • antibodies or binding fragments thereof can be chemically conjugated to other proteins, or expressed as fusion proteins with other proteins.
  • the antibodies can be monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the binding protein of the antibody or binding fragment thereof can include GPRC5D.
  • the antibody or binding fragment thereof can be specific for GPRC5D.
  • antigen-binding fragment refers to a portion of an intact antibody and refers to the antigenically determining variable region of an intact antibody.
  • the antigen-binding fragments may include Fab, Fab', F(ab')2, Fv fragments and single-chain Fv fragments, tandem Fv fragments, VHH, bispecific antibodies.
  • the antigen-binding fragment can be a VHH.
  • the antigen-binding fragment can bind GPRC5D.
  • the antigen-binding fragment can be specific for GPRC5D.
  • VHH generally refers to an antibody comprising the variable antigen-binding domain of a heavy chain antibody.
  • VHHs may also be referred to as Nanobodies (Nb) and/or Single Domain Antibodies.
  • Nb Nanobodies
  • the VHH can bind GPRC5D.
  • the VHH can be specific for GPRC5D.
  • the antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • the term "heavy chain constant region” consists of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region.
  • the term "light chain constant region” consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors.
  • the term "reference antibody” refers to an antibody that can compete with the isolated antigen-binding protein GPRC5D for binding to the same epitope.
  • the reference antibody may comprise a heavy chain variable region VH.
  • the reference antibody may have 3 CDR sequences.
  • the VH of the reference antibody can include HCDR1, HCDR2, and HCDR3.
  • the CDR sequences may be identical to the CDR sequences of the isolated antigen binding protein.
  • G protein-coupled receptor family C group member 5 member D (GPRC5D) protein is an atypical surface orphan receptor.
  • GPRCD5 like other C5 family receptors, has a short amino-terminus, so it is very similar to the C4 family in conformation. Its expression in normal tissues is restricted to hair follicles, but it is also prominently expressed in the bone marrow of patients with multiple myeloma and is highly correlated with plasma cell tumor burden and genetic aberrations.
  • GPRC5D in this application may specifically refer to GPRC5D expressed in MM patients.
  • IgG refers to a polypeptide belonging to the class of antibodies substantially encoded by the recognized immunoglobulin gamma genes. In humans, this class includes IgG1, IgG2, IgG3 and IgG4. In mice, this class includes IgGl, IgG2a, IgG2b and IgG3.
  • chimeric antigen receptor generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically binds an antigen or a target.
  • a hinge region is included between the extracellular domain and the transmembrane region.
  • the chimeric antigen receptor may also include low-density lipoprotein receptor-associated protein or a fragment thereof.
  • the chimeric antigen receptor can include a signal peptide. Binding of the extracellular domain of the CAR to the target antigen on the surface of the target cell results in clustering of the CAR and delivery of an activation stimulus to the CAR-containing cell.
  • the extracellular structure may include an antigen binding protein as described above.
  • the extracellular structure can specifically bind GPRC5D.
  • intracellular domain is meant to include any truncated portion sufficient to transduce an activation signal.
  • the intracellular domain may comprise an intracellular signaling domain and/or a co-stimulatory signaling domain.
  • intracellular signaling region refers to an intracellular region that can generate signals that promote immune effector functions of CAR-containing cells (eg, CART cells or CAR-expressing NK cells).
  • the intracellular signaling region may comprise an intracellular signaling region of one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30 , Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12 and domains containing at least one ITAM.
  • the intracellular signaling region may be a signaling domain derived from CD3 ⁇ .
  • co-stimulatory signaling region refers to a part of the CAR capable of transducing effector signals in the intracellular signaling region.
  • the co-stimulatory signal domain may comprise an intracellular co-stimulatory signal domain derived from one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40 and MyD88.
  • the co-stimulatory signal domain may be an intracellular co-stimulatory signal domain derived from 4-1BB.
  • the term "transmembrane region” refers to a domain of a peptide, polypeptide or protein capable of spanning the plasma membrane of a cell. These domains can be used to anchor the extracellular domain to the cell membrane.
  • the transmembrane region may comprise a transmembrane domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane region
  • the term "hinge region” refers to a part of an antibody heavy chain polypeptide connecting the CH1 domain and the CH2 domain, for example, from about position 216 to about position 230 of the EU numbering system according to Kabat.
  • the hinge region is usually a dimeric molecule composed of two polypeptides with the same amino acid sequence.
  • the hinge region generally comprises about 25 amino acid residues and is flexible, allowing independent movement of the antigen-binding region.
  • the hinge region can be subdivided into 3 domains: upper, middle, and lower hinge domains.
  • the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region may be derived from the hinge region of CD8.
  • the term "low-density lipoprotein receptor-related protein” refers to a cell surface protein belonging to endocytic receptors, which is widely distributed in organisms and has a large interstitial space. Difference, the main function is to take cholesterol into the cell for the synthesis of cell proliferation and sterol hormones and bile salts.
  • the low-density lipoprotein receptor-related protein can be from any vertebrate.
  • the Low-density lipoprotein receptor-associated protein or fragments thereof may be located at the C-terminus of the intracellular signaling region.
  • said low-density lipoprotein receptor-associated proteins or fragments thereof may comprise one or more selected from the following group : Low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 6 or a fragment thereof.
  • signal peptide refers to the leader sequence at the amino terminus (N-terminus) of the nascent CAR protein, which directs the nascent protein to the endoplasmic reticulum and subsequent surface expression upon translation or after translation.
  • the signal peptide is derived from the signal peptide of CD8 protein.
  • said signal peptide comprises the amino acid sequence shown in SEQ ID NO:72.
  • polypeptide polypeptide
  • peptide protein
  • protein polymer of amino acid residues.
  • the term can be used to refer to amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of the corresponding natural amino acid, as well as to natural amino acid polymers, those containing modified residues, and non- Natural amino acid polymer.
  • said polypeptide may comprise said antigen binding protein.
  • nucleic acid molecule includes DNA molecules and RNA molecules.
  • a nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • promoter generally refers to a DNA sequence that regulates the expression of a selected DNA sequence operably linked to the promoter, thereby affecting the expression of the selected DNA sequence in a cell.
  • the nucleic acid molecule may encode an antigen binding protein and/or the chimeric antigen receptor.
  • the nucleic acid molecule can include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the term "vector” generally refers to a molecule to which one or more nucleic acid molecules of the present application can be attached.
  • the vector may be a viral vector.
  • the vector may be a lentiviral vector.
  • the term “cell” refers to a cell into which nucleic acid can be transfected, and the term “cell” includes prokaryotic cells for plasmid propagation, and eukaryotic cells for nucleic acid expression and production of encoded polypeptides.
  • a cell may comprise said antigen binding protein, said nucleic acid molecule and/or said carrier.
  • the cells may be immune effector cells.
  • immune effector cells generally refers to immune cells that participate in the immune response and perform effector functions.
  • the exercising effector functions may include clearing foreign antigens or promoting immune effector responses, etc.
  • immune effector cells can include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells , embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • immune effector cells can be T cells.
  • the term "pharmaceutical composition” generally refers to a chemical or biological composition suitable for administration to a mammalian subject.
  • the pharmaceutical composition may comprise the antigen binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable Carrier.
  • the pharmaceutical composition can be used to prevent, treat and/or alleviate diseases or conditions related to abnormal expression of GPRC5D.
  • the diseases or conditions associated with abnormal expression of GPRC5D may include tumors.
  • the tumors include solid tumors and/or non-solid tumors.
  • the tumor pack may include hematomas and/or lymphomas.
  • the tumor can include myeloma.
  • the present application provides an isolated antigen-binding protein capable of binding G protein-coupled receptor family C group 5 member D (GPRC5D) protein with an EC50 value lower than 1.0 ⁇ g/mL.
  • the antigen binding proteins described herein may be present at less than about 0.9 ⁇ g/mL, less than about 0.8 ⁇ g/mL, less than about 0.7 ⁇ g/mL, less than about 0.6 ⁇ g/mL, less than about 0.5 ⁇ g/mL , an EC50 value of less than about 0.4 ⁇ g/mL, less than about 0.3 ⁇ g/mL, less than about 0.2 ⁇ g/mL, less than about 0.1 ⁇ g/mL or lower binds to the GPRC5D protein.
  • the antigen binding proteins described herein can bind to GPRC5D protein expressed on the cell surface with an EC50 value of less than about 0.9 ⁇ g/mL.
  • the GPRC5D protein expressed on the surface of cells bound to the antigen binding protein of the present application can be detected by flow cytometry fluorescence sorting technique (FACS), for example, using iQue Screener flow cytometry.
  • FACS flow cytometry fluorescence sorting technique
  • the antigen-binding protein may include an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv, VHH and/or dAb.
  • the antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.
  • CDR is the complementarity-determining region of an antibody, which is a part of the variable region of an antibody.
  • the amino acid residues in this region are in contact with the antigen or antigenic epitope, thereby specifically recognizing and binding to the antigen.
  • the isolated antigen-binding protein may comprise at least one CDR in the variable region VH of the antibody heavy chain
  • the VH may comprise such as SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO :8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 , SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO :58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64
  • the antigen binding protein may comprise HCDR3.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 215:
  • X 3 F, G, R, S, T or V
  • X 4 A, G, L, P, R, S or T
  • X 5 A, G, K, R, V or Y
  • X 6 P, R, S, V or Y
  • X 7 A, F, G, I, N, P, S, W or Y
  • X 8 A, F, L, P, R, T or Y
  • X 9 A, G, L, R, S, T or Y
  • X 10 L, P, Q, S, T, W or Y
  • X 11 D, G, N, R, S, T or Y
  • X 12 A, D, E, G, P, R , S or Y
  • X 13 A, G, L, N, R
  • the antigen binding protein may comprise HCDR3.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 218
  • X 3 D, F, G, K, N, R, S, T or V
  • X 4 D, G, I, L, R, S or T
  • X 5 A, F, G, R, V or Y
  • X 6 G, P, R, S, T, W or Y
  • X 7 A, G, L, N, R, S, W or Y
  • X 8 D, F, G, L, N, R, S, T or Y
  • X 9 A, D, G, I, L, P, R, S, T or Y
  • X 10 G, L, N, R, S, T, W or Y
  • the antigen binding protein may comprise HCDR3, and the HCDR3 may comprise such as SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:111, SEQ ID NO:115 , SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 132, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 140, SEQ ID NO:144, SEQ ID NO:147, SEQ ID NO:150, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO :165, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:176, SEQ ID NO:179, SEQ ID NO:182, SEQ ID NO:185, SEQ ID NO:188 , the amino acid sequence shown in any one of
  • the antigen binding protein may comprise HCDR2.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown below:
  • X 1 I or T
  • X 2 N, S or T
  • X 3 A
  • X 4 G, R, S, T or absent
  • X5 D or G
  • X6 G or S
  • X7 D, G, I, N, R, S, T or V.
  • the sequence can be partitioned according to IMGT rules.
  • the antigen binding protein may comprise HCDR2, and the HCDR2 may comprise such as SEQ ID NO:96, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:110, SEQ ID NO:114 , SEQ ID NO: 117, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 131, SEQ ID NO: 134, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO:146, SEQ ID NO:149, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO :171, SEQ ID NO:175, SEQ ID NO:178, SEQ ID NO:181, SEQ ID NO:187, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194 and SEQ ID NO:197
  • the antigen binding protein may include HCDR1.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown below:
  • X 2 F, G, I, L, N, R, S or Y
  • X 3 I or T
  • X 4 F or L
  • X 6 I, L, N, R, S, T or Y
  • X 7 D, N or Y
  • X 8 A, D, G, I, N, R, S, T, V or Y.
  • the sequence can be partitioned according to IMGT rules.
  • the antigen binding protein may comprise HCDR1, and the HCDR1 may comprise such as SEQ ID NO:95, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO:116 , SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO:145, SEQ ID NO:148, SEQ ID NO:152, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:166, SEQ ID NO:169, SEQ ID NO:174, SEQ ID NO :177, SEQ ID NO:180, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:189, SEQ ID NO:193 and the amino acid sequence shown in any one of SEQ ID NO:196.
  • the antigen binding protein may comprise HCDR1, HCDR2, HCDR3, and the HCDR1 may comprise SEQ ID NO:95, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:109, SEQ ID NO : 116, SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 142 , SEQ ID NO: 145, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 166, SEQ ID NO: 169, SEQ ID NO: 174, SEQ The amino acid sequence shown in any one of ID NO:177, SEQ ID NO:180, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:189, SEQ ID NO:193 and SEQ ID NO:196
  • the antigen binding protein may include HCDR1, HCDR2, HCDR3, and the HCDR1, HCDR2, HCDR3 may include any set of amino acid sequences in the following group:
  • HCDR1 SEQ ID NO: 102
  • HCDR2 SEQ ID NO: 103
  • HCDR3 SEQ ID NO: 104;
  • HCDR1 SEQ ID NO: 105
  • HCDR2 SEQ ID NO: 106
  • HCDR3 SEQ ID NO: 107;
  • HCDR1 SEQ ID NO: 109
  • HCDR2 SEQ ID NO: 110
  • HCDR3 SEQ ID NO: 111;
  • HCDR1 SEQ ID NO: 105
  • HCDR2 SEQ ID NO: 114
  • HCDR3 SEQ ID NO: 115;
  • HCDR1 SEQ ID NO: 116
  • HCDR2 SEQ ID NO: 117
  • HCDR3 SEQ ID NO: 118;
  • HCDR1 SEQ ID NO: 119
  • HCDR2 SEQ ID NO: 120
  • HCDR3 SEQ ID NO: 121;
  • HCDR1 SEQ ID NO: 123
  • HCDR2 SEQ ID NO: 124
  • HCDR3 SEQ ID NO: 125;
  • HCDR1 SEQ ID NO: 127
  • HCDR2 SEQ ID NO: 128, and HCDR3: SEQ ID NO: 129;
  • HCDR1 SEQ ID NO: 130
  • HCDR2 SEQ ID NO: 131
  • HCDR3 SEQ ID NO: 132
  • HCDR1 SEQ ID NO:95
  • HCDR2 SEQ ID NO:96
  • HCDR3 SEQ ID NO:97;
  • HCDR1 SEQ ID NO: 133
  • HCDR2 SEQ ID NO: 134
  • HCDR3 SEQ ID NO: 135;
  • HCDR1 SEQ ID NO: 136
  • HCDR2 SEQ ID NO: 134
  • HCDR3 SEQ ID NO: 137;
  • HCDR1 SEQ ID NO: 138
  • HCDR2 SEQ ID NO: 139
  • HCDR3 SEQ ID NO: 140;
  • HCDR1 SEQ ID NO: 142
  • HCDR2 SEQ ID NO: 143
  • HCDR3 SEQ ID NO: 144;
  • HCDR1 SEQ ID NO:145
  • HCDR2 SEQ ID NO:146
  • HCDR3 SEQ ID NO:147;
  • HCDR1 SEQ ID NO: 148
  • HCDR2 SEQ ID NO: 149
  • HCDR3 SEQ ID NO: 150;
  • HCDR1 SEQ ID NO: 152
  • HCDR2 SEQ ID NO: 153
  • HCDR3 SEQ ID NO: 154;
  • HCDR1 SEQ ID NO: 123
  • HCDR2 SEQ ID NO: 155
  • HCDR3 SEQ ID NO: 156;
  • HCDR1 SEQ ID NO:157
  • HCDR2 SEQ ID NO:153
  • HCDR3 SEQ ID NO:158;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:160
  • HCDR3 SEQ ID NO:161;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:162
  • HCDR3 SEQ ID NO:163;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:164
  • HCDR3 SEQ ID NO:165;
  • HCDR1 SEQ ID NO: 166
  • HCDR2 SEQ ID NO: 167
  • HCDR3 SEQ ID NO: 168;
  • HCDR1 SEQ ID NO:169
  • HCDR2 SEQ ID NO:153
  • HCDR3 SEQ ID NO:170
  • HCDR1 SEQ ID NO:157
  • HCDR2 SEQ ID NO:171
  • HCDR3 SEQ ID NO:172
  • HCDR1 SEQ ID NO:174
  • HCDR2 SEQ ID NO:175
  • HCDR3 SEQ ID NO:176;
  • HCDR1 SEQ ID NO:177
  • HCDR2 SEQ ID NO:178
  • HCDR3 SEQ ID NO:179;
  • HCDR1 SEQ ID NO: 180
  • HCDR2 SEQ ID NO: 181
  • HCDR3 SEQ ID NO: 182;
  • HCDR1 SEQ ID NO: 184
  • HCDR2 SEQ ID NO: 106
  • HCDR3 SEQ ID NO: 185;
  • HCDR1 SEQ ID NO: 186
  • HCDR2 SEQ ID NO: 187
  • HCDR3 SEQ ID NO: 188;
  • HCDR1 SEQ ID NO: 189
  • HCDR2 SEQ ID NO: 190
  • HCDR3 SEQ ID NO: 191;
  • HCDR1 SEQ ID NO:159
  • HCDR2 SEQ ID NO:192
  • HCDR3 SEQ ID NO:179;
  • HCDR1 SEQ ID NO: 193
  • HCDR2 SEQ ID NO: 194, and HCDR3: SEQ ID NO: 195;
  • HCDR1 SEQ ID NO: 196
  • HCDR2 SEQ ID NO: 197
  • HCDR3 SEQ ID NO: 198.
  • the antibody framework region FR refers to the part of the antibody variable region that exists between the more divergent (ie hypervariable) CDRs.
  • Such framework regions are typically referred to as Frameworks 1 to 4 (FR1, FR2, FR3, and FR4) and provide the backbone for representing the six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space, to Forms the antigen-binding surface.
  • the antigen binding protein may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:98.
  • the sequence can be partitioned according to IMGT rules.
  • the antigen binding protein may comprise H-FR2, and the H-FR2 may comprise such as SEQ ID NO: 99, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 122, SEQ ID NO: 122, SEQ ID NO: The amino acid sequence shown in any one of ID NO:151 and SEQ ID NO:173.
  • the sequence can be partitioned according to IMGT rules.
  • the antigen binding protein may comprise H-FR3, and the H-FR3 may comprise such as SEQ ID NO: 100, SEQ ID NO: 113, SEQ ID NO: 126, SEQ ID NO: 141 and SEQ ID NO: 141 and SEQ ID NO: 141 The amino acid sequence shown in any one of ID NO:183.
