Chimeric antigen receptor containing GPRC5D antibody and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a chimeric antigen receptor, a chimeric antigen receptor containing a GPRC5D antibody and application of the chimeric antigen receptor, and the chimeric antigen receptor comprises an extracellular domain which can be combined with a tumor antigen, an inflammatory factor or a pathogen antigen; the transmembrane domain is connected with the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein with a hydrophobic helix; the intracellular domain is connected with the transmembrane domain, and the tumor antigen is a tumor specific antigen or a tumor associated antigen. The inventor optimizes the amino acid sequence of the transmembrane domain, so that the chimeric antigen receptor containing the GPRC5D antibody and the engineered immune cell are provided, and the engineered immune cell obtained based on the GPRC5D antibody has specific killing performance on multiple myeloma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a chimeric antigen receptor, a chimeric antigen receptor containing a GPRC5D antibody, a polynucleotide, an expression vector, a cell, a composition, a pharmaceutical use, and a kit for detecting GPRC5D. Background Art

[0002] Multiple myeloma (MM) is a malignant disease characterized by abnormal proliferation of clonal plasma cells. In many countries, it is the second most common malignant tumor of the blood system. It often occurs in the elderly and is still incurable. In my country, the incidence of multiple myeloma has exceeded that of acute leukemia, about one in 100,000. As the aging of the population in my country continues to intensify, the number of patients will increase further. Although treatment strategies have been greatly improved in recent years, such as monoclonal antibody therapy represented by daratumumab (anti-CD38 antibody), bispecific antibody therapy (BCMAxCD3) represented by Tecvayli, and immune cell therapy represented by chimeric antigen receptor T cells (CAR-T).

[0003] G protein coupled receptor family C group 5 member D (GPRC5D) is an orphan receptor and a 7-transmembrane protein. It is highly expressed in multiple myeloma cells, but expressed in small amounts or not at all in normal tissues. Therefore, this target is expected to become a new target for the treatment of MM.

[0004] Chimeric antigen receptors (CARs) can activate immune cells to specifically kill tumors. Studies have found that the transmembrane domain of CAR is crucial for the activation of immune cells (https: / / doi.org / 10.1016 / j.stem.2018.06.002), and the transmembrane domain derived from the NKG2D transmembrane region can make CAR-NK have a better tumor killing effect. In order to further improve the tumor killing effect, it is urgent to optimize the transmembrane domain and the structure of the chimeric antigen receptor. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, one object of the present invention is to provide a chimeric antigen receptor, a chimeric antigen receptor containing a GPRC5D antibody, and an engineered immune cell, wherein the engineered immune cell obtained based on the GPRC5D antibody has specific killing performance against multiple myeloma.

[0006] To this end, in a first aspect of the present invention, the present invention provides a chimeric antigen receptor. According to an embodiment of the present invention, the chimeric antigen receptor comprises:

[0007] An extracellular domain, which is capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0008] A transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and

[0009] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0010] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0011] SNLFVASWIAVMX8IFX9IGX 10 AX 11 AX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 PS

[0012] Wherein, X8 is selected from I or V;

[0013] X9 is selected from R or H;

[0014] X 10 Select from M or L,

[0015] X 11 Selected from V or I;

[0016] X 12 Selected from I or V,

[0017] X 13 Selected from S or F;

[0018] X 14 is selected from C, R or H;

[0019] X 15 Selected from C, T or V,

[0020] X 16 Selected from V, L or absent;

[0021] X 17 Select from F or H,

[0022] X 18 Selected from F or Y,

[0023] X 19 Selected from L or absent,

[0024] X 16 and X 19Cannot exist at the same time, and X 16 and X 19 The tumor antigen cannot be absent at the same time, and the tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0025] The present invention further optimizes the amino acid sequence of the transmembrane domain. When the transmembrane domain of the chimeric antigen receptor has the amino acid sequence in the above general formula, the engineered immune cells containing the chimeric antigen receptor have a higher ability to kill target cells (cells targeted and bound by the extracellular domain).

[0026] According to an embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in any one of SEQ ID NOs: 19-23, 27-29.

[0027] SNLFVASWIAVMIIFRIGMAVAIFCCFFLPS (SEQ ID NO: 19).

[0028] SNLFVASWIAVMVIFRIGMAVAIFRCLFFPS (SEQ ID NO: 20).

[0029] SNLFVASWIAVMIIFRIGMAVAIFCCLFFPS (SEQ ID NO: 21).

[0030] SNLFVASWIAVMIIF H IG L A I AI SHVL FFPS (SEQ ID NO: 22).

[0031] SNLFVASWIAVMIIFHIGMAIAISRCLFFPS (SEQ ID NO: 23).

[0032] SNLFVASWIAVMIIF H IGMA I AI S C TV FFPS (SEQ ID NO: 27).

[0033] SNLFVASWIAVMIIFRIGMAVA VSR C LH FPS (SEQ ID NO: 28).

[0034] SNLFVASWIAVMIIF H IG L A I AI SR C L FY PS (SEQ ID NO: 29).

[0035] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize a tumor antigen, an inflammatory factor or a pathogen antigen.

[0036] In a second aspect of the present invention, the present invention provides a chimeric antigen receptor. According to an embodiment of the present invention, the chimeric antigen receptor comprises:

[0037] an extracellular domain capable of binding to a tumor antigen;

[0038] A transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and

[0039] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0040] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0041] SNLFVASWIAVMX1IFX2IGMAX3AIX4X5CX6FFX7PS

[0042] Wherein, X1 is selected from I or V;

[0043] X2 is selected from R or H;

[0044] X3 is selected from V or I;

[0045] X4 is selected from S or F;

[0046] X5 is selected from C or R;

[0047] X6 is selected from L or is absent;

[0048] X7 is selected from L or is absent,

[0049] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0050] The present invention further optimizes the amino acid sequence of the transmembrane domain. When the transmembrane domain of the chimeric antigen receptor has the amino acid sequence in the above general formula, the engineered immune cells containing the chimeric antigen receptor have a higher ability to kill target cells (cells targeted and bound by the extracellular domain).

[0051] According to an embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in any one of SEQ ID NOs: 19 to 21 and SEQ ID NO: 23.

[0052] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize a tumor antigen.

[0053] In a third aspect of the present invention, the present invention provides a chimeric antigen receptor. According to an embodiment of the present invention, the chimeric antigen receptor comprises:

[0054] An extracellular domain, which is capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0055] A transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and

[0056] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0057] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0058] SNLFVASWIAVMIIFX 20 IGX 21 AX 22 AX 23 SX 24 X 25 X 26 X 27 X 28 PS

[0059] Among them, X 20 Selected from R or H,

[0060] X 21 Select from M or L,

[0061] X 22 Selected from V or I,

[0062] X 23 Selected from V or I,

[0063] X 24 is selected from C, R or H,

[0064] X 25 Selected from C, T or V,

[0065] X 26 Selected from L or V,

[0066] X 27 Select from F or H,

[0067] X 28 Selected from F or Y;

[0068] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0069] The present invention further optimizes the amino acid sequence of the transmembrane domain. When the transmembrane domain of the chimeric antigen receptor has the amino acid sequence in the above general formula, the engineered immune cells containing the chimeric antigen receptor have a higher ability to kill target cells (cells targeted and bound by the extracellular domain).

[0070] According to an embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in any one of SEQ ID NO: 22 and SEQ ID NO: 27-29.

[0071] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize a tumor antigen, an inflammatory factor or a pathogen antigen.

