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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French (fr)
Chinese (zh)
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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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Oricell Therapeutics Co Ltd
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Oricell Therapeutics Co Ltd
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Priority to EP22863407.7A priority Critical patent/EP4397686A4/en
Priority to US18/687,695 priority patent/US20240384001A1/en
Priority to JP2024513196A priority patent/JP2024534847A/ja
Publication of WO2023030272A1 publication Critical patent/WO2023030272A1/zh
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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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PCT/CN2022/115590 2021-08-30 2022-08-29 抗gprc5d抗原结合蛋白及其用途 Ceased WO2023030272A1 (zh)

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EP22863407.7A EP4397686A4 (en) 2021-08-30 2022-08-29 ANTI-GPRC5D ANTIGEN-BINDING PROTEIN AND ITS USE
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US12286475B2 (en) 2023-07-31 2025-04-29 Sanofi Anti-GPRC5D antibodies and compositions

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