  • the antigen binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO: 101.
  • the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 may comprise SEQ Amino acid sequences shown in ID NO:98, SEQ ID NO:216, SEQ ID NO:217 and SEQ ID NO:101.
  • the antigen binding protein may comprise H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 98, the H -FR2 can comprise the amino acid sequence shown in any one of SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:112, SEQ ID NO:122, SEQ ID NO:151 and SEQ ID NO:173, so the H-FR3 may comprise the amino acid sequence shown in any one of SEQ ID NO:100, SEQ ID NO:113, SEQ ID NO:126, SEQ ID NO:141 and SEQ ID NO:183, and the H -FR4 may comprise the amino acid sequence shown in SEQ ID NO: 101.
  • the antigen binding protein may comprise an antibody heavy chain variable region VH
  • the VH may comprise SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO: 44.
  • the antigen-binding fragment may be VHH, and the VHH may comprise SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO: 46.
  • said isolated antigen binding protein may comprise a heavy chain constant region.
  • the heavy chain constant region refers to a region comprising at least three heavy chain constant domains CH1, CH2, and CH3.
  • Non-limiting exemplary heavy chain constant regions include gamma, delta, and alpha.
  • Non-limiting exemplary heavy chain constant regions also include ⁇ and ⁇ .
  • Each heavy chain constant region corresponds to an antibody isotype.
  • an antibody comprising a ⁇ constant region is an IgG antibody
  • an antibody comprising a ⁇ constant region is an IgD antibody
  • an antibody comprising an ⁇ constant region is an IgA antibody.
  • an antibody containing a mu constant region is an IgM antibody
  • an antibody containing an epsilon constant region is an IgE antibody.
  • IgG antibodies include, but are not limited to, IgG1 (comprising the ⁇ 1 constant region), IgG2 (comprising the ⁇ 2 constant region), IgG3 (comprising the ⁇ 3 constant region), and IgG4 (comprising the ⁇ 4 constant region) antibodies
  • IgA antibodies include But not limited to, IgA1 (comprising ⁇ 1 constant region) and IgA2 (comprising ⁇ 2 constant region) antibodies
  • IgM includes but not limited to, IgM1 and IgM2.
  • the antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from IgG.
  • the antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG.
  • the antigen-binding protein may comprise an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from human IgG1.
  • the present application also provides a chimeric antigen receptor (CAR), which may comprise a targeting moiety that binds to the GPRC5D protein, for example, the targeting moiety that binds to the GPRC5D protein may be is the antigen binding protein described in the application.
  • CAR chimeric antigen receptor
  • the CAR of the present application may comprise a VHH, which may comprise SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 , SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO :48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64 , the amino acid sequence shown in any one of SEQ ID NO:66, SEQ ID NO:68 and SEQ ID NO:
  • the CAR includes an extracellular targeting part that binds to the GPRC5D protein, and may also include an intracellular domain.
  • the CAR may include an intracellular co-stimulatory signal region, which can provide a stimulating signal.
  • the co-stimulatory signal domain may comprise an intracellular co-stimulatory signal domain of one or more proteins selected from the group consisting of CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligand for CD83, CD40 and MyD88.
  • the co-stimulatory signal domain may be an intracellular co-stimulatory signal domain derived from 4-1BB.
  • the co-stimulatory signal region may comprise the amino acid sequence shown in SEQ ID NO:78.
  • the CAR may comprise an intracellular signaling region, which may comprise a domain having at least one ITAM motif.
  • the intracellular signaling domain can transmit activation signals to the interior of the cell.
  • the intracellular signaling region may comprise an intracellular signaling region derived from one or more proteins selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia Viral gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and others that contain at least one ITAM domain.
  • EBV Epstein-Barr virus
  • PBj14Nef simian immunodeficiency virus
  • HSKV Kaposi's sarcoma herpesvirus
  • the intracellular signaling region can be a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling region may comprise the amino acid sequence shown in SEQ ID NO:80.
  • the CAR may comprise a transmembrane domain, which is a sequence of a cell surface protein that spans the cell membrane, which may comprise a hydrophobic alpha helix.
  • the transmembrane domain may be derived from any type I transmembrane protein.
  • the transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix.
  • the transmembrane region may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane region may be a transmembrane region derived from CD8.
  • the transmembrane region may comprise the amino acid sequence shown in SEQ ID NO:76.
  • the CAR may comprise a hinge region, which may be located between the extracellular targeting moiety and the transmembrane domain.
  • the hinge region may comprise a hinge region of one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.
  • the hinge region may be a hinge region derived from CD8.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:74.
  • the CAR may further include a signal peptide at the N-terminus of the targeting portion that binds to the GPRC5D protein.
  • the signal peptide may be a signal peptide derived from CD8 protein.
  • the signal peptide may comprise the amino acid sequence shown in SEQ ID NO:72.
  • the CAR may also comprise low-density lipoprotein receptor-related protein or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be located at the C-terminus of the CAR.
  • the low-density lipoprotein receptor-related protein or fragments thereof may include low-density lipoprotein receptor-related protein 1-12 and functional fragments thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may be low-density lipoprotein receptor-related protein 6 or a fragment thereof.
  • the low-density lipoprotein receptor-related protein or a fragment thereof may include the amino acid sequence shown in SEQ ID NO:84.
  • the low-density lipoprotein receptor-associated protein or fragments thereof in the CAR may be linked to the C-terminus of the CAR through a self-cleaving peptide (for example, 2A peptides such as T2A, P2A, E2A, etc.).
  • a self-cleaving peptide for example, 2A peptides such as T2A, P2A, E2A, etc.
  • the low-density lipoprotein receptor-related protein or its fragments can be connected to the C-terminus of the intracellular signaling region through T2A.
  • the CAR may sequentially comprise a targeting moiety that binds to the GPRC5D protein (for example, the antigen-binding protein, and for example, the VHH described in the present application), the hinge region, the The transmembrane domain, the co-stimulatory signal region and the intracellular signal region.
  • GPRC5D protein for example, the antigen-binding protein, and for example, the VHH described in the present application
  • the CAR may sequentially comprise the VHH, the hinge region derived from CD8, the transmembrane region derived from CD8, the co-stimulatory signal region derived from 4-1BB, and the CD3 ⁇ -derived Intracellular signaling region
  • the VHH may comprise SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO :16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32 , SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:50, SEQ ID NO:52
  • the CAR may sequentially comprise a targeting moiety that binds to the CPRC50D protein (for example, the antigen-binding protein, and for example, the VHH described in the present application), the hinge region, the The transmembrane domain, the co-stimulatory signal region, the intracellular signal region and the low-density lipoprotein receptor-associated protein or fragments thereof.
  • the CAR may sequentially comprise the VHH, the hinge region derived from CD8, the transmembrane region derived from CD8, the co-stimulatory signal region derived from 4-1BB, and the cellular region derived from CD3 ⁇ .
  • Inner signal region, and low-density lipoprotein receptor-associated protein or fragment thereof comprising the amino acid sequence shown in SEQ ID NO:84, and the VHH may comprise SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO :8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 , SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO :58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64
  • the CAR may sequentially include a signal peptide, a targeting moiety that binds to the GPRC5D protein (for example, the antigen-binding protein, and for example, the VHH described in this application), the hinge region, the transmembrane domain, the co-stimulatory signal region and the intracellular signal region.
  • a targeting moiety that binds to the GPRC5D protein for example, the antigen-binding protein, and for example, the VHH described in this application
  • the hinge region for example, the antigen-binding protein, and for example, the VHH described in this application
  • the transmembrane domain for example, the co-stimulatory signal region and the intracellular signal region.
  • the CAR may sequentially include a signal peptide, a targeting moiety that binds to the GPRC5D protein (for example, the antigen-binding protein, and for example, the VHH described in this application), the hinge region, the transmembrane domain, the co-stimulatory signal region, the intracellular signal region and the low-density lipoprotein receptor-associated protein or a fragment thereof.
  • a targeting moiety that binds to the GPRC5D protein for example, the antigen-binding protein, and for example, the VHH described in this application
  • the chimeric antigen receptor package may comprise SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:201, The amino acid sequence shown in any one of ID NO:202, SEQ ID NO:203 and SEQ ID NO:204.
  • the chimeric antigen receptor package may comprise SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:210, The amino acid sequence shown in any one of ID NO:211, SEQ ID NO:212, SEQ ID NO:213 and SEQ ID NO:214.
  • the chimeric antigen receptor may be encoded by any one of the nucleotide sequences comprising SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:91.
  • the present application also provides one or more nucleic acid molecules, which can be nucleotides, deoxynucleotides and/ribonucleotides in isolated forms of any length, and can encode said isolated Antigen binding protein and/or said chimeric antigen receptor.
  • the nucleic acid molecule can include a promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be the EF1 ⁇ promoter.
  • the nucleic acid molecule may comprise SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO: 51.
  • nucleic acid molecule may comprise the nucleotide sequence shown in any one of SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89 and SEQ ID NO:91.
  • the present application also provides a carrier, which may include the nucleic acid molecule.
  • the vector can transform, transduce or transfect host cells, so that the genetic material elements carried by it can be expressed in the host cells.
  • vectors can include promoters, transcripts, enhancers, replicons, selection elements, and reporter genes.
  • a carrier may include components that facilitate entry into cells.
  • the 5' and 3' ends of the nucleic acid molecule may also contain long terminal repeats.
  • the vector may be a viral vector.
  • the vector may be a lentiviral vector.
  • the present application also provides cells, which may include the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule and/or the carrier.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell.
  • the cells may be immune effector cells.
  • the cells may include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes , peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells and/or pluripotent stem cells.
  • the cells may be T cells.
  • the present application also provides a pharmaceutical composition, which may include the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the cell, and any Optionally a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition may further comprise one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives.
  • the acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed.
  • Pharmaceutical compositions of the present invention may include liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable adjuvants may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with drug administration, generally safe, non-toxic , and is neither biologically nor otherwise undesirable.
  • the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application also provides a method for preparing the isolated antigen-binding protein and/or the chimeric antigen receptor.
  • the method may comprise culturing said cell under conditions such that said antigen receptor and/or said chimeric antigen receptor is expressed.
  • the present application also provides a method for preparing modified immune effector cells, which may include introducing the carrier into immune cells.
  • the present application also provides the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition
  • the medicaments can be used to prevent, relieve and/or treat tumors.
  • the present application also provides a method for preventing, alleviating and/or treating tumors, the method may include administering the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule to the subject , the carrier, the cell and/or the pharmaceutical composition.
  • the present application also provides the isolated antigen-binding protein, the chimeric antigen receptor, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition for use in for the prevention, mitigation and/or treatment of tumors.
  • the tumor may include solid tumors and/or non-solid tumors.
  • the tumor may include hematoma and/or lymphoma.
  • the tumor may include myeloma. In the present application, the tumor may include multiple myeloma.
  • the subject may include humans or non-human animals.
  • the application provides a polypeptide comprising said isolated antigen binding protein. In another aspect, the application provides an antibody drug conjugate comprising the isolated antigen binding protein.
  • the present application provides a kit or a drug delivery device comprising the isolated antigen binding protein, the chimeric antigen receptor, the nucleic acid molecule, the carrier, the cell and/or the said pharmaceutical composition.
  • CDR3 was three types: 14, 17 and 21 amino acids.
  • Select the already-marketed Nanobody Caplacizumab (Caplacizumab) as the backbone synthesize the nucleotide sequence of Caplacizumab, and then clone it into the HP153 phage vector, then extract the single-stranded DNA of HP153, and use the method of Kunkel Mutagenesis to mutate the Nanobody CDRs, to obtain double-stranded DNA.
  • the double-stranded DNA was electrotransferred into the competent Escherichia coli SS320 pre-infected with M13KO7 helper phage, and the supernatant of the phage was collected after overnight culture. Finally, a synthetic nanobody library NanoOri_1.0 (Shanghai Yuanneng Cell) with a diversity of 1.44 ⁇ 10 10 was constructed. Medical Technology Co., Ltd.), as a seed bank for antibody sequence screening.
  • the GPRC5D full-length sequence (cat:HG24447-UT) purchased from Sino Biological Company was constructed on the company's existing lentiviral vector, and the lentiviral transduction was performed to prepare the overexpression of target cells CHO-GPRC5D and target cells 293-GPRC5D cell line.
  • the target cells CHO-GPRC5D and 293-GPRC5D were used for alternate panning of the artificially synthesized nanobody library (Shanghai Yuanneng Cell Medical Technology Co., Ltd.), and a total of four rounds of panning were carried out:
  • the target cells CHO-GPRC5D and 293-GPRC5D were selected for alternate screening of cell panning. Mix 500 ⁇ L of phage and 500 ⁇ L of 10% FBS/PBS buffer to a final volume of 1 mL, put it in a 2 mL low-adsorption EP tube, and block it for 1 hour at 4°C by gently rotating or periodically mixing. CHO-GPRC5D cells were harvested by centrifugation at 140 g for 10 min, and washed once with 10 mL of PBS.
  • the number of living cells reached more than 95%, and the CHO-GPRC5D cells were washed three times with 5% FBS/PBS buffer. Then resuspend in 1 mL of 5% FBS/PBS buffer to make the number of cells reach 1 ⁇ 10 7 . Cells were kept on ice during this period.
  • Centrifuge CHO-GPRC5D cells at 140g at 4°C for 2min, resuspend in 1mL phage antibody library that has been blocked, rotate gently or mix periodically at 4°C or room temperature for binding for 2 hours, remove supernatant, and harvest cells Resuspend in 1mL of 5% FBS/PBS buffer, repeat washing twice, after resuspending the cells for the last time, change to a new 2mL low-adsorption EP tube to avoid the phage non-specifically adsorbed to the EP tube from being washed take it off.
  • the eluted and neutralized phages were resuspended in CHO cells. Gently swirl at 4°C, or mix periodically for 30 min.
  • cell-based mono phage ELISA Cell-based mono phage ELISA
  • 34 phage antibody clones that is, the isolated GPRC5D antibody described in this application
  • 34 phage antibody clones capable of specifically binding to human GPRC5D
  • the single-chain V H H gene sequence was obtained, and these 10 phage antibody clones were respectively Named 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1, 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506C12, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506F5, 506G1, 506H2 and 506H9.
  • the CDR sequences of the 34 clones are shown in Table 1, and the amino acid sequences of the variable regions are SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68
  • the plasmid was transfected into 293F cells for transient expression, and purified through a protein A affinity column (Protein A column) to obtain 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1 , 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506B12, 506C2, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506H9, 1.
  • the SEC purity of the antibody part is shown in Table 2,
  • VHH-hFc 97.5% 3E7 VHH-hFc 100% 4A2 VHH-hFc 100% 4B3 VHH-hFc 100% 3E9 VHH-hFc 76.5% 3F5 VHH-hFc 100% 3G4 VHH-hFc 100% 4A4 VHH-hFc 100%
  • FACS flow cytometry fluorescence sorting technique
  • iQue Screener flow meter purchased from IntelliCyt Company
  • PBS containing 0.1% BSA as buffer to carry out cell surface target antigen (GPRC5D) and antibody (i.e.
  • Example 2 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12, 4F10, 505A1, 505A3, 505A11, 505B12, 505D2, 505F1, 505H3, 505H8, 505H10, 506A5, 506A8, 506B6, 506B12, 506C5, 506C8, 506C11, 506D2, 506D8, 506E1, 506F5, 506G1, 506H2 and 506H9 antibodies) binding activity assay.
  • target cells M1S myeloma cells
  • concentration of detection antibody was prepared in buffer at 10 ⁇ g/mL mL, and dilute the antibody by 3 times to form 8 concentration gradients
  • add 30 ⁇ L/well of prepared antibodies of different concentrations to the plated target cells and mix well incubate at 4°C for 1 hour; add 150 ⁇ L to each well Buffer, centrifuge at 300g for 5 minutes, discard the supernatant and shake the cells loose; repeat the wash once; use the buffer to prepare the fluorescent secondary antibody (ab98593) at a ratio of 1:200, add 30 ⁇ l per well to the cells and mix well, refrigerate at 4°C Incubate for 30 minutes; add 150 ⁇ L of buffer to each well, centrifuge at 300 g for 5 minutes, discard the supernatant and shake the
  • SphI and NotI endonucleases purchased from NEB were used to double digest the CAR lentiviral core empty plasmid (self-constructed by Shanghai Yuanneng Cell Medical Technology Co., Ltd., containing the new ori2 element, hereinafter referred to as the CAR lentiviral empty vector) 8992bp linearized fragment, recovered from rubber tapping, mixed with the VHH fragments of 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4, 4E12 and 4F10 obtained in Example 1 at a molar ratio of 1:3 for homologous recombination , to transform DH5 ⁇ competent cells. Single colonies were picked for sequencing identification.
  • Example 5 Packaging of lentivirus and determination of virus titer
  • the lentiviral vector system used to construct the present invention belongs to the third generation, and the system is composed of three plasmids, namely the packaging plasmid psPAX2 encoding Gag-Pol protein and Rev protein, and the PMD2 encoding envelope protein VSV-G.
  • each CAR lentiviral plasmid containing VHH sequence in the above-mentioned embodiment 4 ie 3B5VHH-CAR-ori2, 3E7-CAR-ori2, 4A2-CAR-ori2, 4B3-CAR-ori2, 3E9 -CAR-ori2, 3F5-CAR-ori2, 3G4-CAR-ori2, 4A4-CAR-ori2, 4E12-CAR-ori2 and 4F10-CAR-ori2).
  • the expression of the CAR gene in each CAR lentiviral plasmid is regulated by the elongation factor-1 ⁇ (EF-1 ⁇ ) promoter.
  • EF-1 ⁇ elongation factor-1 ⁇
  • the cell culture plate was placed in a 37° C., 5% CO 2 incubator for static culture.
  • NBS solution PBS solution containing 1% newborn bovine serum
  • centrifuge at 500g for 5min at 4°C and discard the supernatant.
  • Results are only available if each test tube is positive - the negative control is 5% - 20% positive.
  • Lentivirus titer number of plated cells ⁇ (positive rate of test tube - positive rate of control tube) / volume of inoculated virus solution (mL).
  • the titer of each of the above-mentioned CAR viruses containing VHH was in the range of 1-10 ⁇ 10 6 IM/mL.