[0072] According to an embodiment of the present invention, the chimeric antigen receptor of the first aspect, the second aspect, and the third aspect may further include at least one of the following additional technical features:

[0073] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize at least one of the following: CD19, BCMA, GPRC5D, CD123, CD138, CD20, CD22, CD30, CD33, CD38, CD4, CD7, CLL-1, CD10, CD34, CS1, CD16, CD5, IL-1-RAP, ITGB7, k-IgG, TACI, TRBC1, MUC1, NKG2DL, PD-L1, CD133, CD117, LeY, CD70, ROR1, CD147, CD171, CD267, AXL, B7-H3, C D44v6, CEA, CAIX, CLDN18.2, CLDN6, DLL3, DR5, EGFR, EpCAM, ErbB, FAP, FBP, FRα, GD2, gp100, HER2, IL-13Rα2, LFA1, LMP1, MAGE-A3, MAGE-A1, MAG E-A4, MART1, MET, MG7, MMP2, MSLN, MUC1, MUC16, MUC16ecto, NECTIN4, NKG2D, PMEL, PSCA, PSMA, ROR1, ROR2, TM4SF1, TnMUC1, VEGFR2, CLD18, and GPC3.

[0074] According to an embodiment of the present invention, the extracellular domain further comprises an antibody or an antigen-binding fragment thereof capable of specifically recognizing a pathogen antigen, and the pathogen comprises EBV.

[0075] According to an embodiment of the present invention, the extracellular domain also includes an antibody or an antigen-binding fragment thereof that can specifically recognize inflammatory factors, and the inflammatory factors include at least one of the following: IL-1, TNF-α, IFN-γ, IL-12, IL-18, GMCSF, IL-6, IL-8, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16 and CCL2-2.

[0076] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize GPRC5D.

[0077] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 90% identity therewith:

[0078] Heavy chain variable region CDR sequences: SEQ ID NOs: 1-3;

[0079] The CDR sequence of the light chain variable region is: SEQ ID NO: 4, the amino acid sequence SAS and SEQ ID NO: 5.

[0080] GYSITSDYA (SEQ ID NO: 1).

[0081] ISYSGSA (SEQ ID NO: 2).

[0082] ARGGIAGRGRWGAMDY (SEQ ID NO: 3).

[0083] QNVGTN (SEQ ID NO: 4).

[0084] CDR 2 sequence of light chain variable region: SAS

[0085] QQYKSYPLT (SEQ ID NO: 5).

[0086] The present invention provides a chimeric antigen receptor, the transmembrane domain of which is optimized, and contains a new GPRC5D antibody outside the cell, which specifically binds to GPRC5D. The engineered immune cells designed based on the GPRC5D antibody of the present invention can specifically target multiple myeloma cells, kill target cells, and avoid recurrence.

[0087] According to an embodiment of the present invention, the antibody or antigen-binding fragment capable of specifically recognizing GPRC5D comprises:

[0088] The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NO: 1, 2 and 3, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NO: 1, 2 and 3.

[0089] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D comprises:

[0090] The light chain variable region CDR1, CDR2, and CDR3 sequences are respectively shown in SEQ ID NO:4, the amino acid sequence SAS and SEQ ID NO:5, or an amino acid sequence that is at least 90% identical to SEQ ID NO:4, the amino acid sequence SAS and SEQ ID NO:5.

[0091] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D comprises:

[0092] The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NO: 1, 2 and 3, respectively, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2 and 3, and the light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NO: 4, the amino acid sequence SAS and SEQ ID NO: 5, respectively, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, the amino acid sequence SAS and SEQ ID NO: 5.

[0093] According to one embodiment of the present invention, a GPRC5D antibody or an antigen-binding fragment thereof has heavy chain variable region CDR1, CDR2, CDR3 as shown in SEQ ID NO: 1, 2 and 3 and light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 4, amino acid sequence SAS and SEQ ID NO: 5. The antibody can specifically bind to GPRC5D on the surface of multiple myeloma cells, and engineered immune cells designed based on the antibody can specifically target multiple myeloma cells, kill target cells, and avoid recurrence.

[0094] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:6.

[0095] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D has a light chain variable region with an amino acid sequence as shown in SEQ ID NO:7.

[0096] According to an embodiment of the present invention, the antibody is a single-chain antibody having an amino acid sequence as shown in SEQ ID NO:8.

[0097] QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKS RITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSS (SEQ ID NO: 6).

[0098] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGS GSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIK (SEQ ID NO: 7).

[0099] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQV QLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSS(SEQ ID NO: 8).

[0100] According to an embodiment of the present invention, the extracellular domain comprises a bispecific binding molecule comprising:

[0101] A first binding region, wherein the first binding region comprises the antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D; and

[0102] The second binding region has the activity of binding to an antigen specifically expressed on lymphocytes.

[0103] According to an embodiment of the present invention, the antigens specifically expressed on the lymphocytes include at least one selected from BCMA, CD38, CD138, CD3E, and FCGR3A.

[0104] According to an embodiment of the present invention, the second binding region comprises a single chain antibody or a single domain antibody.

[0105] According to an embodiment of the present invention, the second binding region has BCMA binding activity.

[0106] According to an embodiment of the present invention, the second binding region comprises a single-chain antibody or a single-domain antibody capable of specifically recognizing BCMA.

[0107] According to an embodiment of the present invention, the second binding region has an amino acid sequence as shown in SEQ ID NO:9.

[0108] QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVK GRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSS (SEQ ID NO: 9).

[0109] According to an embodiment of the present invention, the first binding region and the second binding region are connected via a connecting peptide.

[0110] According to an embodiment of the present invention, the bispecific binding molecule has an amino acid sequence as shown in SEQ ID NO:10.

[0111] DIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTF GQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDT SEQ ID NO: 10).

[0112] Existing bispecific antibodies or CAR-T for BCMA have achieved certain therapeutic effects in the treatment of MM, but due to BCMA antigen escape, MM patients still cannot avoid relapse. The inventors found that the bispecific antibodies composed of the GPRC5D antibody and the BCMA antibody of the present invention and the engineered immune cells designed based on the bispecific antibodies can specifically target multiple myeloma cells, kill target cells, and avoid relapse, which can solve the problem of BCMA antigen escape.

[0113] According to an embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0114] According to an embodiment of the present invention, the hinge domain is derived from the hinge region of at least one protein in the following group:

[0115] 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 and LIGHT.

[0116] According to an embodiment of the present invention, the hinge domain is derived from the hinge region of CD8;

[0117] According to an embodiment of the present invention, the hinge domain has an amino acid sequence as shown in SEQ ID NO:11.

[0118] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 11).

[0119] According to an embodiment of the present invention, the intracellular domain includes a co-stimulatory signal domain and an intracellular signaling domain, and the co-stimulatory signal domain and the intracellular signaling domain are connected by a connecting peptide.

[0120] According to an embodiment of the present invention, the costimulatory signal domain comprises an intracellular costimulatory signal domain of at least one protein selected from the following groups:

[0121] 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, ligand of CD83, CD40 and MyD88.

[0122] According to an embodiment of the present invention, the costimulatory signaling domain is derived from the intracellular costimulatory signaling domain of 2B4.

[0123] According to an embodiment of the present invention, the co-stimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:12.

[0124] WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDVYS (SEQ ID NO: 12).

[0125] According to an embodiment of the present invention, the intracellular signaling domain includes a domain containing at least one ITAM motif.

[0126] According to an embodiment of the present invention, the intracellular signaling domain comprises an intracellular signaling domain derived from at least one protein in the following group;

[0127] CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, DAP10, DAP-12.

[0128] According to an embodiment of the present invention, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ.

[0129] According to an embodiment of the present invention, the intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO:13.

[0130] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 13).