  • PBMC peripheral blood mononuclear cells
  • CD3+ T cells with a purity > 90% were obtained after PBMCs were sorted by CD3 positive selection magnetic beads.
  • T cell sorting please refer to the product manual (MACS, DS130-050-101).
  • Lentiviral transduction is generally performed 20-24 hours after T cell activation.
  • T cell complete medium a certain volume of T cell complete medium, take samples and count them, and add To the virus solution whose titer has been determined in Example 5, polybrene was added to the final concentration of 5 ⁇ g/mL, and then T cell complete medium was added to adjust the cell density to 0.6-1.0 ⁇ 10 6 /mL, and the cells were placed in Cultured in a 37°C, 5% CO 2 incubator, and another part of T cells was not infected with lentivirus, which was used as a negative control group.
  • T cells of each group collect the T cells of each group, centrifuge at 500g for 5 minutes, resuspend the cells with a certain volume of T cell complete medium, take samples and count, add T cell complete medium, and adjust the cells
  • the density is 0.5-0.7 ⁇ 10 6 cells/mL.
  • CAR-T cells were counted every 1-2 days, and T cell complete medium was supplemented according to the actual situation, and the cell density was adjusted to 0.5-1.0 ⁇ 10 6 cells/mL.
  • Dynabeads are removed on the 7th day of culture, and the total culture period is about 12 days.
  • the experimental results are shown in Figures 3A-3D.
  • the total expansion factor of GPRC5D CAR-T cells after 12 days of activation and culture is between 100-600 times, and the CAR-positive rate after infection is between 75% and 95%. It can be used for cytological function experiments.
  • GPRC5D In order to accurately detect the killing ability of CAR-T cells on target cells, we introduced the GPRC5D gene into CHO cells by means of lentivirus, and then sorted GPRC5D positive cells by flow cytometry, that is, CHO-GPRC5D cells.
  • the target cells are CHO-GPRC5D cells, and the negative cells are CHO negative cells.
  • the effector cells cultured to the 9th day were incubated with the target cells for 20 hours at the effect-to-target ratios of 1:3, 1:1 and 3:1, respectively, and the survival of the target cells was judged by the LDH method (i.e. CAR- lethality of T cells).
  • Cell killing toxicity can be calculated with the following formula:
  • Another method for detecting killing is to use MM1S-lucifurase as the target cell, and after co-incubating with effector CAR-T cells for 6 hours, the killing effect is determined by detecting the luciferase value in the remaining target cells.
  • the effect-to-target ratio is 1:1, and the test results are shown in 4D. The results show that compared with T cells, the CAR-T cells of the present application have obvious killing effects.
  • the target cell killing experiment was carried out in a 96-well plate, the ratio of effector cells to target cells was 1:1, and then the number of effector cells in each 96-well plate was fixed at 2 ⁇ 104 , and target cells and effector cells were added in sequence.
  • target cells and effector cells were added in sequence.
  • untreated MM1S cells were selected as target cells for the detection of the targeted proliferation ability of CAR-T cells.
  • the CAR-T cells are cultured for about 9-12 days.
  • the CAR positive rate of each group needs to be detected in advance, and then the blank effector T cells are used to adjust the CAR positive rate of each group to be consistent (the culture medium does not contain any additives. X-VIVO 15 medium).
  • CAR-T cells and MM1S cells were subjected to the first round of targeted stimulation at an effect-to-target ratio of 1:1. After co-incubating for 4-5 days, all the cells were collected, sampled and counted, and then 1:3 (full CAR- The ratio of T cells: new MM1S cells) was used for the second round of targeted stimulation, and after 4-5 days of co-incubation, the third round of targeted stimulation was performed (the ratio between the two cells was the same as that of the second round of targeted stimulation). During the three rounds of targeted stimulation, an appropriate amount of X-VIVO 15 medium was added every 1-2 days according to the growth of the cells.
  • the amplification factor of the CAR-T of this application is comparable to that of the positive control.
  • the co-incubation wells of T cells and the single MM1S target cell wells were basically not amplified.
  • mice were further verified the tumor elimination and tumor suppression effects of GPRC5D CAR-T cells in NSG mice. Specifically, the experiment was divided into three groups, T cell group, positive control group and GPRC5D CAR-T cell group, with 5 mice in each group. First subcutaneously inoculate NSG mice with MM1S cells at a dose of 1 ⁇ 10 7 cells/mouse, and about 2 weeks later (tumor size is 50-150mm 3 ), inject the CARs of the three groups that have been cultured for 10-12 days into the tail vein -T (or T) cells, the dose is 2 ⁇ 10 6 CAR-T cells/mouse (the total number of inoculated cells in each mouse is adjusted to be consistent with T cells).
  • mice After inoculation of CAR-T (or T) cells, the secretion of cytokines in the peripheral blood of mice was detected on the 7th and 14th days after CAR-T (or T) cell inoculation, and the tumor size and body weight of mice were measured 3 times a week , observed for 3 weeks.
  • the GPRC5D CAR-T cell group can produce a larger amount of IFN- ⁇ in mice, and the duration is also longer (IFN- ⁇ detected on the 14th day after CAR-T injection - ⁇ significantly increased compared with the positive control group), but the secretion of other cytokines such as IL-2 was very low, and the difference between the groups was small.
  • GPRC5D CAR-T cells were basically consistent with the positive control group, and compared with the T cell group, they had a significant tumor elimination effect.
  • mice were weighed every 2-3 days, the weight curve of the mice was drawn, and the mouse coat color, excretion, Diet, water, physical exercise, and death of mice.
  • the body weight of GPRC5D CAR-T cells and the positive control group showed a steady growth trend over time, which was consistent with the trend of the body weight curve of the T group mice.
  • the coat color of the GPRC5D CAR-T cells and the positive control group were bright, and the behavior, diet and water intake of the mice, as well as the color of urine and stool were normal, which confirmed that GPRC5D CAR-T cells had better safety in mice.
  • Embodiment 11 Specificity experiment of GPRC5D antibody
  • FACS flow cytometry fluorescence sorting technique
  • iQue Screener flow meter purchased from IntelliCyt Company
  • PBS containing 0.1% BSA as buffer to carry out cell surface target antigen (GPRC5D) and antibody (i.e. Example 2 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4 and 4E12 antibodies) for non-specific binding experiments.
  • GPRC5D cell surface target antigen
  • antibody i.e. Example 2 3B5, 3E7, 4A2, 4B3, 3E9, 3F5, 3G4, 4A4 and 4E12 antibodies
  • target cells cells that do not express GPRC5D antigen, such as A549 cells, KERA cells, MCF-7 cells, and MSC cells
  • concentration of 1x106 cells/mL were prepared with buffer, and added to a 96-well sharp bottom plate (corning 3894), 30 ⁇ L per well.

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Abstract

一种分离的抗原结合蛋白，所述抗原结合蛋白以1.0μg/mL的EC 50值结合G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。包含所述抗原结合蛋白的嵌合抗原受体，以及它们在治疗肿瘤中的用途。

Description

抗GPRC5D抗原结合蛋白及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种抗GPRC5D抗原结合蛋白，包含所述抗原结合蛋白的嵌合抗原受体，以及它们在治疗肿瘤中的用途。
背景技术
多发性骨髓瘤(Multiple Myeloma，MM)是第二常见的恶性血液病，并且在癌症致死率中居于第2位。MM属于浆细胞恶性肿瘤，常伴有多发性溶骨性损害、肾脏功能损害、骨髓浸润、高钙血症和贫血。MM在目前的主要治疗方法是副作用严重的全身化疗，并且无法彻底治愈。
G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白，是一种非典型的表面孤儿受体。GPRCD5与其他C5家族受体一样拥有过短的氨基末端，因此在构象上与C4家族十分类似。其在正常组织中表达仅限于毛囊，但在MM患者的骨髓中也有特异性高表达，并与浆细胞肿瘤负荷与遗传畸变高度相关。
随着蛋白酶抑制剂(PI)、免疫调节药物(IMiD)和单克隆抗体(mAb)等新型药物的介入，MM的治疗方式得到扩充，但依然有着极高的复发率并产生耐药性。而嵌合抗原受体T细胞(CAR-T)治疗对MM产生了显著疗效和可控的毒性，其中主流的以B细胞成熟抗原(BCMA)为靶点的CAR-T细胞疗法对于BCMA阴性或BCMA低表达的MM病患存在靶向逃逸问题。GPRC5D在浆细胞上的特异性高表达，使其成为了MM的理想治疗靶点，但以其为靶点的药物治疗产品尚未成熟。
发明内容
本申请提供一种分离的抗原结合蛋白，其能够特异性结合G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。本申请还提供了包含所述抗原结合蛋白的嵌合抗原受体，以及包含和/或表达该嵌合抗原受体的细胞，所述细胞具有以下一种或多种特点：(1)扩增能力强，(2)能够杀伤表达GPRC5D的靶细胞，(3)在靶细胞刺激下分泌细胞因子，(4)在靶细胞刺激下有较强的增殖能力，以及，(4)抑制肿瘤生长。
一方面，本申请提供了一种分离的抗原结合蛋白，其能够以低于1.0μg/mL的EC50值结合G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。
在某些实施方式中，所述的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab、Fab’、F(ab)2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述抗原结合片段为VHH。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述的抗原结合蛋白能够与参比抗体竞争结合GPRC5D蛋白，其中所述参比抗体包含抗体重链可变区VH，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，且所述参比抗体包含选自下述的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:102，HCDR2：SEQ ID NO:103，和HCDR3：SEQ ID NO:104；
(2)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:107；
(3)HCDR1：SEQ ID NO:109，HCDR2：SEQ ID NO:110，和HCDR3：SEQ ID NO:111；
(4)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:114，和HCDR3：SEQ ID NO:115；
(5)HCDR1：SEQ ID NO:116，HCDR2：SEQ ID NO:117，和HCDR3：SEQ ID NO:118；
(6)HCDR1：SEQ ID NO:119，HCDR2：SEQ ID NO:120，和HCDR3：SEQ ID NO:121；
(7)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:124，和HCDR3：SEQ ID NO:125；
(8)HCDR1：SEQ ID NO:127，HCDR2：SEQ ID NO:128，和HCDR3：SEQ ID NO:129；
(9)HCDR1：SEQ ID NO:130，HCDR2：SEQ ID NO:131，和HCDR3：SEQ ID NO:132；
(10)HCDR1：SEQ ID NO:95，HCDR2：SEQ ID NO:96，和HCDR3：SEQ ID NO:97；
(11)HCDR1：SEQ ID NO:133，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:135；
(12)HCDR1：SEQ ID NO:136，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:137；
(13)HCDR1：SEQ ID NO:138，HCDR2：SEQ ID NO:139，和HCDR3：SEQ ID NO:140；
(14)HCDR1：SEQ ID NO:142，HCDR2：SEQ ID NO:143，和HCDR3：SEQ ID NO:144；
(15)HCDR1：SEQ ID NO:145，HCDR2：SEQ ID NO:146，和HCDR3：SEQ ID NO:147；
(16)HCDR1：SEQ ID NO:148，HCDR2：SEQ ID NO:149，和HCDR3：SEQ ID NO:150；
(17)HCDR1：SEQ ID NO:152，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:154；
(18)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:155，和HCDR3：SEQ ID NO:156；
(19)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:158；
(20)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:160，和HCDR3：SEQ ID NO:161；
(21)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:162，和HCDR3：SEQ ID NO:163；
(22)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:164，和HCDR3：SEQ ID NO:165；
(23)HCDR1：SEQ ID NO:166，HCDR2：SEQ ID NO:167，和HCDR3：SEQ ID NO:168；
(24)HCDR1：SEQ ID NO:169，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:170；
(25)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:171，和HCDR3：SEQ ID NO:172；