[0131] According to an embodiment of the present invention, the chimeric antigen receptor further comprises a signal peptide, and the signal peptide is connected to the N-terminus of the extracellular domain of the chimeric antigen receptor.

[0132] According to an embodiment of the present invention, the signal peptide is a signal peptide derived from CD8 protein.

[0133] According to an embodiment of the present invention, the signal peptide has an amino acid sequence as shown in SEQ ID NO:14.

[0134] MALPVTALLLPLALLLHAARP (SEQ ID NO: 14).

[0135] According to an embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 16, and the transmembrane domain sequence is underlined. The transmembrane domain of the chimeric antigen receptor in the present invention can use the transmembrane domain as shown in SEQ ID NO: 18 to 23 according to different experimental needs, and SEQ ID NO: 18 is the amino acid sequence of the transmembrane domain without mutation.

[0136] MALPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLV KPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIFCCFFLPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDV YSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 15).

[0137] MALPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYK SYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLS SVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAV IGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIFCCFFLPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDV YSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 16).

[0138] In the fourth aspect of the present invention, the present invention provides an isolated polynucleotide. According to an embodiment of the present invention, the polynucleotide encodes the chimeric antigen receptor according to the first, second or third aspect.

[0139] In the fifth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the polynucleotide described in the fourth aspect.

[0140] In the sixth aspect of the present invention, the present invention provides a cell. According to an embodiment of the present invention, the cell carries the polynucleotide described in the fourth aspect or the expression vector described in the fifth aspect or is capable of expressing the chimeric antigen receptor described in the first, second or third aspect.

[0141] According to an embodiment of the present invention, the cells include immune cells.

[0142] According to an embodiment of the present invention, the immune cells are obtained by separation from peripheral blood, umbilical cord blood, tissues, or organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells.

[0143] According to an embodiment of the present invention, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development.

[0144] According to an embodiment of the present invention, the immune cells include at least one selected from NK cells, T cells, and macrophages.

[0145] In the seventh aspect of the present invention, the present invention provides a composition. According to an embodiment of the present invention, the composition contains at least one of the following:

[0146] The chimeric antigen receptor of the first, second or third aspect;

[0147] The polynucleotide according to the fourth aspect;

[0148] The expression vector according to the fifth aspect; and

[0149] The cell described in the sixth aspect.

[0150] In the eighth aspect of the present invention, the present invention proposes use of the composition described in the seventh aspect in preparing a drug for treating a disease.

[0151] According to an embodiment of the present invention, the disease is a hematological tumor or a solid tumor disease, an autoimmune disease or a viral infectious disease.

[0152] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0153] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0154] In a ninth aspect of the present invention, the present invention provides a kit for detecting GPRC5D. According to an embodiment of the present invention, the kit comprises the chimeric antigen receptor described in the first, second or third aspect.

[0155] In the tenth aspect of the present invention, the present invention proposes the use of the antibody or antigen-binding fragment thereof described in the first, second or third aspect, the polynucleotide described in the fourth aspect, and the expression vector described in the fifth aspect in preparing a kit. According to an embodiment of the present invention, the kit is used to detect GPRC5D or diagnose a disease related to GPRC5D.

[0156] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0157] In the eleventh aspect of the present invention, the present invention proposes the use of the chimeric antigen receptor described in the first, second or third aspect, the polynucleotide described in the fourth aspect, the expression vector described in the fifth aspect, the cell described in the sixth aspect and / or the composition described in the seventh aspect in the treatment of individual hematological tumors or solid tumor diseases, autoimmune diseases or viral infectious diseases.

[0158] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0159] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0160] In a twelfth aspect of the present invention, the present invention provides a method for preventing and / or treating a blood tumor or solid tumor disease, an autoimmune disease or a viral infectious disease in an individual. According to an embodiment of the present invention, the method comprises administering to the individual at least one of the following:

[0161] The chimeric antigen receptor of the first, second or third aspect;

[0162] The polynucleotide according to the fourth aspect;

[0163] The expression vector according to the fifth aspect;

[0164] The cell of the sixth aspect; and

[0165] The composition described in the seventh aspect.

[0166] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0167] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0168] In the thirteenth aspect of the present invention, the present invention proposes the use of the chimeric antigen receptor described in the first, second or third aspect, the polynucleotide described in the fourth aspect, and the expression vector described in the fifth aspect in detecting GPRC5D or diagnosing whether an individual suffers from a GPRC5D-related disease.

[0169] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0170] In a fourteenth aspect of the present invention, the present invention provides a method for detecting GPRC5D. According to an embodiment of the present invention, the method comprises detecting GPRC5D in a sample to be tested using at least one of the following:

[0171] The chimeric antigen receptor of the first, second or third aspect;

[0172] The polynucleotide according to the fourth aspect;

[0173] The expression vector described in the fifth aspect.

[0174] The method for detecting GPRC5D according to the embodiment of the present invention can be used in scientific research, and the sample includes but is not limited to tissue, cell, blood, serum, plasma, urine, feces, saliva or sweat.

[0175] According to a specific embodiment of the present invention, the present invention provides a method for diagnosing whether an individual suffers from a GPRC5D-related disease, the method comprising detecting GPRC5D in a sample derived from the individual to be tested using at least one of the following: the chimeric antigen receptor, polynucleotide and expression vector described above.

[0176] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0177] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0178] Figure 1 is an expression frame encoding an anti-GPRC5D CAR polypeptide according to an embodiment of the present invention, wherein,

[0179] A to B show two schematic diagrams of anti-GPRC5D CAR polypeptides; C to E show schematic diagrams of several expression cassettes encoding anti-GPRC5D CAR polypeptides;

[0180] Figure 2 For the piggyBac-mediated anti-GPRC5D CAR expression according to an embodiment of the present invention, wherein,

[0181] A shows several examples of the anti-GPRC5D CAR polypeptide expression cassette.

[0182] B shows a map of a piggyBac plasmid containing the anti-GPRC5D CAR expression cassette;

[0183] Figure 3 This is a schematic diagram of site-directed integration of a DNA template containing anti-GPRC5DCAR by homologous recombination according to an embodiment of the present invention, wherein:

[0184] A shows schematic diagrams of several common forms of expression cassettes encoding anti-GPRC5D CAR polypeptides, and the dotted line portion represents an optional portion that may or may not be present;

[0185] B shows examples of several DNA template components containing anti-GPRC5D CAR polypeptide expression cassettes.

[0186] C shows a DNA template expressing anti-GPRC5D CAR and hnCD16 targeting human genomic site A (referring to any site in the human genome) and a schematic diagram of homologous recombination;

[0187] Figure 4 The general process of obtaining gene-edited iPSC-derived anti-GPRC5D CAR-iNK according to an embodiment of the present invention, wherein "*" means that the DNA encoding anti-GPRC5D CAR can be introduced using different tools, such as donor plasmid, piggyBac, dsDNA, ssDNA, various integrative or non-integrative viruses, etc. According to the selection of different introduction tools, PBase can be selected to assist piggyBac introduction, and nuclease can be selected to form single-strand or double-strand breaks to assist other non-viral introduction methods;

[0188] Figure 5 This is a result diagram of the positive rate detection of CAR molecules in iPSCs and differentiated iNKs introduced with anti-GPCR5D CAR mediated by FACS according to an embodiment of the present invention, wherein the expression ratio of CAR molecules in the cell population to be tested is detected using an anti-GPRC5D scFV antibody, the left peak is an isotype control, and the right peak is a detection marker;

[0189] Figure 6 This is a graph showing the result of detecting the expression levels of anti-GPRC5D CAR-iNK CD56 and CD45 derived from iPSC by FACS according to an embodiment of the present invention, wherein the left peak is the isotype control and the right peak is the detection marker;

[0190] Figure 7This is a result diagram of the FACS detection of the NKG2D expression level of the iPSC-derived anti-GPRC5D CAR-iNK according to an embodiment of the present invention, wherein the left peak is the isotype control (isotype control) and the right peak is the detection marker;

[0191] Figure 8 This is a result diagram of the FACS detection of TCRαβ and TCRY expression levels of iPSC-derived anti-GPRC5D CAR-iNK according to an embodiment of the present invention, wherein the left peak is the isotype control (isotype control) and the right peak is the detection marker;

[0192] Fig. 9 This is a graph showing the results of detecting the killing activity of anti-GPRC5D or anti-BCMACAR-iNK cells derived from iPSCs against K562-Luc cells according to an embodiment of the present invention, wherein:

[0193] A shows the results of the cytotoxic activity test of different NK cells against K562-Luc under the conditions of 4 hours incubation time and 4:1 effector-target ratio.