(26)HCDR1：SEQ ID NO:174，HCDR2：SEQ ID NO:175，和HCDR3：SEQ ID NO:176；
(27)HCDR1：SEQ ID NO:177，HCDR2：SEQ ID NO:178，和HCDR3：SEQ ID NO: 179；
(28)HCDR1：SEQ ID NO:180，HCDR2：SEQ ID NO:181，和HCDR3：SEQ ID NO:182；
(29)HCDR1：SEQ ID NO:184，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:185；
(30)HCDR1：SEQ ID NO:186，HCDR2：SEQ ID NO:187，和HCDR3：SEQ ID NO:188；
(31)HCDR1：SEQ ID NO:189，HCDR2：SEQ ID NO:190，和HCDR3：SEQ ID NO:191；
(32)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:192，和HCDR3：SEQ ID NO:179；
(33)HCDR1：SEQ ID NO:193，HCDR2：SEQ ID NO:194，和HCDR3：SEQ ID NO:195；以及
(34)HCDR1：SEQ ID NO:196，HCDR2：SEQ ID NO:197，和HCDR3：SEQ ID NO:198。
在某些实施方式中，所述的抗原结合蛋白包括源自抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包括HCDR3，且所述HCDR3包含SEQ ID NO:215或SEQ ID NO:218所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包括HCDR3，且所述HCDR3包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:115、SEQ ID NO:118、SEQ ID NO:121、SEQ ID NO:125、SEQ ID NO:129、SEQ ID NO:132、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:140、SEQ ID NO:144、SEQ ID NO:147、SEQ ID NO:150、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:176、 SEQ ID NO:179、SEQ ID NO:182、SEQ ID NO:185、SEQ ID NO:188、SEQ ID NO:191、SEQ ID NO:195和SEQ ID NO:198中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包括HCDR2，且所述HCDR2包含X 1X 2X 3X 4X 5X 6X 7T所示的氨基酸序列，其中，X 1为I或T，X 2为N、S或T，X 3为A、P、R、S或W，X 4为G、R、S、T或不存在，X 5为D或G，X 6为G或S，且X 7为D、G、I、N、R、S、T或V。
在某些实施方式中，所述的抗原结合蛋白包括HCDR2，且所述HCDR2包含SEQ ID NO:96、SEQ ID NO:103、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:114、SEQ ID NO:117、SEQ ID NO:120、SEQ ID NO:124、SEQ ID NO:128、SEQ ID NO:131、SEQ ID NO:134、SEQ ID NO:139、SEQ ID NO:143、SEQ ID NO:146、SEQ ID NO:149、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:167、SEQ ID NO:171、SEQ ID NO:175、SEQ ID NO:178、SEQ ID NO:181、SEQ ID NO:187、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194和SEQ ID NO:197中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包括HCDR1，且所述HCDR1包含GX 2X 3X 4SX 6X 7X 8所示的氨基酸序列，其中，X 2为F、G、I、L、N、R、S或Y，X 3为I或T，X 4为F或L，X 6为I、L、N、R、S、T或Y，X 7为D、N或Y，且X 8为A、D、G、I、N、R、S、T、V或Y。
在某些实施方式中，所述的抗原结合蛋白包括HCDR1，且所述HCDR1包含SEQ ID NO:95、SEQ ID NO:102、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:116、SEQ ID NO:119、SEQ ID NO:123、SEQ ID NO:127、SEQ ID NO:130、SEQ ID NO:133、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:142、SEQ ID NO:145、SEQ ID NO:148、SEQ ID NO:152、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:166、SEQ ID NO:169、SEQ ID NO:174、SEQ ID NO:177、SEQ ID NO:180、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:189、SEQ ID NO:193和SEQ ID NO:196中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包括HCDR1、HCDR2、HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下组的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:102，HCDR2：SEQ ID NO:103，和HCDR3：SEQ ID NO:104；
(2)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO: 107；
(3)HCDR1：SEQ ID NO:109，HCDR2：SEQ ID NO:110，和HCDR3：SEQ ID NO:111；
(4)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:114，和HCDR3：SEQ ID NO:115；
(5)HCDR1：SEQ ID NO:116，HCDR2：SEQ ID NO:117，和HCDR3：SEQ ID NO:118；
(6)HCDR1：SEQ ID NO:119，HCDR2：SEQ ID NO:120，和HCDR3：SEQ ID NO:121；
(7)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:124，和HCDR3：SEQ ID NO:125；
(8)HCDR1：SEQ ID NO:127，HCDR2：SEQ ID NO:128，和HCDR3：SEQ ID NO:129；
(9)HCDR1：SEQ ID NO:130，HCDR2：SEQ ID NO:131，和HCDR3：SEQ ID NO:132；
(10)HCDR1：SEQ ID NO:95，HCDR2：SEQ ID NO:96，和HCDR3：SEQ ID NO:97；
(11)HCDR1：SEQ ID NO:133，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:135；
(12)HCDR1：SEQ ID NO:136，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:137；
(13)HCDR1：SEQ ID NO:138，HCDR2：SEQ ID NO:139，和HCDR3：SEQ ID NO:140；
(14)HCDR1：SEQ ID NO:142，HCDR2：SEQ ID NO:143，和HCDR3：SEQ ID NO:144；
(15)HCDR1：SEQ ID NO:145，HCDR2：SEQ ID NO:146，和HCDR3：SEQ ID NO:147；
(16)HCDR1：SEQ ID NO:148，HCDR2：SEQ ID NO:149，和HCDR3：SEQ ID NO:150；
(17)HCDR1：SEQ ID NO:152，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:154；
(18)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:155，和HCDR3：SEQ ID NO: 156；
(19)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:158；
(20)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:160，和HCDR3：SEQ ID NO:161；
(21)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:162，和HCDR3：SEQ ID NO:163；
(22)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:164，和HCDR3：SEQ ID NO:165；
(23)HCDR1：SEQ ID NO:166，HCDR2：SEQ ID NO:167，和HCDR3：SEQ ID NO:168；
(24)HCDR1：SEQ ID NO:169，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:170；
(25)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:171，和HCDR3：SEQ ID NO:172；
(26)HCDR1：SEQ ID NO:174，HCDR2：SEQ ID NO:175，和HCDR3：SEQ ID NO:176；
(27)HCDR1：SEQ ID NO:177，HCDR2：SEQ ID NO:178，和HCDR3：SEQ ID NO:179；
(28)HCDR1：SEQ ID NO:180，HCDR2：SEQ ID NO:181，和HCDR3：SEQ ID NO:182；
(29)HCDR1：SEQ ID NO:184，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:185；
(30)HCDR1：SEQ ID NO:186，HCDR2：SEQ ID NO:187，和HCDR3：SEQ ID NO:188；
(31)HCDR1：SEQ ID NO:189，HCDR2：SEQ ID NO:190，和HCDR3：SEQ ID NO:191；
(32)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:192，和HCDR3：SEQ ID NO:179；
(33)HCDR1：SEQ ID NO:193，HCDR2：SEQ ID NO:194，和HCDR3：SEQ ID NO:195；以及
(34)HCDR1：SEQ ID NO:196，HCDR2：SEQ ID NO:197，和HCDR3：SEQ ID NO:198。
在某些实施方式中，所述的抗原结合蛋白包含抗体重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:98所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白中所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:216所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白中所述H-FR2包含SEQ ID NO:99、SEQ ID NO:108、SEQ ID NO:112、SEQ ID NO:122、SEQ ID NO:151和SEQ ID NO:173中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白中所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:217所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白中所述H-FR3包含SEQ ID NO:100、SEQ ID NO:113、SEQ ID NO:126、SEQ ID NO:141和SEQ ID NO:183中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白中所述VH包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:101所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白中所述VH包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白为VHH，且所述VHH包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、 SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在某些实施方式中，所述的抗原结合蛋白包含抗体重链恒定区，所述抗体重链恒定区源自IgG。
在某些实施方式中，所述的抗原结合蛋白包含抗体重链恒定区，所述抗体重链恒定区源自人IgG。
在某些实施方式中，所述的抗原结合蛋白包含抗体重链恒定区，所述抗体重链恒定区源自人IgG1。
另一方面，本申请提供了一种嵌合抗原受体，其包含靶向部分，所述靶向部分包含所述的抗原结合蛋白。
在某些实施方式中，所述的嵌合抗原受体包含共刺激信号区域，其中所述胞共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述的嵌合抗原受体中所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
在某些实施方式中，所述的嵌合抗原受体中所述共刺激信号区域包含SEQ ID NO:78所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包括胞内信号区域，所述胞内信号区域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。
在某些实施方式中，所述的嵌合抗原受体中所述胞内信号区域为源自CD3ζ的信号传导结构域。
在某些实施方式中，所述的嵌合抗原受体中所述胞内信号区域包含SEQ ID NO:80所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
在某些实施方式中，所述的嵌合抗原受体中所述跨膜区为源自CD8的跨膜区。
在某些实施方式中，所述的嵌合抗原受体中所述跨膜区包含SEQ ID NO:76所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体在靶向部分和跨膜区之间包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
在某些实施方式中，所述的嵌合抗原受体中所述铰链区为源自CD8的铰链区。
在某些实施方式中，所述的嵌合抗原受体中所述铰链区包含SEQ ID NO:74所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体还包含低密度脂蛋白受体相关蛋白或其片段，所述低密度脂蛋白受体相关蛋白或其片段位于所述胞内信号区域的C端。
在某些实施方式中，所述的嵌合抗原受体中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
在某些实施方式中，所述的嵌合抗原受体中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白6或其片段。
在某些实施方式中，所述的嵌合抗原受体中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:84所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体还包含信号肽。
在某些实施方式中，所述的嵌合抗原受体中所述信号肽源自CD8蛋白的信号肽。
在某些实施方式中，所述的嵌合抗原受体中所述信号肽包含SEQ ID NO:72所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包含SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:202、SEQ ID NO: 203和SEQ ID NO:204中任一项所示的氨基酸序列。
在某些实施方式中，所述的嵌合抗原受体包含SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89和SEQ ID NO:91中任一项所示的核苷酸序列。
另一方面，本申请还提供了一种多肽，其包含所述的抗原结合蛋白。
另一方面，本申请还提供了一种或多种分离的核酸分子，其编码所述的抗原结合蛋白和/或所述的嵌合抗原受体。
在某些实施方式中，所述的核酸分子还包含启动子。
在某些实施方式中，所述的核酸分子中所述启动子为组成型启动子。
在某些实施方式中，所述的核酸分子中所述启动子为EF1α启动子。
在某些实施方式中，所述的核酸分子包含SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89和SEQ ID NO:91中任一项所示的核苷酸序列。
另一方面，本申请还提供了一种载体，其包含所述的核酸分子。
在某些实施方式中，所述的载体为病毒载体。
在某些实施方式中，所述的载体为慢病毒载体。
另一方面，本申请还提供一种细胞，其包含所述的抗原结合蛋白，所述的嵌合抗原受体，所述的核酸分子，和/或所述的载体。
在某些实施方式中，所述的细胞为免疫效应细胞。
在某些实施方式中，所述的细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
在某些实施方式中，所述的细胞为T细胞。
另一方面，本申请还提供了一种制备所述的抗原结合蛋白和/或所述的嵌合抗原受体的方法，所述方法包括在使得所述的抗原结合蛋白和/或所述的嵌合抗原受体表达的条件下，培养根所述的细胞。
另一方面，本申请还提供了一种制备经修饰的免疫效应细胞的方法，其包括向免疫效应细胞中引入所述的载体。
另一方面，本申请还提供了一种药物组合物，其包含所述的抗原结合蛋白，所述的嵌合抗原受体，所述的多肽，中任一项所述的核酸分子，中任一项所述的载体，和/或权所述的细胞，以及可选地药学上可接受的载体。
另一方面，本申请还提供了所述的抗原结合蛋白，所述的抗原嵌合受体，所述的核酸分 子，所述的载体，所述的细胞，和/或所述的药物组合物，在制备药物中的用途，所述药物用于预防、治疗和/或缓解与GPRC5D异常表达相关的疾病或病症。
在某些实施方式中，所述的用途中所述与GPRC5D异常表达相关的疾病或病症包括肿瘤。
在某些实施方式中，所述的用途中所述肿瘤包括实体瘤。
在某些实施方式中，所述的用途中所述肿瘤包括非实体瘤。
在某些实施方式中，所述的用途中所述肿瘤包括血液瘤和/或淋巴瘤。
在某些实施方式中，所述的用途中所述肿瘤包括骨髓瘤。
另一方面，本申请还提供了一种预防、治疗和/或缓解与GPRC5D异常表达相关的疾病或病症的方法，所述方法包括向有需要的受试者施用所述的抗原结合蛋白，所述的抗原嵌合受体，所述的核酸分子，所述的载体，所述的细胞，和/或所述的药物组合物。
在某些实施方式中，所述的方法中所述与GPRC5D异常表达相关的疾病或病症包括肿瘤。
在某些实施方式中，所述的方法中所述肿瘤包括实体瘤。
在某些实施方式中，所述的方法中所述肿瘤包括非实体瘤。
在某些实施方式中，所述的方法中所述肿瘤包括血液瘤和/或淋巴瘤。
在某些实施方式中，所述的方法中所述肿瘤包括骨髓瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1A至图1J显示的是通过流式分析本申请所述抗CPRC5D抗体与细胞表面GPRC5D的结合活性。
图2显示的是本申请所述嵌合抗原受体慢病毒表达载体和GPRC5D核心质粒中CAR各部分元件及连接顺序。
图3A至图3C显示的是经激活培养的GPRC5D CAR-T细胞的扩增倍数和感染后T细胞的CAR阳性率。
图4A至图4D显示的是GPRC5D CAR-T细胞对于靶细胞(CHO细胞/MM1S细胞)的杀伤能力。
图5A至图5B显示的是GPRC5D CAR-T细胞体外杀伤后的细胞因子(IL2\IFN-γ)分泌情况。
图6显示的是GPRC5D CAR-T细胞的靶向增殖倍数。
图7A至图7D显示的是GPRC5D CAR-T细胞在小鼠体内的细胞因子分泌情况、消瘤能力和安全性。
图8A至图8D和图9A至图9D显示的是抗GPRC5D抗体不结合不表达GPRC5D抗原的细胞，A：A549细胞，B：KERA细胞，C：MCF-7细胞，D：MSC细胞。
图10A至图10D显示的是抗GPRC5D的内吞效应实验结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“分离的抗原结合蛋白”通常是指脱离了其天然存在状态的具有抗原结合能力的蛋白。所述“分离的抗原结合蛋白”可以包含结合抗原的部分和任选的，允许抗原结合部分采用促进其结合抗原的构象的框架或构架部分。抗原结合蛋白可以包含例如抗体来源的蛋白框架区(FR)或具有移植的可变区(CDR)或CDR衍生物的备选蛋白框架区或人工框架区。例如，所述抗原结合蛋白可以包括抗体或其抗原结合片段。例如，所述抗原结合蛋白可以结合GPRC5D蛋白。例如，所述抗原结合蛋白可以与参比抗体竞争结合GPRC5D蛋白。例如，所述抗原结合蛋白可以包括抗体重链可变区VH。例如，所述抗原结合蛋白可以包括源自抗体重链可变区VH中的至少一个CDR。例如，所述VH可以包含HCDR3、HCDR2和/或HCDR1。例如，所述VH可以包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连。例如，所述VH可以包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间。例如，所述VH可以包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间。例如，所述VH可以包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连。例如，所述抗原结合蛋白可以为VHH。例如，所述的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自IgG。例如，所述抗体重链恒定区可以源自人IgG。例如，所述抗体重链恒定区可以源自人IgG1。
使用的术语“抗体”包括完整抗体和其结合片段。通常，片段与其来源的完整抗体竞争性地与抗原特异性结合。任选，抗体或其结合片段可以与其它蛋白质化学结合，或者与其它蛋白质以融合蛋白质的形式表达。例如，所述抗体可以是单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。例如，所述抗体或其结合片段的结合蛋白质可以包括GPRC5D。例如，所述抗体或其结合片段对GPRC5D可以有特异性。
术语“抗原结合片段”是指完整抗体的一部分并且是指完整抗体的抗原决定可变区。例如，所述抗原结合片段的可以包括Fab、Fab'、F(ab')2、Fv片段和单链Fv片段、串联Fv片段、VHH、双特异性抗体。例如，所述抗原结合片段可以是VHH。例如，所述抗原结合片段可以结合GPRC5D。例如，所述抗原结合片段对GPRC5D可以有特异性。
在本申请中，术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体。VHH也可称为纳米抗体(Nanobody)(Nb)和/或单域抗体。例如，所述VHH可以结合GPRC5D。例如，所述VHH对GPRC5D可以有特异性。
在本申请中，所述抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链。每条重链由重链可变区(VH)和重链恒定区组成。术语“重链恒定区”由三个结构域CH1，CH2和CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区组成。术语“轻链恒定区”由一个结构域CL组成。VH和VL区可以进一步细分为高变区，称为互补决定区(CDR)，散布有更保守的区域，称为构架区(FR)。每个VH和VL由以下列顺序从氨基末端到羧基末端排列的三个CDR和四个FR组成:FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合。
在本申请中，术语“参比抗体”是指可与所述分离的抗原结合蛋白GPRC5D竞争性结合于相同表位的抗体。所述参比抗体可以包括重链可变区VH。例如，所述参比抗体可以具有3个CDR序列。例如，所述参比抗体的VH可以包括HCDR1、HCDR2和HCDR3。例如，所述的CDR序列可以与所述分离的抗原结合蛋白的CDR序列一致。
在本申请中，术语“G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白”是一种非典型的表面孤儿受体。GPRCD5与其他C5家族受体一样拥有过短的氨基末端，因此在构象上与C4家族十分类似。其在正常组织中表达仅限于毛囊，但在多发性骨髓瘤患者的骨髓中也有显著表达，并与浆细胞肿瘤负荷与遗传畸变高度相关。例如，在本申请中的GPRC5D可以特指在MM患者体内表达的GPRC5D。
在本申请中，术语“IgG”是指属于基本上由公认的免疫球蛋白γ基因编码的抗体类别的多肽。在人类中，此类别包括IgG1、IgG2、IgG3和IgG4。在小鼠中，此类别包括IgG1、IgG2a、 IgG2b和IgG3。
在本申请中，术语“嵌合抗原受体”(CAR)通常是指至少包含特异性地结合抗原或靶标的胞外结构域、跨膜区以及胞内结构域的重组多肽。例如，所述胞外结构域与所述跨膜区之间包括铰链区。例如，所述嵌合抗原受体还可以包括低密度脂蛋白受体相关蛋白或其片段。例如，所述嵌合抗原受体可以包括信号肽。CAR的胞外结构域与靶细胞表面上的靶抗原结合导致CAR聚簇并将激活刺激传送给含CAR细胞。CAR重定向免疫效应细胞的特异性，并且触发了增殖、细胞因子产生、能够以不依赖主要组织相容性(MHC)的方式介导表达靶抗原的细胞死亡的分子的吞噬作用和/或产生。例如，所述胞外结构可以包括上述的抗原结合蛋白。例如，所述胞外结构可以特异性结合GPRC5D。
在本申请中，术语“胞内结构域”指意思是包括足以转导活化信号的任何截短部分的细胞内结构域。所述胞内结构域可以包括细胞内信号区域和/或共刺激信号区域。术语“细胞内信号区域”指可以产生促进含CAR细胞(例如CART细胞或表达CAR的NK细胞)的免疫效应子功能的信号的胞内区域。例如，所述胞内信号区域可以包括选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和至少包含一个ITAM的结构域。例如，所述胞内信号区域可以是源自CD3ζ的信号传导结构域。术语“共刺激信号区域”是指所述胞内信号区域中能够转导效应信号的CAR的一部分。例如，所述共刺激信号区域可以包括源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。例如，所述共刺激信号区域可以是源自4-1BB的胞内共刺激信号区域。
在本申请中，术语“跨膜区”指能够跨越细胞质膜的肽、多肽或蛋白质的结构域。可采用这些结构域将胞外结构域锚定在细胞膜上。例如，所述跨膜区可以包含选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。例如，所述跨膜区可以源自CD8的跨膜区。
在本申请中，术语“铰链区”表示抗体重链多肽中连接CH1结构域和CH2结构域的一部分，例如，从根据Kabat的EU编号系统的约第216位至约第230位。铰链区通常是由两条具有相同氨基酸序列的多肽组成的二聚体分子。铰链区一般包括约25个氨基酸残基，是柔性的，允许抗原结合区独立的移动。铰链区可以细分为3个结构域：上、中、下铰链结构域。例如，所述铰链区可以包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。例如，所述铰链区可以源自CD8的铰链区。
在本申请中，术语“低密度脂蛋白受体相关蛋白(Low-density lipoproteinreceptor-related protein)指一种属于内吞性受体的细胞表面蛋白，在生物体内广泛分布并有很大的组织间差异性，主要功能是摄取胆固醇进入细胞内用以细胞增殖和固醇类激素及胆汁酸盐的合成。例如，所述低密度脂蛋白受体相关蛋白可以来自于任何脊椎动物。例如，所述低密度脂蛋白受体相关蛋白或其片段可以位于所述胞内信号区域的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以为低密度脂蛋白受体相关蛋白6或其片段。
在本申请中，术语“信号肽”是指处于新生CAR蛋白的氨基末端(N-末端)的前导序列，其在翻译时或在翻译后将新生蛋白引导到内质网并后续表面表达。例如，所述信号肽源自CD8蛋白的信号肽。例如，其中所述信号肽包括SEQ ID NO:72所示的氨基酸序列。
在本申请中，术语“多肽”、“肽”和“蛋白”在本文中可以互换，是指氨基酸残基的聚合物。这个术语可用于指一个或多个氨基酸残基是其相应的天然氨基酸的人工合成化学模拟物的氨基酸聚合物，也可用于指天然的氨基酸聚合物，那些含修饰残基的氨基酸聚合物以及非天然的氨基酸聚合物。例如，所述多肽可以包括所述的抗原结合蛋白。
在本申请中，术语“核酸分子”包括DNA分子和RNA分子。一个核酸分子可以是单链或双链的，但优选为双链DNA。术语“启动子”通常是指一个DNA序列，可以调节与启动子操作性连接的所选DNA序列的表达，从而影响细胞中所选DNA序列的表达。例如，所述核酸分子可以编码包括所属抗原结合蛋白和/或所述嵌合抗原受体。例如，所述核酸分子可以包括启动子。例如，所述启动子可以是组成型启动子。例如，所述启动子可以是EF1α启动子。
在本申请中，术语“载体”通常是指可以附着本申请的一种或多种核酸分子的分子。例如，所述载体可以是病毒载体。例如，所述载体可以是慢病毒载体。
在本申请中，术语“细胞”指可以向其转染核酸的细胞，术语“细胞”包括用于质粒繁殖的原核细胞，和用于核酸表达和编码多肽产生的真核细胞。例如，细胞可以包括所述的抗原结合蛋白，所述的核酸分子和/或所述的载体。例如，所述细胞可以是免疫效应细胞。术语“免疫效应细胞”通常是指参与免疫应答，行使效应功能的免疫细胞。例如，所述行使效应功能可以包括清除异物抗原或促进免疫效应子应答等。例如，免疫效应细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如，免疫效应细胞可以是T细胞。
在本申请中，术语“药物组合物”通常指适合施用于哺乳动物个体的化学或生物组合物。例如，所述药物组合物可以包括所述抗原结合蛋白，所述嵌合抗原受体，所述多肽，所述核酸分子，所述载体和/或所述细胞，以及可选的药学上可接受的载体。所述药物组合物可以用于预防、治疗和/或缓解与GPRC5D异常表达相关的疾病或病症。例如，所述与GPRC5D异常表达相关的疾病或病症可以包括肿瘤。例如，所述肿瘤包括实体瘤和/或非实体瘤。例如，所述肿瘤包可以括血液瘤和/或淋巴瘤。例如，所述肿瘤可以包括骨髓瘤。
发明详述
分离的抗原结合的蛋白
一方面，本申请提供一种分离的抗原结合蛋白，其能够以低于1.0μg/mL的EC50值结合G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。例如，本申请所述抗原结合蛋白可以以低于约0.9μg/mL、低于约0.8μg/mL、低于约0.7μg/mL、低于约0.6μg/mL、低于约0.5μg/mL、低于约0.4μg/mL、低于约0.3μg/mL、低于约0.2μg/mL、低于约0.1μg/mL或更低的EC50值结合GPRC5D蛋白。例如，本申请所述抗原结合蛋白可以以低于约0.9μg/mL的EC50值结合细胞表面表达的GPRC5D蛋白。例如，本申请的抗原结合蛋白结合细胞表面表达的GPRC5D蛋白可以通过流式细胞荧光分选技术(FACS)检测，例如，使用iQue Screener流式仪检测。
在本申请中，所述抗原结合蛋白可以包括抗体或其抗原结合片段。在本申请中，所述抗原结合片段可以包括Fab、Fab’、F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。在本申请中，所述抗体可以包括单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
CDR
CDR是抗体的互补决定区，是抗体可变区的一部分，该区域的氨基酸残基与抗原或抗原表位接触，从而特异性识别并结合抗原。
在本申请中，所述分离的抗原结合蛋白可以包括抗体重链可变区VH中的至少一个CDR， 例如，所述VH可以包括如SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包含HCDR3。例如，所述抗原结合蛋白的HCDR3可以包含SEQ ID NO:215所示的氨基酸序列：
AAX 3X 4X 5X 6X 7X 8X 9X 10X 11X 12X 13X 14YX 16Y(SEQ ID NO:215)，其中，X 3＝F、G、R、S、T或V，X 4＝A、G、L、P、R、S或T，X 5＝A、G、K、R、V或Y，X 6＝P、R、S、V或Y，X 7＝A、F、G、I、N、P、S、W或Y，X 8＝A、F、L、P、R、T或Y，X 9＝A、G、L、R、S、T或Y，X 10＝L、P、Q、S、T、W或Y，X 11＝D、G、N、R、S、T或Y，X 12＝A、D、E、G、P、R、S或Y，X 13＝A、G、L、N、R、S或Y，X 14＝E、G、L、N、R、T或V，且X 16＝D或N。例如，所述序列可以根据IMGT规则划分。
在本申请中，所述抗原结合蛋白可以包含HCDR3。例如，所述抗原结合蛋白的HCDR3可以包含SEQ ID NO:218所示的氨基酸序列
AAX 3X 4X 5X 6X 7X 8X 9X 10X 11YX 13Y(SEQ ID NO:218)，其中，X 3＝D、F、G、K、N、R、S、T或V，X 4＝D、G、I、L、R、S或T，X 5＝A、F、G、R、V或Y，X 6＝G、P、R、S、T、W或Y，X 7＝A、G、L、N、R、S、W或Y，X 8＝D、F、G、L、N、R、S、T或Y，X 9＝A、D、G、I、L、P、R、S、T或Y，X 10＝G、L、N、R、S、T、W或Y，X 11＝D、G、L、N、P、R或T，X 13＝D或N。
在本申请中，所述抗原结合蛋白可以包括HCDR3，且所述HCDR3可包含如SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:115、SEQ ID NO:118、SEQ ID NO:121、SEQ ID NO:125、SEQ ID NO:129、SEQ ID NO:132、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:140、SEQ ID NO:144、SEQ ID NO:147、SEQ ID NO:150、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:176、SEQ ID NO:179、SEQ ID NO:182、SEQ ID NO:185、SEQ ID NO:188、SEQ ID NO:191、SEQ ID NO:195和SEQ ID NO:198中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包含HCDR2。例如，所述抗原结合蛋白的HCDR2可以包括如下所示的氨基酸序列：
X 1X 2X 3X 4X 5X 6X 7T，其中，X 1＝I或T，X 2＝N、S或T，X 3＝A、P、R、S或W，X 4＝G、R、S、T或不存在，X 5＝D或G，X 6＝G或S，且X 7＝D、G、I、N、R、S、T或V。例如，所述序列可以根据IMGT规则划分。
在本申请中，所述抗原结合蛋白可以包括HCDR2，且所述HCDR2可以包括如SEQ ID NO:96、SEQ ID NO:103、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:114、SEQ ID NO:117、SEQ ID NO:120、SEQ ID NO:124、SEQ ID NO:128、SEQ ID NO:131、SEQ ID NO:134、SEQ ID NO:139、SEQ ID NO:143、SEQ ID NO:146、SEQ ID NO:149、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:167、SEQ ID NO:171、SEQ ID NO:175、SEQ ID NO:178、SEQ ID NO:181、SEQ ID NO:187、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194和SEQ ID NO:197中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括HCDR1。例如，所述抗原结合蛋白的HCDR1可包含如下所示的氨基酸序列：
GX 2X 3X 4SX 6X 7X 8，其中，X 2＝F、G、I、L、N、R、S或Y，X 3＝I或T，X 4＝F或L，X 6＝I、L、N、R、S、T或Y，X 7＝D、N或Y，且X 8＝A、D、G、I、N、R、S、T、V或Y。例如，所述序列可以根据IMGT规则划分。
在本申请中，所述抗原结合蛋白可以包括HCDR1，且所述HCDR1可以包括如SEQ ID NO:95、SEQ ID NO:102、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:116、SEQ ID NO:119、SEQ ID NO:123、SEQ ID NO:127、SEQ ID NO:130、SEQ ID NO:133、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:142、SEQ ID NO:145、SEQ ID NO:148、SEQ ID NO:152、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:166、SEQ ID NO:169、SEQ ID NO:174、SEQ ID NO:177、SEQ ID NO:180、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:189、SEQ ID NO:193和SEQ ID NO:196中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括HCDR1、HCDR2、HCDR3，所述HCDR1可包含SEQ ID NO:95、SEQ ID NO:102、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:116、SEQ ID NO:119、SEQ ID NO:123、SEQ ID NO:127、SEQ ID NO:130、SEQ ID NO:133、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:142、SEQ ID NO:145、SEQ ID NO:148、SEQ ID NO:152、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:166、SEQ ID NO:169、 SEQ ID NO:174、SEQ ID NO:177、SEQ ID NO:180、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:189、SEQ ID NO:193和SEQ ID NO:196中任一项所示的氨基酸序列，所述HCDR2可以包含SEQ ID NO:96、SEQ ID NO:103、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:114、SEQ ID NO:117、SEQ ID NO:120、SEQ ID NO:124、SEQ ID NO:128、SEQ ID NO:131、SEQ ID NO:134、SEQ ID NO:139、SEQ ID NO:143、SEQ ID NO:146、SEQ ID NO:149、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:167、SEQ ID NO:171、SEQ ID NO:175、SEQ ID NO:178、SEQ ID NO:181、SEQ ID NO:187、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194和SEQ ID NO:197中任一项所示的氨基酸序列，且所述HCDR3可以包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:115、SEQ ID NO:118、SEQ ID NO:121、SEQ ID NO:125、SEQ ID NO:129、SEQ ID NO:132、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:140、SEQ ID NO:144、SEQ ID NO:147、SEQ ID NO:150、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:176、SEQ ID NO:179、SEQ ID NO:182、SEQ ID NO:185、SEQ ID NO:188、SEQ ID NO:191、SEQ ID NO:195和SEQ ID NO:198中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括HCDR1、HCDR2、HCDR3，且所述HCDR1、HCDR2、HCDR3可以包括下组的任意一组氨基酸序列：
(1)HCDR1：SEQ ID NO:102，HCDR2：SEQ ID NO:103，和HCDR3：SEQ ID NO:104；
(2)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:107；
(3)HCDR1：SEQ ID NO:109，HCDR2：SEQ ID NO:110，和HCDR3：SEQ ID NO:111；
(4)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:114，和HCDR3：SEQ ID NO:115；
(5)HCDR1：SEQ ID NO:116，HCDR2：SEQ ID NO:117，和HCDR3：SEQ ID NO:118；
(6)HCDR1：SEQ ID NO:119，HCDR2：SEQ ID NO:120，和HCDR3：SEQ ID NO:121；
(7)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:124，和HCDR3：SEQ ID NO:125；
(8)HCDR1：SEQ ID NO:127，HCDR2：SEQ ID NO:128，和HCDR3：SEQ ID NO:129；
(9)HCDR1：SEQ ID NO:130，HCDR2：SEQ ID NO:131，和HCDR3：SEQ ID NO:132；
(10)HCDR1：SEQ ID NO:95，HCDR2：SEQ ID NO:96，和HCDR3：SEQ ID NO:97；
(11)HCDR1：SEQ ID NO:133，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:135；
(12)HCDR1：SEQ ID NO:136，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:137；
(13)HCDR1：SEQ ID NO:138，HCDR2：SEQ ID NO:139，和HCDR3：SEQ ID NO:140；
(14)HCDR1：SEQ ID NO:142，HCDR2：SEQ ID NO:143，和HCDR3：SEQ ID NO:144；
(15)HCDR1：SEQ ID NO:145，HCDR2：SEQ ID NO:146，和HCDR3：SEQ ID NO:147；
(16)HCDR1：SEQ ID NO:148，HCDR2：SEQ ID NO:149，和HCDR3：SEQ ID NO:150；
(17)HCDR1：SEQ ID NO:152，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:154；
(18)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:155，和HCDR3：SEQ ID NO:156；
(19)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:158；
(20)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:160，和HCDR3：SEQ ID NO:161；
(21)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:162，和HCDR3：SEQ ID NO:163；
(22)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:164，和HCDR3：SEQ ID NO:165；
(23)HCDR1：SEQ ID NO:166，HCDR2：SEQ ID NO:167，和HCDR3：SEQ ID NO:168；
(24)HCDR1：SEQ ID NO:169，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:170；
(25)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:171，和HCDR3：SEQ ID NO:172；
(26)HCDR1：SEQ ID NO:174，HCDR2：SEQ ID NO:175，和HCDR3：SEQ ID NO:176；
(27)HCDR1：SEQ ID NO:177，HCDR2：SEQ ID NO:178，和HCDR3：SEQ ID NO:179；
(28)HCDR1：SEQ ID NO:180，HCDR2：SEQ ID NO:181，和HCDR3：SEQ ID NO:182；
(29)HCDR1：SEQ ID NO:184，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:185；
(30)HCDR1：SEQ ID NO:186，HCDR2：SEQ ID NO:187，和HCDR3：SEQ ID NO:188；
(31)HCDR1：SEQ ID NO:189，HCDR2：SEQ ID NO:190，和HCDR3：SEQ ID NO:191；
(32)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:192，和HCDR3：SEQ ID NO:179；
(33)HCDR1：SEQ ID NO:193，HCDR2：SEQ ID NO:194，和HCDR3：SEQ ID NO:195；以及
(34)HCDR1：SEQ ID NO:196，HCDR2：SEQ ID NO:197，和HCDR3：SEQ ID NO:198。
FR
本申请中，抗体框架区FR指抗体可变区中存在于分歧性更高的(即高变)CDR之间的部分。此类框架区典型地称为框架1至4(FR1、FR2、FR3和FR4)且提供用于在三维空间中呈现六个CDR(三个来自重链且三个来自轻链)的骨架，以形成抗原结合表面。