[0194] B. A graph showing the detection results of the effects of WT-iNK and anti-GPRC5D or anti-BCMACAR-iNK on the killing activity of K562-Luc under different effector-target ratios;

[0195] Fig.10 The killing activity of the anti-GPRC5D cells derived from iPSCs according to an embodiment of the present invention on H929-Luc2 cells, wherein,

[0196] The left figure shows the test results of the effects of different NK cells on the killing activity of H929-Luc2 at an effector-target ratio of 4:1 after a 4-hour incubation time, and the right figure shows the test results of the effects of WT-iNK and anti-GPRC5D or anti-BCMACAR-iNK on the killing activity of H929-Luc2 at different effector-target ratios;

[0197] Fig.11 It is a graph showing the results of detecting the killing activity of anti-GPRC5D / BCMACAR-iNK, anti-GPRC5DCAR-iNK and anti-BCMACAR-iNK cells derived from iPSCs on H929-Luc2 cells at different effector-target ratios according to an embodiment of the present invention, wherein the extracellular domain of anti-GPRC5D / BCMACAR-iNK contains antibody sequences that recognize GPRC5D and BCMA;

[0198] Fig.12The figure is a test result diagram comparing the killing ability of iPSC-derived WT iNK and anti-GPRC5D CAR-iNK against K562-Luc cells and H929-Luc2 after cryopreservation and resuscitation according to an embodiment of the present invention;

[0199] Fig.13 This is a graph showing the results of detecting the change in killing ability of fresh (Fresh) and cryopreserved (Cryopreserved) anti-GPRC5DCAR-iNK against K562-Luc and H929-Luc2 according to an embodiment of the present invention;

[0200] Fig.14 The graph is a result of detecting the killing activity of anti-GPRC5D CAR-iNK and WT iNK with different effector-target ratios on MM.1S-Luc cells and OPM-2Luc2 cells after cryopreservation and resuscitation according to an embodiment of the present invention;

[0201] Fig.15 This is a result diagram of detecting the proportion of cells expressing CAR molecules using anti-anti-GPRC5D scFV antibodies after CAR molecules (TM control) and variants thereof (TM1-TM14) loaded with anti-GPRC5D scFV were transfected into 293T cells according to an embodiment of the present invention. The expression of CAR molecules was detected using anti-anti-GPRC5D scFV antibodies;

[0202] Fig.16 The results of detecting the expression levels of CD34 and CD43 on day 10 of iPSCs differentiated into iHSPCs by flow cytometry using CAR molecules (TM control) and variants thereof (TM1 to TM8) loaded with anti-GPRC5D scFV according to an embodiment of the present invention, wherein:

[0203] The left and right figures respectively show the expression percentages of CD34 and CD43 in iHSPCs obtained by differentiation of each group in this example;

[0204] Fig.17 The CD56 and CD314 expression levels of iPSCs differentiated into CAR-iNKs at day 23 loaded with anti-GPRC5D scFV CAR molecules (TM control) and their variants (TM 1-TM8) according to an embodiment of the present invention, wherein,

[0205] The left and right figures respectively show the expression percentages of CD56 and CD314 in the CAR-iNK obtained by differentiation of each group in this example;

[0206] Fig.18 This is a result diagram of the expansion multiple of the CD56+CAR-iNK cell population relative to the iPSC cell population at the beginning of differentiation at 23 days during the differentiation of iPSCs loaded with anti-GPRC5D scFV CAR molecules (TM control) or variants thereof (TM1-TM8) into CAR-iNKs according to an embodiment of the present invention;

[0207] FIG. 19 is a graph showing the expression of CD45, CD56, and anti-GPRC5DscFV in the obtained cells after further expansion of CAR molecules (TM control) and variants thereof (TM 1 to TM 8) according to an embodiment of the present invention for 13 days (i.e., Day 36 CAR-iNK), wherein:

[0208] Figures 19A, 19B, and 19C respectively show the results of flow cytometry detection of the expression percentage of each marker (CD45, CD56, anti-GPRC5D scFV) in this embodiment. The left peak in the figure is the detection result of Isotype control, and the right peak is the detection result of each marker (CD45, CD56, anti-GPRC5D scFV).

[0209] Figure 19D shows the statistical results of the expression percentage of each marker (CD45, CD56, anti-GPRC5DscFV) in this example detected by flow cytometry;

[0210] Fig. 20 The figure is a graph showing the antigen-dependent specific killing ability of CAR-iNK loaded with anti-GPRC5D scFV CAR molecules (TM control) and their variants (TM 1 to TM 8) against K562-GPRC5D-Luc according to an embodiment of the present invention, wherein the statistical analysis was performed using One Way ANOVA with multiple comparisons performed by Dunnett's test (GraphPadPrism 9.5.0 (525)). DETAILED DESCRIPTION

[0211] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0212] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0213] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0214] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0215] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0216] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0217] the term

[0218] "scFv" stands for "Single-Chain Fragment Variable", which is translated into Chinese as "single-chain variable region fragment". It is a genetically engineered fusion protein. It is a small molecule composed of the variable region of the antibody heavy chain and the variable region of the light chain connected by a peptide chain.

[0219] "Antibodies" include polyclonal and monoclonal antibodies, including complete antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specific binding to antigens, single-chain antibody fragments, including single-chain variable fragments (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. For example, bispecific antibodies, double antibodies, three antibodies and four antibodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to include its functional antibody fragments.

[0220] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody capable of binding to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy chain regions, single-chain antibody molecules (such as scFv) and single-domain antibodies; and multispecific antibodies formed from antibody fragments. In a specific embodiment, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as scFv.

[0221] As used herein, "heavy chain variable region" refers to a region of an antibody molecule that includes at least one complementarity determining region (CDR) of the antibody heavy chain variable domain. The heavy chain variable region may contain one, two or three CDRs of the antibody heavy chain.

[0222] Herein, the term "single domain antibody (sdAb for short)" refers to a heavy chain antibody naturally lacking a light chain found in camel blood / alpaca blood, and only includes a heavy chain H chain with a heavier molecular weight. Among them, the amino acid sequence at the amino terminal (N-terminus) of the peptide chain varies greatly, which is called the variable region (V region), and the carboxyl terminal (C-terminus) is relatively stable and has little variation, which is called the constant region (C region). The V region of the H chain is called VH. In the variable region, the amino acid composition and arrangement order of certain regions have a higher degree of variation, which is called the hypervariable region (HVR). The hypervariable region is the location where the antigen and antibody bind, so it is also called the complementarity-determining region (CDR). There are three CDR regions in the heavy chain variable region. CDR1 and CDR3 are slightly longer than those of humans, and CDR3 protrudes outward in the tertiary structure. Therefore, it is speculated that single domain antibodies have higher antigen binding specificity and affinity than traditional antibodies. In certain embodiments, the single domain antibody is a human single domain antibody or an alpaca single domain antibody with or without humanization. The single-domain antibody variable domain exhibits the same general structure of a relatively conservative framework region (FR) connected by three hypervariable regions or CDRs. From N-terminal to C-terminal, the heavy chain comprises domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid distribution of each domain conforms to the definition of Kabat immune target protein sequence (Kabat Sequences of Proteins of Immunological Interest) (National Institutes of Health, Bethesda, Md., 1987 and 1991 in Bethesda, Maryland); or Chothia and Lesk, Journal of Molecular Biology, 196: 901-917, 1987; Chothia et al., Nature, 342: 878-883, 1989).