在本申请中，所述抗原结合蛋白可以包括H-FR1，所述H-FR1可包含SEQ ID NO:98所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括H-FR2，所述H-FR2可包含如SEQ ID NO:216所示的氨基酸序列：MGWFRQAPGKGRELVAX 17(SEQ ID NO:216)，其中，所述X 17＝A、 F、G、R、S或V。例如，所述序列可以根据IMGT规则划分。
在本申请中，所述抗原结合蛋白可以包括H-FR2，且所述H-FR2可包含如SEQ ID NO:99、SEQ ID NO:108、SEQ ID NO:112、SEQ ID NO:122、SEQ ID NO:151和SEQ ID NO:173中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以H-FR3，且所述H-FR3可包括如SEQ ID NO:217所示的氨基酸序列：X 1YPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYC(SEQ ID NO:217)，其中，所述X 1＝H、F、S、N或Y。例如，所述序列可以根据IMGT规则划分。
在本申请中，所述抗原结合蛋白可以包括H-FR3，且所述H-FR3可包含如SEQ ID NO:100、SEQ ID NO:113、SEQ ID NO:126、SEQ ID NO:141和SEQ ID NO:183中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括H-FR4，且所述H-FR4可包含如SEQ ID NO:101所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4可依次包含SEQ ID NO:98、SEQ ID NO:216、SEQ ID NO:217和SEQ ID NO:101所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1可以包含SEQ ID NO:98所示的氨基酸序列，所述H-FR2可以包含SEQ ID NO:99、SEQ ID NO:108、SEQ ID NO:112、SEQ ID NO:122、SEQ ID NO:151和SEQ ID NO:173中任一项所示的氨基酸序列，所述H-FR3可以包含如SEQ ID NO:100、SEQ ID NO:113、SEQ ID NO:126、SEQ ID NO:141和SEQ ID NO:183中任一项所示的氨基酸序列，且所述H-FR4可包含如SEQ ID NO:101所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可以包括抗体重链可变区VH，且所述VH可以包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在本申请中，所述抗原结合片段可以是VHH，且所述VHH可以包含SEQ ID NO:4、SEQ  ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
重链恒定区
在本申请中，所述的分离的抗原结合蛋白可以包括重链恒定区。所述重链恒定区是指包含至少三个重链恒定结构域CH1、CH2、和CH3的区域。非限制性示例性重链恒定区包括γ、δ、和α。非限制性示例性重链恒定区还包括ε和μ。每个重链恒定区对应于一种抗体同种型。例如，包含γ恒定区的抗体为IgG抗体，包含δ恒定区的抗体为IgD抗体，包含α恒定区的抗体为IgA抗体。此外，包含μ恒定区的抗体为IgM抗体，包含ε恒定区的抗体为IgE抗体。某些同种型可以进一步细分为亚类。例如，IgG抗体包括但不限于，IgG1(包含γ 1恒定区)、IgG2(包含γ 2恒定区)、IgG3(包含γ 3恒定区)、和IgG4(包含γ 4恒定区)抗体；IgA抗体包括但不限于，IgA1(包含α 1恒定区)和IgA2(包含α 2恒定区)抗体；IgM包括但不限于，IgM1和IgM2。
在本申请中，所述的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自IgG。在本申请中，所述的抗原结合蛋白可以包括抗体重链恒定区，所述抗体重链恒定区可以源自人IgG。在本申请中，所述的抗原结合蛋白可以包含抗体重链恒定区，所述抗体重链恒定区可以源自人IgG1。
嵌合抗原受体
另一方面，本申请还提供了一种嵌合抗原受体(CAR)，所述嵌合抗原受体(CAR)可以包含结合GPRC5D蛋白的靶向部分，例如，结合GPRC5D蛋白的靶向部分可以是本申请所述的抗原结合蛋白。
例如，本申请的CAR可包含VHH，所述VHH可包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID  NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在本申请中，所述CAR包含胞外的结合GPRC5D蛋白的靶向部分外，还可包含胞内结构域。
在本申请中，所述CAR可以包括胞内的共刺激信号区域，其可提供刺激信号。例如，所述共刺激信号区域可以包括选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
例如，所述共刺激信号区域可以为源自4-1BB的胞内共刺激信号区域。例如，所述共刺激信号区域可以包含SEQ ID NO:78所示的氨基酸序列。
在某些情形中，所述CAR可包含胞内信号区域，其可以包含至少有一个ITAM基序的结构域。所述胞内信号传导结构域可以将激活信号传输到细胞内部。例如，所述胞内信号区域可以包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、DAP10、DAP-12和其他至少包含一个ITAM的结构域。
例如，所述胞内信号区域可以为源自CD3ζ的信号传导结构域。例如，所述胞内信号区域可以包含SEQ ID NO:80所示的氨基酸序列。
在某些情形中，所述CAR可包含跨膜域，所述跨膜域是细胞表面蛋白中一段跨越细胞膜的序列，其可以包含疏水性alpha螺旋。所述跨膜域可以源自任意的I型跨膜蛋白。跨膜域可以是预测为形成疏水螺旋的合成序列。例如，所述跨膜区可以包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
例如，所述跨膜区可以为源自CD8的跨膜区。例如，所述跨膜区可以包含SEQ ID NO:76所示的氨基酸序列。
在某些情形中，所述CAR可包含铰链区，所述铰链区可以位于所述胞外的靶向部分和所述跨膜域之间。例如，所述铰链区可以包含选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
例如，所述铰链区可以为源自CD8的铰链区。例如，所述铰链区可以包含SEQ ID NO:74所示的氨基酸序列。
在本申请中，所述CAR在所述结合GPRC5D蛋白的靶向部分的N端还可包含信号肽。例如，所述信号肽可以为源自CD8蛋白的信号肽。例如，所述信号肽可以包含SEQ ID NO:72所示的氨基酸序列。
在本申请中，所述CAR还可包含低密度脂蛋白受体相关蛋白或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以位于所述CAR的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包括低密度脂蛋白受体相关蛋白1-12和其功能性片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以是低密度脂蛋白受体相关蛋白6或其片段。例如，所述低密度脂蛋白受体相关蛋白或其片段可以包括SEQ ID NO:84所示的氨基酸序列。
在本申请中，所述CAR中的所述低密度脂蛋白受体相关蛋白或其片段可通过自剪切肽(例如，T2A、P2A、E2A等2A肽)连接至CAR的C端。例如，所述低密度脂蛋白受体相关蛋白或其片段可以通过T2A与胞内信号区域的C端连接。
在本申请中，自N端至C端，所述CAR可依次包含结合GPRC5D蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激信号区域和所述胞内信号区域。例如，自N端至C端，所述CAR可依次包含所述VHH，源自CD8的铰链区，源自CD8的跨膜区，源自4-1BB的共刺激信号区域，和源自CD3ζ的胞内信号区域，且所述VHH可包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在本申请中，自N端至C端，所述CAR可依次包含结合CPRC50D蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激 信号区域、所述胞内信号区域和所述低密度脂蛋白受体相关蛋白或其片段。例如，自N端至C端，所述CAR可依次包含所述VHH，源自CD8的铰链区，源自CD8的跨膜区，源自4-1BB的共刺激信号区域，源自CD3ζ的胞内信号区域，和包含SEQ ID NO:84所示的氨基酸序列的低密度脂蛋白受体相关蛋白或其片段，且所述VHH可包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
在本申请中，自N端至C端，所述CAR可依次包含信号肽、结合GPRC5D蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激信号区域和所述胞内信号区域。
在本申请中，自N端至C端，所述CAR可依次包含信号肽、结合GPRC5D蛋白的靶向部分(例如，所述抗原结合蛋白，又例如，本申请所述VHH)、所述铰链区、所述跨膜域、所述共刺激信号区域、所述胞内信号区域和所述低密度脂蛋白受体相关蛋白或其片段。
例如，所述的嵌合抗原受体包可以含SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:202、SEQ ID NO:203和SEQ ID NO:204中任一项所示的氨基酸序列。例如，所述的嵌合抗原受体包可以含SEQ ID NO:205、SEQ ID NO:206、SEQ ID NO:207、SEQ ID NO:208、SEQ ID NO:209、SEQ ID NO:210、SEQ ID NO:211、SEQ ID NO:212、SEQ ID NO:213和SEQ ID NO:214中任一项所示的氨基酸序列。
例如，所述的嵌合抗原受体可以由包含SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89和SEQ ID NO:91中任一项所示的核苷酸序列所编码。
核酸分子
另一方面，本申请还提供了一种或多种核酸分子，所述核酸分子可以是任意长度的分离形式的核苷酸、脱氧核苷酸和/核糖核苷酸，可以编码所述分离的抗原结合蛋白和/或所述的嵌合抗原受体。
例如，所述核酸分子可以包括启动子。例如，所述启动子可以是组成型启动子。例如， 所述启动子可以是EF1α启动子。
例如，所述核酸分子可包含SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67和SEQ ID NO:69中任一项所示的核苷酸序列。例如，所述核酸分子可以包括SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89和SEQ ID NO:91中任一项所示的核苷酸序列。
载体
另一方面，本申请还提供了一种载体，所述载体可以包括所述的核酸分子。所述载体可以转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达。例如，载体可以包括启动子、转录子、增强子、复制子、选择元件和报告基因。例如，载体可以包括协助进入细胞的成分。为了使所述核酸分子在载体中复制，所述核酸分子的5’端和3’端还可以包含长末端重复序列。
例如，所述载体可以是病毒载体。例如，所述载体可以是慢病毒载体。
细胞
另一方面，本申请还提供了细胞，所述细胞可以包括所述的分离的抗原结合蛋白、所述的嵌合抗原受体、所述的核酸分子和/或所述的载体。所述细胞可以包括单个细胞的后代。由于天然、偶然或有意的突变，后代可以不一定与原始母细胞完全相同(在总DNA互补体的形态上或在基因组上)。
在某些实施方案中，所述的细胞可以是免疫效应细胞。在某些实施方案中，所述的细胞可以包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。例如,
在某些实施方案中，所述的细胞可以是T细胞。
药物组合物
另一方面，本申请还提供了一种药物组合物，可以包括所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体和/或所述细胞，以及任选地药学上可接受的佐剂。
在某些实施方案中，所述药物组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、 赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下优选地对接受者无毒。本发明的药物组合物可以包括液体、冷冻和冻干组合物。
在某些实施方案中，所述药学上可接受的佐剂可以包括与药物给药相容的任何和所有的溶剂、分散介质、包衣、等渗剂和吸收延迟剂，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。
在某些实施方案中，所述药物组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述药物组合物可以通过输注或注射施用于患者或者受试者。在某些实施方案中，所述药物组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
制备方法
另一方面，本申请还提供了制备所述分离的抗原结合蛋白和/或所述嵌合抗原受体的方法。所述方法可以包括在使得所述抗原受体和/或所述嵌合抗原受体表达的条件下，培养所述细胞。
本申请还提供了制备经修饰的免疫效应细胞的方法，所述方法可以包括向免疫细胞中引入所述载体。
用途
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述的嵌合抗原受体、所述的核酸分子、所述的载体、所述的细胞和/或所述的药物组合物在制备药物中的用途，所述药物可以用于预防、缓解和/或治疗肿瘤。
另一方面，本申请还提供了预防、缓解和/或治疗肿瘤的方法，所述方法可以包括向受试者施用所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物。
另一方面，本申请还提供了所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物，其用于预防、缓解和/或治疗肿瘤。
在本申请中，所述肿瘤可以包括实体瘤和/或非实体瘤。
在本申请中，所述肿瘤可以包括血液瘤和/或淋巴瘤。
在本申请中，所述肿瘤可以包括骨髓瘤。在本申请中，所述肿瘤可以包括多发性骨髓瘤。
在本申请中，所述受试者可以包括人类或非人类动物。
另一方面，本申请提供了多肽，其包含所述分离的抗原结合蛋白。另一方面，本申请提 供了抗体药物偶联物，其包含所述分离的抗原结合蛋白。
另一方面，本申请提供了试剂盒或给药装置，其包括所述分离的抗原结合蛋白、所述嵌合抗原受体、所述核酸分子、所述载体、所述的细胞和/或所述的药物组合物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的抗原结合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1.利用人工合成纳米抗体库NanoOri_1.0筛选抗GPRC5D抗体
首先比对PDB数据库(Protein Data Bank)中296条无重复序列的纳米抗体及其结构，确定CDRs突变策略，其中CDR3的长度为三种：14，17和21个氨基酸。选取已经上市的纳米抗体卡拉西单抗(Caplacizumab)为骨架，全基因合成Caplacizumab的核苷酸序列，然后将其克隆至HP153噬菌体载体，之后提取HP153的单链DNA，利用Kunkel Mutagenesis的方法突变纳米抗体的CDRs，获得双链DNA。将双链DNA电转至M13KO7辅助噬菌体预感染的感受态大肠杆菌SS320，过夜培养后收集噬菌体上清，最终构建出多样性为1.44×10 10的人工合成纳米抗体库NanoOri_1.0(上海原能细胞医学技术有限公司)，作为种子库进行抗体序列筛选。
将从Sino Biological公司购买的GPRC5D全长序列(cat:HG24447-UT)构建到公司已有的慢病毒载体上，进行慢病毒转导，制备靶细胞CHO-GPRC5D和靶细胞293-GPRC5D的过表达细胞株。利用靶细胞CHO-GPRC5D和293-GPRC5D进行人工合成纳米抗体库(上海原能细胞医学技术有限公司)的交替淘选，共进行四轮淘选：
从人工合成纳米抗体库NanoOri_1.0里取一支加入PEG/NaCl，重新沉淀使用。选取靶细胞CHO-GPRC5D和293-GPRC5D用作细胞淘选的交替筛选。取500μL的噬菌体和500μL的10％FBS/PBS缓冲液混合到终体积1mL，装于2mL的低吸附的EP管中，4℃下轻轻旋转或者周期性混匀进行封闭1小时。140g离心10min收获CHO-GPRC5D细胞，用10mL的PBS洗涤一次。进行计数，活细胞数达到95％以上，用5％FBS/PBS缓冲液洗涤CHO-GPRC5D细胞三次。然后重悬于1mL的5％FBS/PBS缓冲液，使细胞数目达到1×10 7个。期间细胞置于冰上。4℃下140g离心CHO-GPRC5D细胞2min，重悬于已经封闭好的1mL的噬菌体抗体库中，4℃或者室温下轻轻旋转或者周期性混匀进行结合2小时，移去上清，收获细胞重悬于1mL的5％FBS/PBS缓冲液中，重复洗涤二次，最后一次重悬细胞以后，换到新的2mL的低吸附的EP管中，避免非特异性吸附到EP管的噬菌体被洗脱下来。
用1mL的100nM的Gly-HCl(pH2.2)重悬细胞，室温孵育10min后4℃下140g离心2min，把上清转移到新的EP管中，加入25μL的2M Tris中和液。把中和液约1mL再用约500μL的7.5％FBS/PBS缓冲液中和。
离心3份相同的吸附细胞CHO，处理方法同CHO-GPRC5D细胞。把洗脱下来后中和好的噬菌体重悬于CHO细胞中。4℃下轻轻旋转，或者周期性混匀进行结合30min。4℃下140g离心2min，取上清用于下一次吸附过程，一共3次。最后一次的噬菌体上清用于感染TG1细胞。
同样的方法进行第二轮细胞淘选，靶细胞CHO-GPRC5D的数目为5×10 6个，洗涤次数为3次。采用同样的操作方法进行第3轮和第4轮细胞淘选，靶细胞换成293-GPRC5D，细胞数目为1×10 6个，洗涤次数增加为10次。
4轮淘洗之后，观察到输出的噬菌体针对靶细胞有显著的特异性富集。利用基于细胞的单噬菌体ELISA(Cell-based mono phage ELISA)对淘选得到的噬菌体抗体克隆进行鉴定：在96孔ELISA板上以3×10 4细胞/孔包被CHO-GPRC5D靶细胞，4℃过夜。然后用5％脱脂奶粉封闭非特异性结合位点，充分洗涤后，取单克隆的噬菌体上清(预混脱脂奶粉)加入到96孔板，孵育2小时，充分洗涤后，加入Anti-M13-HRP，反应45min，充分洗涤后加入TMB显色，室温下作用5-10min后，用硫酸终止反应，于450nm处测定各孔OD值。
通过ELISA鉴定获得了34株能特异性结合人GPRC5D的噬菌体抗体克隆(也就是本申请所述的分离的GPRC5D抗体)，测序后获得单链V HH基因序列，将这10株噬菌体抗体克隆分别命名为3B5，3E7，4A2，4B3，3E9，3F5，3G4，4A4，4E12，4F10，505A1，505A3，505A11，505B12，505D2，505F1，505H3，505H8，505H10，506A5，506A8，506B6，506B12，506C2，506C5，506C8，506C11，506D2，506D8，506E1，506F5，506G1，506H2和506H9。34株克隆的CDR序列如表1所示，可变区的氨基酸序列分别为SEQ ID NO:4，SEQ ID NO:6，SEQ ID NO:8，SEQ ID NO:10，SEQ ID NO:12，SEQ ID NO:14，SEQ ID NO:16，SEQ ID NO:18，SEQ ID NO:20，SEQ ID NO:22，SEQ ID NO:24，SEQ ID NO:26，SEQ ID NO:28，SEQ ID NO:30，SEQ ID NO:32，SEQ ID NO:34，SEQ ID NO:36，SEQ ID NO:38，SEQ ID NO:40，SEQ ID NO:42，SEQ ID NO:44，SEQ ID NO:46，SEQ ID NO:48，SEQ ID NO:50，SEQ ID NO:52，SEQ ID NO:54，SEQ ID NO:56，SEQ ID NO:58，SEQ ID NO:60，SEQ ID NO:62，SEQ ID NO:64，SEQ ID NO:66，SEQ ID NO:68和SEQ ID NO:70。
表1 34株克隆的CDR序列(根据IMGT划分)
克隆号 HCDR1 HCDR2 HCDR3
3B5 GFIFSRYG INWSDGTT AADRRLRSQTGYDY
3E7 GRIFSRDA INSSGGNT AARGVRWTSTTAYGYNY
4A2 GFIFSRYD ITWGGGST AARSASAYRTGRLEYNY
4B3 GLIFSRYV INSSGGST AASSRTSTTRGYNY
3E9 GFIFSRYD INWGDSST AARRYYSYYPSSGGYNY
3F5 GFILSRYV ITWSDGST AAKSRTYRGGLYDY
3G4 GSIFSTYR ISPGGSDT AATPGRFALLSPRGYNY
4A4 GFIFSRNA INWSGSST AAGSRTNTGTDWSRTRYTYNY
4E12 GFIFSRNS ISWRDGST AAFRGWNNGRNYNY
4F10 GFIFSLNA ISPGDGTT AARRRSLSGYGYNY
505A1 GYIFSRYN ISAGGSST AARRRRATDSPYDY
505A3 GFIFSRYS ISAGGSST AAFARSPRTSTEYLYDY
505A11 GIIFSRYV ITSGGGST AAGPRPNYGQRYNTYNY
505B12 GFIFSRYI INAGGGST AAGSASGWSTYSPRRLYRYNY
505D2 GYIFSRYA INRGGSTT AAGRFGLGSRTYDY
505F1 GRTLSNNT ISASGSRT AAVSRVGFYTRGATYNY
505H3 GNILSINI ISRTGGST AASSRRSSLNTYNY
505H8 GFIFSRNA ITWSGGTT AARIAYSSTWTYDY
505H10 GFIFSRYY ISRTGGST AADLYPGFRRGYNY
506A5 GFIFSRYA ISRGDSDT AAVGRYYYGYYANNYNY
506A8 GFIFSRYA INRGDSVT AARGRTSLAYTYDY
506B6 GFIFSRYA INAGGGTT AAFTKRSLLTGSAVYNY
506B12 GLIFSRNA INWGGGTT AATTRTSRPYTYNY
506C2 GYIFSNDV ISRTGGST AASTRRRYATVDYRRDYTYDY
506C5 GFIFSRYY ITSGDGST AARSFRRDIYDYNY
506C8 GYIFSNYA INRSGSIT AANDVRGYYRRYNY
506C11 GLIFSSYA INRRGGST AAGSRSSSRLGYNY
506D2 GIIFSRYA INRSGGRT AAFLASWDGTTYDY
506D8 GYTLSSYY ITWGGGST AAARKRSVTYTTLSYAYSYDY
506E1 GYIFSRYS ISSSGSST AATPRRSTGNTF
506F5 GGIFSSYA INRGGGGT AAFGRYSRGSDDSRYNY
506G1 GFIFSRYA ISWSGGST AAGSRSSSRLGYNY
506H2 GRTFSYYT TSRDGST AAVRRWSSDSTYNY
506H9 GRTFSRYA ISSRGSTT AASLYVIPAWNEYGYNY
实施例2.抗GPRC5D V HH_hFc融合抗体的构建及其真核表达和纯化
将噬菌体抗体克隆3B5，3E7，4A2，4B3，3E9，3F5，3G4，4A4，4E12，4F10，505A1，505A3，505A11，505B12，505D2，505F1，505H3，505H8，505H10，506A5，506A8，506B6，506B12，506C2，506C5，506C8，506C11，506D2，506D8，506E1，506F5，506G1，506H2和506H9以及阳性对照抗体ET150-5scFv(专利CN107428829A)重新设计构建到含Fc(IgG1)的真核表达载体上：设计引物对噬菌体抗体克隆3B5，3E7，4A2，4B3，3E9，3F5，3G4，4A4，4E12，4F10，505A1，505A3，505A11，505B12，505D2，505F1，505H3，505H8，505H10，506A5，506A8，506B6，506B12，506C2，506C5，506C8，506C11，506D2，506D8，506E1，506F5，506G1，506H2和506H9的V HH进行PCR扩增，将PCR产物通过重组克隆至经SfiI和NotI限制性内切酶双酶切的pcDNA3.4-hFc载体上。测序正确后将其质粒转染293F细胞进行瞬时表达，并通过蛋白A亲和柱(Protein A柱)纯化，获得3B5，3E7，4A2，4B3，3E9，3F5，3G4，4A4，4E12，4F10，505A1，505A3，505A11，505B12，505D2，505F1，505H3，505H8，505H10，506A5，506A8，506B6，506B12，506C2，506C5，506C8，506C11，506D2，506D8，506E1，506F5，506G1，506H2和506H9抗体。抗体部分SEC纯度如表2所示，
表2.V HH抗体的SEC纯度
名称 SEC纯度
3B5 VHH-hFc 97.5％
3E7 VHH-hFc 100％
4A2 VHH-hFc 100％
4B3 VHH-hFc 100％
3E9 VHH-hFc 76.5％
3F5 VHH-hFc 100％
3G4 VHH-hFc 100％
4A4 VHH-hFc 100％
4E12VHH-hFc 97.1％
4F10 VHH-hFc 98.7％
505A1 VHH-hFc 94.11％
505A3 VHH-hFc 93.37％
506D8 VHH-hFc 95.92％
506A8 VHH-hFc 96.52％
506B12 VHH-hFc 94.18％
506C5 VHH-hFc 90.90％
506C11 VHH-hFc 91.70％
实施例3.抗GPRC5D抗体与细胞表面GPRC5D结合活性检测
通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液进行细胞表面靶抗原(GPRC5D)与抗体(即实施例2获得的3B5，3E7，4A2，4B3，3E9，3F5，3G4,4A4，4E12，4F10，505A1，505A3，505A11，505B12，505D2，505F1，505H3，505H8，505H10，506A5，506A8，506B6，506B12，506C2，506C5，506C8，506C11，506D2，506D8，506E1，506F5，506G1，506H2和506H9抗体)结合活性检测。简言之，使用缓冲液配置浓度为1x10 6细胞/mL的靶细胞(即MM1S骨髓瘤细胞)，加入96孔尖底板(corning 3894)，每孔30μL；使用缓冲液配置检测抗体浓度为10μg/mL，并按3倍比稀释抗体，形成8个浓度梯度；将配置好的不同浓度的抗体按30μL/孔加入已铺好的靶细胞中混匀；4℃冰箱孵育1小时；每孔加150μL缓冲液，300g离心5分钟，弃上清后将细胞震松散；重复洗涤一次；使用缓冲液按1:200配比配置荧光二抗(ab98593)，每孔30μl加入细胞中混匀，4℃冰箱孵育30分钟；每孔加150μL缓冲液，300g离心5分钟，弃上清后将细胞震松散；重复洗涤一次；每孔加35μL缓冲液混匀后用流式仪器检测。流式亲和力结合实验的分析结果如表3和图1所示，本申请的抗GPRC5D VHH均能结合细胞表面的GPRC5D，且结合能力与阳性对照ET150-5scFv相当，甚至高于阳性对照。
表3.抗GPRC5D抗体与细胞表面GPRC5D蛋白的结合
Figure PCTCN2022115590-appb-000001
Figure PCTCN2022115590-appb-000002
实施例4.嵌合抗原受体慢病毒表达载体的构建
用SphI和NotI内切酶(购自NEB)对CAR慢病毒核心空载质粒(上海原能细胞医学技术有限公司自构，包含ori2新元件，以下简称CAR慢病毒空载体)进行双酶切产生8992bp线性化片段，割胶回收后与实施例1所得的3B5，3E7，4A2，4B3，3E9，3F5，3G4，4A4，4E12和4F10的VHH片段，按1:3的摩尔比例混合进行同源重组后，转化DH5α感受态细胞。挑取单菌落，进行测序鉴定。CAR慢病毒空载体和GPRC5D核心质粒(3B5VHH-CAR-ori2，3E7-CAR-ori2，4A2-CAR-ori2，4B3-CAR-ori2，3E9-CAR-ori2，3F5-CAR-ori2，3G4-CAR-ori2，4A4-CAR-ori2，4E12-CAR-ori2和4F10-CAR-ori2)中CAR各部分元件及连接顺序见说明书附图2，氨基酸序列依次如SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:199、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:202、SEQ ID NO:203和SEQ ID NO:204所示。
实施例5.慢病毒的包装及病毒滴度测定
作为示例，用于构建本发明的慢病毒载体系统属于第三代，该系统共由三个质粒组成，即编码Gag-Pol蛋白和Rev蛋白的包装质粒psPAX2、编码包膜蛋白VSV-G的PMD2.G质粒以及核心质粒，即上述实施例4中的含VHH序列的各个CAR慢病毒质粒(即3B5VHH-CAR-ori2，3E7-CAR-ori2，4A2-CAR-ori2，4B3-CAR-ori2，3E9-CAR-ori2，3F5-CAR-ori2，3G4-CAR-ori2，4A4-CAR-ori2，4E12-CAR-ori2和4F10-CAR-ori2)。各个CAR慢病毒质粒中CAR基因由延长因子-1α(EF-1α)启动子调控其表达。
5.1慢病毒的包装过程如下：
(1)取生长状态良好(一般选取20代以内的细胞)、约2.5×10 6个293T细胞于5mL293T细胞完全培养基(含10％FBS的高糖DMEM)中，混匀后接种于6cm培养皿中(确保20-24h后细胞汇合度在80-90％)，于37℃，5％CO 2的培养箱内培养过夜。
(2)约20-24h后，取300μL Opti-MEM培养基，加入总共6.3μg的三种质粒(按质量比CAR慢病毒核心质粒：psPAX2：PMD2.G＝4:3:2的比例加入)，混匀后加入18.9μL的FuGENEHD转染试剂(Promega，E2311)，小心混匀后，室温静置15-20min，然后将混合液小心滴加至6cm培养皿中，小心摇动培养皿，于37℃，5％CO 2的培养箱内培养过夜。
(3)转染约20-24h后，小心弃去6cm培养皿中的上层液体，然后缓慢加入5mL 293T细胞完全培养基。
(4)转染约48h后，收取6cm培养皿中的上层液体(含死细胞和碎片)，3000g离心5min 后，将去除细胞碎片的上层病毒液转移至新的离心管中，分装后，保存于-80℃待用。
5.2慢病毒滴度的检测按以下方法进行：
(1)快速解冻以上制备的病毒液。
(2)取生长状态良好的293T细胞(一般培养20代以内的细胞)，弃去上层废液，PBS洗涤细胞后，用0.25％的胰酶(GIBICO)，于37℃消化约3分钟，待细胞消化完全后加入一定体积的293T细胞完全培养基终止反应，取样计数，然后将细胞密度调整为2.0×10 5/mL，并加入终浓度为10μg/mL的聚凝胺(polybrene)，再按每孔2.5mL的接种量，将细胞接种于六孔板中。
(3)然后往六孔板中分别加入上述病毒液，一般每种病毒液做三个上样梯度：2μL，5μL和10μL，并且需增加1-2个不加入病毒液的空白孔作为流式检测时的阴性对照。
(4)细胞培养板置于37℃，5％CO 2培养箱中静置培养。
(5)细胞静置培养48h后，弃去上层废液，PBS洗涤细胞后，用0.25％的胰酶(GIBICO)，于37℃消化约3min，待细胞消化完全后加入一定体积的293T细胞完全培养基终止反应，取样计数，然后取约5×10 5个细胞至1.5mL无菌离心管，做好标记。
(6)用NBS溶液(含1％新生牛血清的PBS溶液)补齐至1.2-1.5mL，4℃下500g离心5min，弃上清。
(7)每管加入1mL NBS，轻轻吹打混匀，4℃下500g离心5min，充分弃去上清。
(8)每管加入50μL NBS和0.8μg Biotin-Goat anti-human F(ab)2抗体(109-066-006)，用移液枪轻轻吹打混匀(阴性对照不加一抗)，4℃孵育60min。
(9)孵育完毕直接加入1mL NBS重悬细胞，4℃下500g离心5min，弃去上清。
(10)每管加入50μL NBS和0.5μg APC streptavidin(554067)荧光二抗(阴性对照不加此蛋白)，4℃孵育30min。
(11)重复步骤(9)。
(12)每管加入200μL NBS重悬细胞。
(13)BD FACS CantoII流式仪上检测荧光表达百分比，数据处理。
仅当各测试管阳性率-阴性对照阳性率为5％-20％时，结果可用。
慢病毒滴度(IΜ/mL)＝铺板细胞个数×(测试管阳性率-对照管阳性率)/接种病毒液体积(mL)。
经检测，上述各个含VHH的CAR病毒滴度范围在1-10×10 6IΜ/mL。
实施例6. T细胞的感染和BCMA CAR-T细胞体外扩增
含有GPRC5D抗体序列及阳性对照序列109 scFv(专利CN107428829A)的CAR-T细胞的制备方法如下：
(1)通过Ficoll密度梯度离心法，从单采机采集的血样中分离得到人外周血单个核细胞(PBMC)(单采血由上海原能细胞医学技术有限公司方舟计划志愿者提供)。
(2)PBMC经CD3阳选磁珠分选后得到纯度>90％的CD3+T细胞，T细胞分选具体方法见产品说明书(MACS，DS130-050-101)。
(3)用T细胞完全培养基(X-VIVO 15(Lonza)+5％FBS+细胞因子)重悬CD3+T细胞，再按3倍细胞量加入已经洗涤的CD3/CD28 Dynabeads(Gibco，40203D)，然后补加T细胞完全培养基，调整细胞密度为1.0-1.2×10 6细胞/mL，并将细胞置于37℃，5％CO 2培养箱中激活培养(记录为D0)。
(4)一般在T细胞激活20-24h后，进行慢病毒转导。
(5)收集激活20-24h的T细胞，500g离心5min后，用一定体积的T细胞完全培养基重悬细胞后取样计数，按照病毒复染指数(MOI)为2-5的比例，分别加入实施例5中已测定滴度的病毒液，然后加入polybrene至终浓度为5μg/mL，再补加T细胞完全培养基，调整细胞密度为0.6-1.0×10 6/mL，再将细胞置于37℃，5％CO 2培养箱中培养，另取一份T细胞不做慢病毒感染，作为阴性对照组。
(6)病毒转导约20-24h后，收集各组的T细胞，500g离心5min后，用一定体积的T细胞完全培养基重悬细胞后取样计数，补加T细胞完全培养基，调整细胞密度为0.5-0.7×10 6细胞/mL。
(7)此后，每1-2天对CAR-T细胞进行计数处理，并根据实际情况补加T细胞完全培养基，将细胞密度调整为0.5-1.0×10 6细胞/mL。
(8)一般情况下，在培养第7天时去除Dynabeads，总培养周期为12天左右。
实验结果如图3A-3D所示，经激活培养12天的GPRC5D CAR-T细胞的总扩增倍数介于100-600倍之间，感染后的CAR阳性率均介于75％至95％之间，可以用于细胞学功能实验。
实施例7. GPRC5D CAR-T细胞的体外靶向杀伤活性评估
为了准确地检测CAR-T细胞对于靶细胞的杀伤能力，我们将GPRC5D基因通过慢病毒的方式导入CHO细胞中，再通过了流式分选仪分选出GPRC5D阳性细胞，即CHO-GPRC5D细胞。靶细胞即为CHO-GPRC5D细胞，阴性细胞即为CHO阴性细胞。将培养到第9天的效 应细胞，分别以效靶比为1：3，1：1以及3:1的比例，与靶细胞孵育20小时，通过LDH方法判断靶细胞的存活情况(即CAR-T细胞的杀伤力)。
细胞的杀伤毒性可以用以下公式进行计算：
Figure PCTCN2022115590-appb-000003
如图4A至图4C所示，和空白效应细胞T细胞相比，结果显示，抗GPRC5D CAR-T细胞与109阳性对照对靶细胞有明显的杀伤效果，而且是梯度依赖型的。而对非靶细胞无杀伤效果。
另一种检测杀伤的方法，用MM1S-lucifurase作为靶细胞，与效应CAR-T细胞共孵育6小时以后，通过检测剩余靶细胞中的荧光素酶值，来确定杀伤效果。效靶比为1：1，检测结果如4D所示，结果显示与T细胞相比，本申请的CAR-T细胞有明显的杀伤效果。
实施例8. GPRC5D CAR-T细胞体外杀伤后的细胞因子分泌
参照实施例7，在96孔板中进行靶细胞杀伤实验，效应细胞和靶细胞的比例为1:1，然后固定每个96孔的效应细胞数2×10 4个，依次加入靶细胞和效应细胞，每组每个效靶比下设置2个重复孔，另外每组均需设置2个单独效应细胞孔，检测效应细胞的本底因子分泌量。
在靶细胞杀伤后的24小时左右，将96孔板离心，收集各个孔的上层清液(若不立即检测，需将上层清液保存于-80℃冰箱中)。
将效应细胞离心去上清，用X-VIVO稀释至3×10 5细胞/mL，每孔取100μL使用。将无靶细胞(即不与肿瘤细胞共培养的CAR-T细胞)及与MM1S细胞均按效靶比为1：1的比例混合于圆底96孔板中，共孵育24小时后，吸取上清，用R&D Cytokine Kit检测上清液中IL-2和IFN-γ的含量。
如图5所示，在没有MM1S细胞刺激下，各组CAR-T细胞分泌少量的IL-2和IFN-γ，而当在MM1S细胞刺激下，与本申请选取的阳性对照109-ori2相比，本申请的在杀伤靶细胞时能产生更多的IFN-γ分泌。
实施例9. GPRC5D CAR-T细胞的靶向增殖能力
本实施例CAR-T细胞的靶向增殖能力检测选用未经处理的MM1S细胞作为靶细胞。
一般在CAR-T细胞培养9-12天左右进行，需提前检测各组的CAR阳性率，再用空白效应T细胞将各组的CAR阳性率调成一致(培养基为不含任何添加物的X-VIVO 15培养基)。
将CAR-T细胞和MM1S细胞，以效靶比1:1的比例进行第一轮靶向刺激，在共孵育4-5 天后，收集所有细胞，取样计数，再按照1:3(全CAR-T细胞:新MM1S细胞)的比例，进行第二轮靶向刺激，再次共孵育4-5天后，进行第三轮靶向刺激(两种细胞间的比例与第二轮靶向刺激相同)。三轮靶向刺激期间，按照细胞生长情况每1-2天补加适量X-VIVO 15培养基。
如图6所示，GPRC5D CAR-T细胞经两三轮靶向刺激后，本申请的CAR-T增殖扩增倍数与阳性对照相当。而T细胞的共孵育孔和单独的MM1S靶细胞孔基本都不扩增。
实施例10. GPRC5D CAR-T小鼠体内实验
我们进一步在NSG小鼠体内验证了GPRC5D CAR-T细胞的消瘤、抑瘤作用。具体而言实验分为三组，T细胞组、阳性对照组和GPRC5D CAR-T细胞组，每组5只小鼠。先在NSG小鼠皮下，按1×10 7细胞/只的剂量接种MM1S细胞，约2周后(肿瘤大小为50-150mm 3)，尾静脉注射已培养10-12天的三个组的CAR-T(或T)细胞，剂量均为2×10 6CAR-T细胞/只的剂量(以T细胞调整每只小鼠的总接种细胞数一致)。接种CAR-T(或T)细胞后，CAR-T(或T)细胞接种后第7天和第14天检测小鼠外周血中细胞因子分泌，每周测量小鼠瘤体大小和体重3次，观察3周。
细胞因子分泌方面，如图7B和7C所示，GPRC5D CAR-T细胞组在小鼠体内能产生更大量的IFN-γ，且持续时间也更长(CAR-T注射后第14天检测的IFN-γ比阳性对照组显著增多)，不过其它细胞因子比如IL-2的分泌量很低，组间差别很小。
在消瘤速度方面，如图7E所示，GPRC5D CAR-T细胞与阳性对照组基本趋于一致，与T细胞组对比，有很显著的消瘤作用。
如图7D所述，在NSG小鼠接种CAR-T(或T)细胞后，每2-3天称量小鼠体重，绘制小鼠的体重曲线，并观察和记录小鼠毛色、排泄物、饮食进水、躯体运动以及小鼠死亡情况。GPRC5D CAR-T细胞与阳性对照组的小鼠体重随着时间成稳定增长趋势，与T组的小鼠体重曲线趋势一致。实验期间GPRC5D CAR-T细胞与阳性对照组的小鼠毛色光亮，小鼠行为、饮食进水以及大小便颜色均正常，证实GPRC5D CAR-T细胞在小鼠体内具有较好的安全性。
实施例11. GPRC5D抗体的特异性实验
通过流式细胞荧光分选技术(FACS)，使用iQue Screener流式仪(购自IntelliCyt公司)，用含有0.1％BSA的PBS作为缓冲液进行细胞表面靶抗原(GPRC5D)与抗体(即实施例2获得的3B5，3E7，4A2，4B3，3E9，3F5，3G4,4A4和4E12抗体)非特异性结合实验。即使用缓冲液配置浓度为1x10 6细胞/mL的靶细胞(即不表达GPRC5D抗原的细胞，如A549细 胞、KERA细胞、MCF-7细胞和MSC细胞)，加入96孔尖底板(corning 3894)，每孔30μL。使用缓冲液按20μg/mL浓度配置检测抗体，并按3倍比稀释抗体，形成8个浓度梯度，将配置好的不同浓度的抗体按30μL/孔加入已铺好的靶细胞中混匀，4℃冰箱孵育1小时，每孔加150μL缓冲液，300g离心5分钟，弃上清后将细胞震松散，重复洗涤一次。使用缓冲液按1:200配比配置荧光二抗(ab98593)，每孔30μL加入细胞中混匀，4℃冰箱孵育30分钟；每孔加150μL缓冲液，300g离心5分钟，弃上清后将细胞震松散，重复洗涤一次。每孔加35μL缓冲液混匀后用流式仪器检测。
流式亲和力结合实验的分析结果如图8A至图8D，以及图9A至图9D所示，抗GPRC5D的3B5，3E7，4A2，4B3，3E9，3F5，3G4,4A4和4E12抗体都不与以上细胞非特异性结合，说明抗GPRC5D抗体特异性较强。
实施例12. GPRC5D抗体内吞效应实验
选择野生型细胞系MM.1S细胞及过表达GPRC5D靶抗原的NCIH929-GPRC5D细胞，具体操作如下：5x10 4细胞/孔的靶细胞铺到96孔V底板，30μL/孔，共两块板(板A和板B)；抗体稀释浓度是20μg/mL起始，3倍比梯度稀释，共7个浓度梯度，30μL/孔加入到铺有靶细胞的96孔V底板中，混匀孵育1h，含0.1％BSA的PBS清洗1次。参考抗体稀释浓度，按1：200稀释，加入Anti-Human IgG-Fc(Dylight650)二抗，30μL/孔，4℃孵育30min后，含0.1％BSA的PBS清洗1次，再加50μL的完全培养基(RPMI1640+10％FBS)，分别将板A和板B于4℃和37℃培养箱孵育2个小时，加含0.1％BSA的PBS清洗1次；每孔加入30μL的荧光淬灭buffer，4℃放置10min后，加含0.1％BSA的PBS清洗1次，30μL/well含0.1％BSA的PBS重悬，intellicyt iQue3流式细胞仪检测。
结果如图10A-10D所示，结果显示，本申请提供的GPRC5D抗体在37℃下均存在不同程度的内吞效应。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (75)

  1. 分离的抗原结合蛋白，其能够以低于1.0μg/mL的EC 50值结合G蛋白偶联受体家族C组5成员D(GPRC5D)蛋白。
  2. 根据权利要求1所述的抗原结合蛋白，其包括抗体或其抗原结合片段。
  3. 根据权利要求1-2中任一项所述的抗原结合蛋白，其中所述抗原结合片段包括Fab、Fab’、F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。
  4. 根据权利要求2-3中任一项所述的抗原结合蛋白，其中所述抗原结合片段为VHH。
  5. 根据权利要求2-4中任一项所述的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  6. 根据权利要求1-5中任一项所述的抗原结合蛋白，其能够与参比抗体竞争结合GPRC5D蛋白，其中所述参比抗体包含抗体重链可变区VH，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，且所述参比抗体包含选自下述的任意一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:102，HCDR2：SEQ ID NO:103，和HCDR3：SEQ ID NO:104；
    (2)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:107；
    (3)HCDR1：SEQ ID NO:109，HCDR2：SEQ ID NO:110，和HCDR3：SEQ ID NO:111；
    (4)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:114，和HCDR3：SEQ ID NO:115；
    (5)HCDR1：SEQ ID NO:116，HCDR2：SEQ ID NO:117，和HCDR3：SEQ ID NO:118；
    (6)HCDR1：SEQ ID NO:119，HCDR2：SEQ ID NO:120，和HCDR3：SEQ ID NO:121；
    (7)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:124，和HCDR3：SEQ ID NO:125；
    (8)HCDR1：SEQ ID NO:127，HCDR2：SEQ ID NO:128，和HCDR3：SEQ ID NO:129；
    (9)HCDR1：SEQ ID NO:130，HCDR2：SEQ ID NO:131，和HCDR3：SEQ ID NO:132；
    (10)HCDR1：SEQ ID NO:95，HCDR2：SEQ ID NO:96，和HCDR3：SEQ ID NO:97；
    (11)HCDR1：SEQ ID NO:133，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO: 135；
    (12)HCDR1：SEQ ID NO:136，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:137；
    (13)HCDR1：SEQ ID NO:138，HCDR2：SEQ ID NO:139，和HCDR3：SEQ ID NO:140；
    (14)HCDR1：SEQ ID NO:142，HCDR2：SEQ ID NO:143，和HCDR3：SEQ ID NO:144；
    (15)HCDR1：SEQ ID NO:145，HCDR2：SEQ ID NO:146，和HCDR3：SEQ ID NO:147；
    (16)HCDR1：SEQ ID NO:148，HCDR2：SEQ ID NO:149，和HCDR3：SEQ ID NO:150；
    (17)HCDR1：SEQ ID NO:152，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:154；
    (18)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:155，和HCDR3：SEQ ID NO:156；
    (19)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:158；
    (20)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:160，和HCDR3：SEQ ID NO:161；
    (21)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:162，和HCDR3：SEQ ID NO:163；