[0223] "Hypervariable region of an antibody" refers to the CDR amino acid residues responsible for antigen binding of an antibody. A hypervariable region comprises amino acid residues from CDRs, e.g., 31-35 (H1), 50-65 (H2), and 95-102 (H3) in a heavy chain variable domain (as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Maryland (1991)); and / or residues from hypervariable loops, e.g., 26-32 (H1), 53-55 (H2), and 96-101 (H3) in a heavy chain variable domain (as described by Chothia et al., J. Molecular Biology 196: 901-917 (1987)).

[0224] "Monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, wherein all antibodies in the mixture have a single amino acid sequence derived from a single clone. Monoclonal antibodies are generally highly specific and are directed against a single antigenic site or epitope. In contrast, polyclonal antibody preparations generally include a mixture of antibodies with different amino acid sequences directed against the same or different determinants (epitopes). In addition to its specificity, the advantage of monoclonal antibodies is that they are synthesized in homogenous cultures and are not contaminated by other immunoglobulins with different specificities and characteristics.

[0225] As used herein, the term "hinge domain" refers to the region between the CH1 region and the CH2 region of the antibody heavy chain, which includes the inter-H chain disulfide bond, is rich in proline, does not form an alpha helix, is prone to stretching and twisting to a certain extent, and is conducive to the complementary binding between the antigen binding site of the antibody and the antigen epitope. The antigen binding domain in the present invention is connected to the transmembrane domain through the hinge domain.

[0226] In the present invention, "antigen" refers specifically to a marker on the surface of tumor cells.

[0227] "Chimeric antigen receptor" is abbreviated as CAR. It is an artificial receptor molecule manufactured by genetic engineering technology, including an antigen binding domain (usually from a single-chain antibody variable region gene fragment, scFV; or from a single-domain antibody), a hinge domain, a transmembrane domain, a co-stimulatory signal domain, and an intracellular signal transduction domain.

[0228] "Reduced expression" As described herein, "reduced expression" of gene expression refers to the elimination or reduction of the expression of one or more gene products encoded by the gene in the cell compared to the expression level of the gene mRNA or protein under normal circumstances. In some cases, this effect on gene expression is temporary or reversible, such as inhibiting the expression of the gene by small molecule compounds, RNAi, siRNA, shRNA and other nucleic acids or specific proteins. In some cases, this effect on gene expression is permanent, such as introducing base insertion or base deletion in the coding region of the target gene through gene editing methods, causing frameshift mutations, or inserting a DNA sequence in the coding region or non-coding region of the target gene through homologous recombination, causing the gene frameshift mutation or premature termination of transcription and / or translation, or translating a completely different polypeptide, which can permanently inactivate the gene. The proportion of gene expression reduction caused by different methods can be 100%, more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, more than 30%, more than 20%.

[0229] "Inactive" means loss of function, or loss of protein function.

[0230] As used herein, the term "nucleic acid" is used interchangeably with the term "polynucleotide" herein, and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form, covering nucleic acids containing known nucleotide analogs or modified backbone residues or connections, the nucleic acids are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to reference nucleic acids, and are metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleic acids encoding polypeptides or fusion proteins refer to one or more nucleic acid molecules encoding polypeptides or fusion proteins, including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present in one or more positions in a host cell. Unless otherwise indicated, a specific nucleic acid sequence also implicitly covers conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated.

[0231] As used herein, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phage or M13 phage, and animal viruses, etc. The vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including, but not limited to, viral particles, liposomes or protein shells. The vector may be an expression vector or a cloning vector. In some embodiments, the vector provided by the present invention (e.g., expression vector) contains the nucleic acid sequence encoding the fusion protein described in the present invention, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0232] As used herein, the term "cell" in the present invention refers to a eukaryotic cell, particularly a mammalian cell, such as a human cell.

[0233] As used herein, the term "immune cell" refers to a cell from a human in the present invention. More specifically, the immune cells in the present invention can be derived from pluripotent stem cells by directed differentiation. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells and macrophages. The immune cells in the present invention can also be derived from human hematopoietic stem cells. These hematopoietic stem cells can be separated from peripheral blood, umbilical cord blood, placental blood, or differentiated from pluripotent stem cells. Depending on the different differentiation conditions provided, these immune cells can be T cells, NK cells and macrophages. The immune cells in the present invention can also be separated from human blood, bone marrow, lymph or lymphoid organs, specific tissues (including but not limited to tumor tissue, abdominal cavity, liver, lungs and other organs, muscles, etc.). For example, cells of innate or adaptive immunity, for example, bone marrow or lymphocytes, are usually T cells, NK cells, macrophages, etc. Preferably, according to the present invention, immune cells include T cells, NK cells and macrophages. The immune cells of the present invention can also be differentiated from immune cell progenitor cells, such as lymphoid progenitor cells.

[0234] "Pluripotent stem cells" are generally human embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC). Human ESCs are isolated from blastocysts during the development of human fertilized eggs, and can also be isolated from embryos obtained by nuclear transplantation (embryonic stem cells within 14 days of fertilization that have not undergone in vivo development); human iPSCs can be reprogrammed from human cells by overexpressing specific transcription factors (such as OCT4, SOX2, KLF4, MYC). Both human ESCs and iPSCs express OCT4, NANOG, Tra-1-60, SSEA-4, and do not express SSEA-1. A notable feature of pluripotent stem cells is that they can differentiate into three germ layer tissues or cells in vitro or in vivo.

[0235] "Hematopoietic stem cells" in the present invention refer to CD34 + These cells can be isolated from peripheral blood, umbilical cord blood, placental blood, bone marrow, spleen, lymph or other tissues, or differentiated from pluripotent stem cells.

[0236] "Progenitor cells" can differentiate into various types of immune cells, such as myeloid stem cells, lymphoid stem cells, and further, also refer to erythroid stem cells, granulocytes, monocytic stem cells, megakaryotic stem cells, precursor B cells, precursor T cells, NK progenitor cells, and macrophage progenitor cells.

[0237] As used herein, "NK cells" refer to natural killer cells, which are generally CD56 + CD45 + Double positive, expressing multiple activating receptors at the same time, such as NKG2D, NKp44, NKp46, etc. NK cells can be differentiated from pluripotent stem cells (such as iPSC), or isolated from peripheral blood, or differentiated from CD34+ cells (such as umbilical cord blood or placental blood), or obtained from tumor cell lines (such as NK92).

[0238] In this article, "T cells" refers to CD4 + or CD8 + T cells, or double negative T cells (CD3 + CD4 - CD8 - ), or γδT cells (expressing γδTCR), or NKT cells and NKT-like cells (CD3 + CD56 +). The cell can be a naive T cell (TN cell), a T memory stem cell (TSCM cell), a memory T cell (TCM cell), a regulatory T cell (Treg cell), a TCR-T cell, a tumor infiltrating lymphocyte (TIL) or an effector memory T cell (TEM cell) and a combination. "CBNK" refers to NK cells derived from cord blood, and "WTiNK" refers to NK cells derived from wild-type iPSCs. "Anti-BCMACAR-iNK" refers to NK cells expressing anti-BCMACARs derived from iPSCs. "Anti-GPRC5D CAR-iNK" refers to NK cells expressing anti-GPRC5D CARs derived from iPSCs.

[0239] As used herein, the term "composition" is in a form that permits the biological activity of the active ingredients to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0240] As used herein, the term "treat," ...

[0241] As used herein, the term "diagnosis" refers to the identification, disclosure, identification and / or definition of the location of the pathogenic process of a pathological state, disease or condition. In some specific embodiments, the pharmaceutical composition of the present invention, when administered to a subject or contacted with a sample from a subject, helps diagnose cancer, tumor formation or condition.

[0242] In this article, autoimmune diseases are diseases caused by the immune system mistakenly attacking the body's own tissues, and there are more than 80 known autoimmune diseases. These diseases can be divided into systemic autoimmune diseases and organ-specific autoimmune diseases. The autoimmune diseases include, but are not limited to, at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, arthritis syndrome, autoimmune anemia, rheumatoid heart disease, Sjögren's syndrome, systemic vasculitis, pemphigus, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, Hashimoto's thyroiditis, type 1 diabetes and multiple sclerosis.

[0243] In this article, viral infectious diseases refer to diseases caused by viral infection, and viral infection refers to the process in which viruses invade the body through various pathways and proliferate in susceptible host cells. The viral infectious diseases include but are not limited to EBV infectious diseases.

[0244] In many embodiments, the terms "individual", "subject" and "patient" are used interchangeably, regardless of whether the subject has been or is currently receiving any form of treatment. As used herein, the terms "individual", "subject" or "patient" refer to a mammalian individual, subject or patient. Unless otherwise indicated, the terms "individual", "patient" or "subject" are used interchangeably herein. Exemplary individuals include, but are not limited to, humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, birds, goats and sheep. In certain embodiments, the individual is a human. In some embodiments, the individual is a person suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0245] As used herein, the term "tumor antigen" is a general term for newly appearing and highly expressed proteins and polypeptide molecules during the process of malignant transformation of cells. According to the relationship between tumor antigens and tumors, tumor antigens are divided into two categories: "tumor-specific antigens" (TSA) and "tumor-associated antigens" (TAA). TSA refers to antigens that are unique to tumor cells and do not exist on normal tissue cells. TAA refers to antigens that are not unique to tumor cells and can also exist on normal tissue cells, especially embryonic tissues. Therefore, between tumor cells and normal tissues, TAA only shows quantitative changes. In terms of the source or characteristics of the antigen, TAA is generally a protein expressed by a tumor induced by chemical and physical carcinogens, a protein expressed by a tumor induced by a virus, an abnormal protein encoded by an oncogene and a mutant tumor suppressor gene, or a protein expressed after the activation of a dormant gene. TAAs are generally embryonic proteins, differentiation proteins, highly expressed oncogene-encoded proteins, or over- or abnormally expressed glycolipids and glycoproteins (Principles of Immunology (4th edition), edited by Zhou Guangyan, Beijing: Science Press, 2018.1; ISBN978-7-03-053131-5; p339).

[0246] Chimeric Antigen Receptor

[0247] The present invention provides a chimeric antigen receptor, which comprises:

[0248] An extracellular domain, which is capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0249] A transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and

[0250] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0251] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0252] SNLFVASWIAVMX8IFX9IGX 10 AX 11 AX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 PS

[0253] Wherein, X8 is selected from I or V;

[0254] X9 is selected from R or H;

[0255] X 10 Select from M or L,

[0256] X 11 Selected from V or I;

[0257] X 12 Selected from I or V,

[0258] X 13 Selected from S or F;

[0259] X 14 is selected from C, R or H;

[0260] X 15 Selected from C, T or V,

[0261] X 16 Selected from V, L or absent;

[0262] X 17 Select from F or H,

[0263] X 18 Selected from F or Y,

[0264] X 19 Selected from L or absent,

[0265] X 16 and X 19 Cannot exist at the same time, and X 16 and X 19 Can't exist at the same time.

[0266] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0267] The present invention further optimizes the amino acid sequence of the transmembrane domain. When the transmembrane domain of the chimeric antigen receptor has the amino acid sequence in the above general formula, the engineered immune cells containing the chimeric antigen receptor have a higher ability to kill target cells (cells targeted and bound by the extracellular domain).

[0268] According to an embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in any one of SEQ ID NOs: 19-23, 27-29.

[0269] The present invention provides a chimeric antigen receptor (CAR), comprising:

[0270] an extracellular domain capable of binding to a tumor antigen;

[0271] A transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and

[0272] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0273] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0274] SNLFVASWIAVMX1IFX2IGMAX3AIX4X5CX6FFX7PS

[0275] Wherein, X1 is selected from I or V;

[0276] X2 is selected from R or H;

[0277] X3 is selected from V or I;

[0278] X4 is selected from S or F;

[0279] X5 is selected from C or R;

[0280] X6 is selected from L or is absent;

[0281] X7 is selected from L or is absent,

[0282] The tumor antigen may be a tumor-specific antigen or a tumor-associated antigen.

[0283] According to an embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in any one of SEQ ID NOs: 19 to 21 and SEQ ID NO: 23.

[0284] The present invention provides a chimeric antigen receptor, which comprises:

[0285] An extracellular domain, which is capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0286] A transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and

[0287] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0288] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0289] SNLFVASWIAVMIIFX 20 IGX 21 AX 22 AX 23 SX 24 X 25 X 26 X 27 X 28 PS

[0290] Among them, X 20 Selected from R or H,

[0291] X 21 Select from M or L,

[0292] X 22 Selected from V or I,

[0293] X 23 Selected from V or I,

[0294] X 24 is selected from C, R or H,

[0295] X 25 Selected from C, T or V,

[0296] X 26 Selected from L or V,

[0297] X 27 Select from F or H,

[0298] X 28 Selected from F or Y;

[0299] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0300] The present invention further optimizes the amino acid sequence of the transmembrane domain. When the transmembrane domain of the chimeric antigen receptor has the amino acid sequence in the above general formula, the engineered immune cells containing the chimeric antigen receptor have a higher ability to kill target cells (cells targeted and bound by the extracellular domain).

[0301] According to an embodiment of the present invention, the transmembrane domain has an amino acid sequence as shown in any one of SEQ ID NO: 22 and SEQ ID NO: 27-29.

[0302] According to an embodiment of the present invention, the chimeric antigen receptor proposed above may also include at least one of the following additional technical features:

[0303] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize a tumor antigen, an inflammatory factor or a pathogen antigen.

[0304] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize at least one of the following: CD19, BCMA, GPRC5D, CD123, CD138, CD20, CD22, CD30, CD33, CD38, CD4, CD7, CLL-1, CD10, CD34, CS1, CD16, CD5, IL-1-RAP, ITGB7, k-IgG, TACI, TRBC1, MUC1, NKG2DL, PD-L1, CD133, CD117, LeY, CD70, ROR1, CD147, CD171, CD267, AXL, B7-H3, C D44v6, CEA, CAIX, CLDN18.2, CLDN6, DLL3, DR5, EGFR, EpCAM, ErbB, FAP, FBP, FRα, GD2, gp100, HER2, IL-13Rα2, LFA1, LMP1, MAGE-A3, MAGE-A1, MAG E-A4, MART1, MET, MG7, MMP2, MSLN, MUC1, MUC16, MUC16ecto, NECTIN4, NKG2D, PMEL, PSCA, PSMA, ROR1, ROR2, TM4SF1, TnMUC1, VEGFR2, CLD18, and GPC3.