    (22)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:164，和HCDR3：SEQ ID NO:165；
    (23)HCDR1：SEQ ID NO:166，HCDR2：SEQ ID NO:167，和HCDR3：SEQ ID NO:168；
    (24)HCDR1：SEQ ID NO:169，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:170；
    (25)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:171，和HCDR3：SEQ ID NO:172；
    (26)HCDR1：SEQ ID NO:174，HCDR2：SEQ ID NO:175，和HCDR3：SEQ ID NO:176；
    (27)HCDR1：SEQ ID NO:177，HCDR2：SEQ ID NO:178，和HCDR3：SEQ ID NO:179；
    (28)HCDR1：SEQ ID NO:180，HCDR2：SEQ ID NO:181，和HCDR3：SEQ ID NO:182；
    (29)HCDR1：SEQ ID NO:184，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:185；
    (30)HCDR1：SEQ ID NO:186，HCDR2：SEQ ID NO:187，和HCDR3：SEQ ID NO:188；
    (31)HCDR1：SEQ ID NO:189，HCDR2：SEQ ID NO:190，和HCDR3：SEQ ID NO:191；
    (32)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:192，和HCDR3：SEQ ID NO:179；
    (33)HCDR1：SEQ ID NO:193，HCDR2：SEQ ID NO:194，和HCDR3：SEQ ID NO:195；以及
    (34)HCDR1：SEQ ID NO:196，HCDR2：SEQ ID NO:197，和HCDR3：SEQ ID NO:198。
  7. 根据权利要求1-6中任一项所述的抗原结合蛋白，其包括源自抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
  8. 根据权利要求1-7中任一项所述的抗原结合蛋白，其包括HCDR3，且所述HCDR3包含SEQ ID NO:215或SEQ ID NO:218所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的抗原结合蛋白，其包括HCDR3，且所述HCDR3包含SEQ ID NO:97、SEQ ID NO:104、SEQ ID NO:107、SEQ ID NO:111、SEQ ID NO:115、SEQ ID NO:118、SEQ ID NO:121、SEQ ID NO:125、SEQ ID NO:129、SEQ ID NO:132、SEQ ID NO:135、SEQ ID NO:137、SEQ ID NO:140、SEQ ID NO:144、SEQ  ID NO:147、SEQ ID NO:150、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:158、SEQ ID NO:161、SEQ ID NO:163、SEQ ID NO:165、SEQ ID NO:168、SEQ ID NO:170、SEQ ID NO:172、SEQ ID NO:176、SEQ ID NO:179、SEQ ID NO:182、SEQ ID NO:185、SEQ ID NO:188、SEQ ID NO:191、SEQ ID NO:195和SEQ ID NO:198中任一项所示的氨基酸序列。
  10. 根据权利要求1-9中任一项所述的抗原结合蛋白，其包括HCDR2，且所述HCDR2包含X 1X 2X 3X 4X 5X 6X 7T所示的氨基酸序列，其中，X 1为I或T，X 2为N、S或T，X 3为A、P、R、S或W，X 4为G、R、S、T或不存在，X 5为D或G，X 6为G或S，且X 7为D、G、I、N、R、S、T或V。
  11. 根据权利要求1-10中任一项所述的抗原结合蛋白，其包括HCDR2，且所述HCDR2包含SEQ ID NO:96、SEQ ID NO:103、SEQ ID NO:106、SEQ ID NO:110、SEQ ID NO:114、SEQ ID NO:117、SEQ ID NO:120、SEQ ID NO:124、SEQ ID NO:128、SEQ ID NO:131、SEQ ID NO:134、SEQ ID NO:139、SEQ ID NO:143、SEQ ID NO:146、SEQ ID NO:149、SEQ ID NO:153、SEQ ID NO:155、SEQ ID NO:160、SEQ ID NO:162、SEQ ID NO:164、SEQ ID NO:167、SEQ ID NO:171、SEQ ID NO:175、SEQ ID NO:178、SEQ ID NO:181、SEQ ID NO:187、SEQ ID NO:190、SEQ ID NO:192、SEQ ID NO:194和SEQ ID NO:197中任一项所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的抗原结合蛋白，其包括HCDR1，且所述HCDR1包含GX 2X 3X 4SX 6X 7X 8所示的氨基酸序列，其中，X 2为F、G、I、L、N、R、S或Y，X 3为I或T，X 4为F或L，X 6为I、L、N、R、S、T或Y，X 7为D、N或Y，且X 8为A、D、G、I、N、R、S、T、V或Y。
  13. 根据权利要求1-12中任一项所述的抗原结合蛋白，其包括HCDR1，且所述HCDR1包含SEQ ID NO:95、SEQ ID NO:102、SEQ ID NO:105、SEQ ID NO:109、SEQ ID NO:116、SEQ ID NO:119、SEQ ID NO:123、SEQ ID NO:127、SEQ ID NO:130、SEQ ID NO:133、SEQ ID NO:136、SEQ ID NO:138、SEQ ID NO:142、SEQ ID NO:145、SEQ ID NO:148、SEQ ID NO:152、SEQ ID NO:157、SEQ ID NO:159、SEQ ID NO:166、SEQ ID NO:169、SEQ ID NO:174、SEQ ID NO:177、SEQ ID NO:180、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:189、SEQ ID NO:193和SEQ ID NO:196中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的抗原结合蛋白，其包括HCDR1、HCDR2、HCDR3，且所述HCDR1、HCDR2、HCDR3包含选自下组的任意一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:102，HCDR2：SEQ ID NO:103，和HCDR3：SEQ ID NO:104；
    (2)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:107；
    (3)HCDR1：SEQ ID NO:109，HCDR2：SEQ ID NO:110，和HCDR3：SEQ ID NO:111；
    (4)HCDR1：SEQ ID NO:105，HCDR2：SEQ ID NO:114，和HCDR3：SEQ ID NO:115；
    (5)HCDR1：SEQ ID NO:116，HCDR2：SEQ ID NO:117，和HCDR3：SEQ ID NO:118；
    (6)HCDR1：SEQ ID NO:119，HCDR2：SEQ ID NO:120，和HCDR3：SEQ ID NO:121；
    (7)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:124，和HCDR3：SEQ ID NO:125；
    (8)HCDR1：SEQ ID NO:127，HCDR2：SEQ ID NO:128，和HCDR3：SEQ ID NO:129；
    (9)HCDR1：SEQ ID NO:130，HCDR2：SEQ ID NO:131，和HCDR3：SEQ ID NO:132；
    (10)HCDR1：SEQ ID NO:95，HCDR2：SEQ ID NO:96，和HCDR3：SEQ ID NO:97；
    (11)HCDR1：SEQ ID NO:133，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:135；
    (12)HCDR1：SEQ ID NO:136，HCDR2：SEQ ID NO:134，和HCDR3：SEQ ID NO:137；
    (13)HCDR1：SEQ ID NO:138，HCDR2：SEQ ID NO:139，和HCDR3：SEQ ID NO:140；
    (14)HCDR1：SEQ ID NO:142，HCDR2：SEQ ID NO:143，和HCDR3：SEQ ID NO:144；
    (15)HCDR1：SEQ ID NO:145，HCDR2：SEQ ID NO:146，和HCDR3：SEQ ID NO:147；
    (16)HCDR1：SEQ ID NO:148，HCDR2：SEQ ID NO:149，和HCDR3：SEQ ID NO:150；
    (17)HCDR1：SEQ ID NO:152，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:154；
    (18)HCDR1：SEQ ID NO:123，HCDR2：SEQ ID NO:155，和HCDR3：SEQ ID NO:156；
    (19)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:158；
    (20)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:160，和HCDR3：SEQ ID NO:161；
    (21)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:162，和HCDR3：SEQ ID NO:163；
    (22)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:164，和HCDR3：SEQ ID NO:165；
    (23)HCDR1：SEQ ID NO:166，HCDR2：SEQ ID NO:167，和HCDR3：SEQ ID NO:168；
    (24)HCDR1：SEQ ID NO:169，HCDR2：SEQ ID NO:153，和HCDR3：SEQ ID NO:170；
    (25)HCDR1：SEQ ID NO:157，HCDR2：SEQ ID NO:171，和HCDR3：SEQ ID NO:172；
    (26)HCDR1：SEQ ID NO:174，HCDR2：SEQ ID NO:175，和HCDR3：SEQ ID NO:176；
    (27)HCDR1：SEQ ID NO:177，HCDR2：SEQ ID NO:178，和HCDR3：SEQ ID NO:179；
    (28)HCDR1：SEQ ID NO:180，HCDR2：SEQ ID NO:181，和HCDR3：SEQ ID NO:182；
    (29)HCDR1：SEQ ID NO:184，HCDR2：SEQ ID NO:106，和HCDR3：SEQ ID NO:185；
    (30)HCDR1：SEQ ID NO:186，HCDR2：SEQ ID NO:187，和HCDR3：SEQ ID NO:188；
    (31)HCDR1：SEQ ID NO:189，HCDR2：SEQ ID NO:190，和HCDR3：SEQ ID NO:191；
    (32)HCDR1：SEQ ID NO:159，HCDR2：SEQ ID NO:192，和HCDR3：SEQ ID NO: 179；
    (33)HCDR1：SEQ ID NO:193，HCDR2：SEQ ID NO:194，和HCDR3：SEQ ID NO:195；以及
    (34)HCDR1：SEQ ID NO:196，HCDR2：SEQ ID NO:197，和HCDR3：SEQ ID NO:198。
  15. 根据权利要求1-14中任一项所述的抗原结合蛋白，其包含抗体重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:98所示的氨基酸序列。
  16. 根据权利要求15所述的抗原结合蛋白，其中所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:216所示的氨基酸序列。
  17. 根据权利要求16所述的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:99、SEQ ID NO:108、SEQ ID NO:112、SEQ ID NO:122、SEQ ID NO:151和SEQ ID NO:173中任一项所示的氨基酸序列。
  18. 根据权利要求15-17中任一项所述的抗原结合蛋白，其中所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:217所示的氨基酸序列。
  19. 根据权利要求18所述的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:100、SEQ ID NO:113、SEQ ID NO:126、SEQ ID NO:141和SEQ ID NO:183中任一项所示的氨基酸序列。
  20. 根据权利要求15-19中任一项所述的抗原结合蛋白，其中所述VH包括框架区H-FR4，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:101所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的抗原结合蛋白，其包括抗体重链可变区VH，且所述VH包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的 氨基酸序列。
  22. 根据权利要求1-21中任一项所述的抗原结合蛋白，其为VHH，且所述VHH包含SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:56、SEQ ID NO:58、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68和SEQ ID NO:70中任一项所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的抗原结合蛋白，其包含抗体重链恒定区，所述抗体重链恒定区源自IgG。
  24. 根据权利要求1-23中任一项所述的抗原结合蛋白，其包含抗体重链恒定区，所述抗体重链恒定区源自人IgG。
  25. 根据权利要求1-24中任一项所述的抗原结合蛋白，其包含抗体重链恒定区，所述抗体重链恒定区源自人IgG1。
  26. 嵌合抗原受体，其包含靶向部分，所述靶向部分包含权利要求1-25中任一项所述的抗原结合蛋白。
  27. 根据权利要求26所述的嵌合抗原受体，其包含共刺激信号区域，其中所述胞共刺激信号区域包含源自选自下组中的一种或多种蛋白的胞内共刺激信号区域：CD28、4-1BB、CD27、CD2、CD7、CD8、OX40、CD226、DR3、SLAM、CDS、ICAM-1、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  28. 根据权利要求27所述的嵌合抗原受体，其中所述共刺激信号区域为源自4-1BB的胞内共刺激信号区域。
  29. 根据权利要求27-28中任一项所述的嵌合抗原受体，其中所述共刺激信号区域包含SEQ ID NO:78所示的氨基酸序列。
  30. 根据权利要求27-29中任一项所述的嵌合抗原受体，其包括胞内信号区域，所述胞内信号区域包含源自选自下组中的一种或多种蛋白的胞内信号区域：CD3ζ、CD3δ、CD3γ、CD3ε、CD79a、CD79b、FcεRIγ、FcεRIβ、FcγRIIa、牛白血病病毒gp30、Epstein-Barr病毒(EBV)LMP2A、猿免疫缺陷病毒PBj14Nef、卡波西肉瘤疱疹病毒(HSKV)、 DAP10、DAP-12和至少包含一个ITAM的结构域。
  31. 根据权利要求30所述的嵌合抗原受体，其中所述胞内信号区域为源自CD3ζ的信号传导结构域。
  32. 根据权利要求31所述的嵌合抗原受体，其中所述胞内信号区域包含SEQ ID NO:80所示的氨基酸序列。
  33. 根据权利要求26-32中任一项所述的嵌合抗原受体，其包括跨膜区，所述跨膜区包含源自选自下组中的一种或多种蛋白的跨膜域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TRAC、TRBC、CD3ε、CD3ζ、CTLA-4、LAG-3、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154和SLAM。
  34. 根据权利要求33所述的嵌合抗原受体，其中所述跨膜区为源自CD8的跨膜区。
  35. 根据权利要求34所述的嵌合抗原受体，其中所述跨膜区包含SEQ ID NO:76所示的氨基酸序列。
  36. 根据权利要求34-35中任一项所述的嵌合抗原受体，其在靶向部分和跨膜区之间包括铰链区，所述铰链区包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、CD40L、TIM1、CD226、SLAM、CD30和LIGHT。
  37. 根据权利要求36所述的嵌合抗原受体，其中所述铰链区为源自CD8的铰链区。
  38. 根据权利要求37所述的嵌合抗原受体，其中所述铰链区包含SEQ ID NO:74所示的氨基酸序列。
  39. 根据权利要求26-38中任一项所述的嵌合抗原受体，其还包含低密度脂蛋白受体相关蛋白或其片段，所述低密度脂蛋白受体相关蛋白或其片段位于所述胞内信号区域的C端。
  40. 根据权利要求39所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含选自下组的一种或多种：低密度脂蛋白受体相关蛋白1-12和其功能性片段。
  41. 根据权利要求39-40中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段为低密度脂蛋白受体相关蛋白6或其片段。
  42. 根据权利要求39-41中任一项所述的嵌合抗原受体，其中所述低密度脂蛋白受体相关蛋白或其片段包含SEQ ID NO:84所示的氨基酸序列。
  43. 根据权利要求26-42中任一项所述的嵌合抗原受体，其还包含信号肽。
  44. 根据权利要求43所述的嵌合抗原受体，其中所述信号肽源自CD8蛋白的信号肽。
  45. 根据权利要求44所述的嵌合抗原受体，其中所述信号肽包含SEQ ID NO:72所示的氨基酸序列。
  46. 根据权利要求26-45中任一项所述的嵌合抗原受体，其包含SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:202、SEQ ID NO:203和SEQ ID NO:204中任一项所示的氨基酸序列。
  47. 根据权利要求26-46中任一项所述的嵌合抗原受体，其包含SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89和SEQ ID NO:91中任一项所示的核苷酸序列。
  48. 多肽，其包含权利要求1-25中任一项所述的抗原结合蛋白。
  49. 一种或多种分离的核酸分子，其编码权利要求1-25中任一项所述的抗原结合蛋白和/或权利要求26-47中任一项所述的嵌合抗原受体。
  50. 根据权利要求49所述的核酸分子，其还包含启动子。
  51. 根据权利要求50所述的核酸分子，其中所述启动子为组成型启动子。
  52. 根据权利要求50-51中任一项所述的核酸分子，其中所述启动子为EF1α启动子。
  53. 根据权利要求49-52中任一项所述的核酸分子，其包含SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89和SEQ ID NO:91中任一项所示的核苷酸序列。
  54. 载体，其包含权利要求49-53中任一项所述的核酸分子。
  55. 根据权利要求54所述的载体，其为病毒载体。
  56. 根据权利要求54-55中任一项所述的载体，其为慢病毒载体。
  57. 细胞，其包含权利要求1-25中任一项所述的抗原结合蛋白，权利要求26-47中任一项所述的嵌合抗原受体，权利要求49-53中任一项所述的核酸分子，和/或权利要求54-56中任一项所述的载体。
  58. 根据权利要求57所述的细胞，其为免疫效应细胞。
  59. 根据权利要求57-58中任一项所述的细胞，其包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。
  60. 根据权利要求57-59中任一项所述的细胞，其为T细胞。
  61. 制备权利要求1-25中任一项所述的抗原结合蛋白和/或权利要求26-47中任一项所述的嵌合抗原受体的方法，所述方法包括在使得权利要求1-25中任一项所述的抗原结合蛋白和/或权利要求26-47中任一项所述的嵌合抗原受体表达的条件下，培养根据权利要求57-60中任一项所述的细胞。
  62. 制备经修饰的免疫效应细胞的方法，其包括向免疫效应细胞中引入权利要求55-56中任一项所述的载体。
  63. 药物组合物，其包含权利要求1-25中任一项所述的抗原结合蛋白，权利要求26-47中任一项所述的嵌合抗原受体，权利要求48所述的多肽，权利要求49-53中任一项所述的核酸分子，权利要求54-56中任一项所述的载体，和/或权利要求57-60中任一项所述的细胞，以及可选地药学上可接受的载体。
  64. 权利要求1-25中任一项所述的抗原结合蛋白，权利要求26-47中任一项所述的嵌合抗原受体，权利要求48所述的多肽，权利要求49-53中任一项所述的核酸分子，权利要求54-56中任一项所述的载体，权利要求57-60中任一项所述的细胞，和/或权利要求63所述的药物组合物，在制备药物中的用途，所述药物用于预防、治疗和/或缓解与GPRC5D异常表达相关的疾病或病症。
  65. 根据权利要求64所述的用途，其中所述与GPRC5D异常表达相关的疾病或病症包括肿瘤。
  66. 根据权利要求65所述的用途，其中所述肿瘤包括实体瘤。
  67. 根据权利要求65所述的用途，其中所述肿瘤包括非实体瘤。
  68. 根据权利要求65所述的用途，其中所述肿瘤包括血液瘤和/或淋巴瘤。
  69. 根据权利要求65所述的用途，其中所述肿瘤包括骨髓瘤。
  70. 一种预防、治疗和/或缓解与GPRC5D异常表达相关的疾病或病症的方法，所述方法包括向有需要的受试者施用权利要求1-25中任一项所述的抗原结合蛋白，权利要求26-47中任一项所述的嵌合抗原受体，权利要求48所述的多肽，权利要求49-53中任一项所述的核酸分子，权利要求54-56中任一项所述的载体，权利要求57-60中任一项所述的细胞，和/或权利要求63所述的药物组合物。
  71. 根据权利要求70所述的方法，其中所述与GPRC5D异常表达相关的疾病或病症包括肿瘤。
  72. 根据权利要求71所述的方法，其中所述肿瘤包括实体瘤。
  73. 根据权利要求71所述的方法，其中所述肿瘤包括非实体瘤。
  74. 根据权利要求71所述的方法，其中所述肿瘤包括血液瘤和/或淋巴瘤。
  75. 根据权利要求71所述的方法，其中所述肿瘤包括骨髓瘤。
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