[0305] According to an embodiment of the present invention, the extracellular domain further comprises an antibody or an antigen-binding fragment thereof capable of specifically recognizing a pathogen antigen, and the pathogen comprises EBV.

[0306] According to an embodiment of the present invention, the extracellular domain also includes an antibody or an antigen-binding fragment thereof that can specifically recognize inflammatory factors, and the inflammatory factors include at least one of the following: IL-1, TNF-α, IFN-γ, IL-12, IL-18, GMCSF, IL-6, IL-8, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16 and CCL2-2.

[0307] According to an embodiment of the present invention, the extracellular domain includes an antibody or an antigen-binding fragment thereof that can specifically recognize GPRC5D.

[0308] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 90% identity therewith:

[0309] Heavy chain variable region CDR sequences: SEQ ID NOs: 1-3;

[0310] The CDR sequence of the light chain variable region is: SEQ ID NO: 4, the amino acid sequence SAS and SEQ ID NO: 5.

[0311] According to a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically recognizes GPRC5D provided by the present invention comprises:

[0312] The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NO: 1, 2 and 3, respectively, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, 2 and 3, and the light chain variable region CDR1, CDR2, CDR3 sequences are shown in SEQ ID NO: 4, the amino acid sequence SAS and SEQ ID NO: 5, respectively, or an amino acid sequence that is at least 90% identical to SEQ ID NO: 4, the amino acid sequence SAS and SEQ ID NO: 5.

[0313] It should be noted that any antibody or antibody fragment having the above heavy chain variable region CDR1, CDR2, CDR3 sequence and light chain variable region CDR1, CDR2, CDR3 sequence is included in the protection scope of the present invention, such as IgG, scFv, Fab, Fab', etc.

[0314] According to a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically recognizes GPRC5D has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:6; and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:7.

[0315] According to a preferred embodiment of the present invention, the antibody is a single-chain antibody having an amino acid sequence as shown in SEQ ID NO: 8. The GPRC5D single-chain antibody of the present invention is also abbreviated as GPRC5D scFV.

[0316] According to an embodiment of the present invention, the extracellular domain comprises a bispecific binding molecule comprising:

[0317] A first binding region, wherein the first binding region comprises the antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D; and

[0318] The second binding region has the activity of binding to an antigen specifically expressed on lymphocytes.

[0319] According to an embodiment of the present invention, antibodies that have the ability to bind to antigens specifically expressed on lymphocytes are all included within the scope of the second binding region species of the bispecific binding molecules of the present invention.

[0320] According to a specific embodiment of the present invention, the antigen specifically expressed on the lymphocytes includes at least one selected from BCMA, CD38, CD138, CD3E, and FCGR3A. The antigen specifically expressed on the lymphocytes mentioned in the present invention is not limited to the above types, and can also be any type of antigen specifically expressed on lymphocytes known in the art, including the aforementioned antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D and a bispecific binding molecule having the activity of binding to an antigen specifically expressed on lymphocytes, which are all included in the protection scope of the present invention. Preferably, the second binding region can be a complete antibody or a single-chain antibody or a single-domain antibody.

[0321] According to a preferred embodiment of the present invention, the second binding region has BCMA binding activity, and the second binding region comprises a single-chain antibody or a single-domain antibody that can specifically recognize BCMA.

[0322] According to a preferred embodiment of the present invention, the second binding region has an amino acid sequence as shown in SEQ ID NO:9.

[0323] According to a preferred embodiment of the present invention, the bispecific binding molecule comprises an antibody or an antigen-binding fragment thereof that can specifically recognize GPRC5D and a single-chain antibody or a single-domain antibody that can specifically recognize BCMA, and the GPRC5D single-chain antibody and the single-chain antibody or the single-domain antibody of BCMA are connected by a connecting peptide, and can target and kill multiple myeloma cells. Preferably, the bispecific binding molecule has an amino acid sequence as shown in SEQ ID NO: 10.

[0324] In the present invention, CAR includes an extracellular targeting portion that can recognize GPRC5D protein, and may also include a hinge domain. The hinge domain may be located between the extracellular antigen recognition domain and the transmembrane domain. For example, the hinge domain may include a hinge domain 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, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region may be a hinge region derived from CD8. For example, the hinge domain has an amino acid sequence as shown in SEQ ID NO: 11.

[0325] In the present invention, the intracellular domain includes a costimulatory signal domain and an intracellular signaling domain, and the costimulatory signal domain and the intracellular signaling domain are connected by a connecting peptide. The CAR includes an extracellular targeting portion that can recognize the GPRC5D protein, and may also include a costimulatory signal domain. The costimulatory signal domain can provide a stimulation signal. For example, the costimulatory signal domain may include an intracellular costimulatory 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, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligands, CD40 and MyD88. For example, the costimulatory signal region may be an intracellular costimulatory signal region derived from 2B4. For example, the costimulatory signal domain has an amino acid sequence as shown in SEQ ID NO:12.

[0326] It should be noted that there is no particular limitation on the selection of the "connecting peptide" in the present invention, and it can be (GGGGS) n In the form of a connecting peptide, n can be 4 to 10. All connecting peptide forms known in the art that can be used to connect functional amino acids and do not affect the functions of the functional amino acids themselves are included in the protection scope of the present invention and can be used as the connecting peptide in the present invention.

[0327] In the present invention, the intracellular signaling domain comprises a domain having at least one ITAM motif to transmit the activation signal to the interior of the cell. For example, the intracellular signaling domain may comprise an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, DAP10, DAP-12 and other domains comprising at least one ITAM. For example, the intracellular signaling domain may be a signaling domain derived from CD3ζ. For example, the intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO: 13.

[0328] In the present invention, the CAR may further comprise a signal peptide at the N-terminus of the targeting portion that binds to the GPRC5D protein. For example, the signal peptide may be a signal peptide derived from a CD8 protein. For example, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 14.

[0329] According to a specific embodiment of the present invention, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO: 15 or SEQ ID NO: 16, wherein the transmembrane domain of the chimeric antigen receptor in the present invention can use the transmembrane domain shown in SEQ ID NO: 18 to 23 according to different experimental needs.

[0330] According to a specific embodiment of the present invention, the polypeptide encoding the anti-GPRC5D CAR comprises: a) an extracellular antigen domain comprising a GPRC5D binding portion, wherein the GPRC5D binding domain may comprise a single-chain variable region fragment (scFV) or sdAb of anti-GPRC5D; b) a hinge region; c) a transmembrane domain; d) a co-stimulatory signaling domain; and (d) an intracellular signaling domain,

[0331] The polypeptide encoding the anti-GPRC5D CAR, wherein the amino acid sequence of the anti-GPRC5D scFV is shown in SEQ ID NO: 8.

[0332] According to a specific embodiment of the present invention, from the N-terminus to the C-terminus, the CAR may sequentially comprise a targeting portion that binds to the GPRC5D protein (e.g., the GPRC5D scFV of the present invention), the hinge region, the transmembrane domain, the co-stimulatory signaling domain, the intracellular signaling domain ( Figure 1 (as shown in Figure A).

[0333] According to a more specific embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise the GPRC5D scFV, a hinge region derived from CD8, a transmembrane domain derived from reverse NKG2D, a co-stimulatory signaling domain derived from 2B4, and an intracellular signaling domain derived from CD3ζ.

[0334] In the present invention, from the N-terminus to the C-terminus, the CAR may sequentially comprise a targeting portion that binds to the GPRC5D protein (e.g., the GPRC5D scFV of the present invention) and a targeting portion that binds to a second antigen (antigen 2) (e.g., anti-antigen 2 scFV or sdAb), the hinge region, the transmembrane domain, the costimulatory signaling domain, the intracellular signaling domain, such as Figure 1 As shown in Figure B. The anti-antigen 2 scFV or sdAb can be a scFV or sdAb that targets and binds to an antigen specifically expressed on lymphocytes.

[0335] For example, from N-terminus to C-terminus, the CAR may sequentially comprise the GPRC5D scFV and scFV or sdAb that can bind to antigen 2, a hinge region derived from CD8, a transmembrane domain derived from NKG2D in reverse, a costimulatory signaling domain derived from 2B4, and an intracellular signaling domain derived from CD3ζ.

[0336] According to a more specific embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a signal peptide, a GPRC5D scFV, a BCMA scFV or a BCMA sdAb, the hinge region, the transmembrane domain, the co-stimulatory signal region and the intracellular signal region.

[0337] The BCMA sdAb has the amino acid sequence shown in SEQ ID NO:9.

[0338] Patent CN 109153731 B provides an anti-BCMA sdAb sequence and CAR molecule, and assembles them on T cells to develop CAR-T therapy for multiple myeloma. The present invention compares the performance of the anti-BCMA sdAb sequence and the anti-GPRC5DscFV sequence and CAR on NK cells after assembly, and finds that the anti-GPRC5D CARNK obtained by the present invention has better killing activity against MM tumor cell lines than anti-BCMACAR NK, that is, the anti-GPRC5D CAR NK cells provided by the present invention have better anti-tumor activity. Further, the anti-BCMA antibody sequence is introduced into the extracellular domain of the anti-GPRC5D CAR (anti-GPRC5D / BCMACAR), and it is found that the anti-tumor activity of the anti-GPRC5DCAR-iNK can be further improved.

[0339] Polynucleotides, expression vectors, cells and compositions

[0340] The present invention provides an isolated polynucleotide encoding the chimeric antigen receptor described above.

[0341] The present invention provides an expression vector, which carries the polynucleotide described above.

[0342] The present invention provides a cell, which carries the aforementioned polynucleotide or the aforementioned expression vector or is capable of expressing the aforementioned chimeric antigen receptor. According to an embodiment of the present invention, the cell can efficiently express the aforementioned chimeric antigen receptor under appropriate conditions.

[0343] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the chimeric antigen receptor described in this application. It is easy for those skilled in the art to understand that conditions suitable for the expression of the chimeric antigen receptor include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the chimeric antigen receptor according to the specific environment of the laboratory.

[0344] The nucleic acid of the antibody, or antigen-binding fragment thereof, or the bispecific binding molecule described above, or the chimeric antigen receptor described above can be introduced into cells using different tools, such as donor plasmid, piggyBac, dsDNA, ssDNA, various integrating or non-integrating viruses, etc. If necessary, according to the selection of different introduction tools, PBase can be selected to assist piggyBac introduction, and nuclease can be selected to form single-strand or double-strand breaks to assist other non-viral introduction methods.

[0345] The cell provided by the present invention may be a recombinant cell carrying the aforementioned polynucleotide or the aforementioned expression vector, or may be an engineered immune cell capable of expressing the aforementioned chimeric antigen receptor.

[0346] According to an embodiment of the present invention, immune cells with enhanced functions can be obtained by using any one of the following methods or a combination thereof:

[0347] 1) Randomly integrate one or more circular or linear exogenous DNA fragments into target cells;

[0348] 2) One or more circular or linear exogenous DNA fragments are site-specifically integrated into the target cell. Site-specific integration can improve the efficiency of homologous recombination of exogenous DNA by introducing nucleases into the target site; the efficiency of homologous recombination can also be improved by other physical means, such as electroporation or nuclear transfection.

[0349] 3) one or more endonucleases that can recognize specific sites introduce one or more double-strand breaks into target cells at the specific sites, and the target cells generate base insertions or base deletions at the specific sites by non-homologous end joining;

[0350] 4) One or more nucleases that can recognize specific sites introduce one or more double-strand breaks into target cells at specific sites, and exogenous circular or linear DNA fragments are provided at the same time, so that the cells produce base insertions or base deletions at the selected sites through homologous recombination.

[0351] At this time, the target cell is the immune cell or stem cell or progenitor cell mentioned above, or a donor cell for reprogramming into iPSC, or any derivative cell in the process of differentiation of stem cells or progenitor cells into immune cells. The exogenous DNA fragment may contain a gene expression frame, that is, at least components such as a promoter and an open reading frame; or it may not contain a gene expression frame.

[0352] According to an embodiment of the present invention, the promoter in the gene expression frame can be CAG, CMV, EF1α, PGK, UBC or other constitutive, inducible, time, tissue- or cell type-specific promoters; the open reading frame in the gene expression frame includes one or more polypeptides or proteins to form a chimeric antigen receptor or enhance immune cell function. The exogenous DNA fragment insertion site can be the AAVS1 site or other sites that are not affected by the insertion of exogenous genes and affect cell function, or a site where the gene of the target site is destroyed after insertion, or a stop codon position of a functional gene.

[0353] The construction of CAR expression vector can be in various forms, such as Figure 1 and 2 As shown, the 2A or IRES sequence can be inserted into the exon position of the target gene in-frame, and the promoter of the target gene can be used to start expression ( Figure 1 In some embodiments, the expression cassette may further include a promoter and a polyadenylation sequence ( Figure 1 D in the middle). When integrated into the genome, the expression cassette uses the expression elements it carries to start expression. The expression cassette can also express multiple polypeptides simultaneously, wherein two or more polypeptide coding sequences can be connected by 2A or IRES sequences. As an example, an expression cassette comprising a promoter, anti-GPRC5D CAR-2A / IRES-hnCD16, and polyadenylation, anti-GPRC5D CAR and hnCD16 can be simultaneously expressed under the drive of an exogenous promoter ( Figure 1 (Fig. E).

[0354] In some embodiments, the genetically modified immune cells or cells derived therefrom produced by the above method express genetically engineered antigen receptors, which may be chimeric antigen receptors (CARs), T cell receptors (TCRs), or NK cell receptors (NCRs).

[0355] In some embodiments, the genetically modified immune cells or cells derived therefrom produced using the methods provided herein, wherein the immune cells are NK cells, and the cells derived therefrom are pluripotent stem cells or cells derived therefrom, in particular, wherein the pluripotent stem cells are iPSCs, cloned iPSCs or iPSC cell lines, and the cells derived therefrom include CD34 + Cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells (HSPC), hematopoietic multipotent progenitor cells, NK cell progenitor cells, NK cells. The source cells can also be NK cells, NK progenitor cells, hematopoietic multipotent progenitor cells, CD34 isolated from peripheral blood, umbilical cord blood, or other tissues (including tumor tissues) or organs. + Cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells.

[0356] In some embodiments, the genetically modified immune cells or cells derived therefrom produced using the methods provided herein, wherein the immune cells are T cells, including CD4 + or CD8 + T cells, or CD3 + CD4 - CD8 - T cells, or CD3 + CD56 + T cells. Preferred cells can be selected from naive T cells (TN cells), T memory stem cells (TSCM cells), memory T cells (TCM cells), regulatory T cells (Treg cells), TCR-T cells, tumor infiltrating lymphocytes (TIL) or effector memory T cells (TEM cells) and combinations thereof. The source cells are pluripotent stem cells or hematopoietic stem cells derived therefrom, in particular, wherein the pluripotent stem cells are iPSCs, cloned iPSCs or iPSC cell lines, and the derived cells of the pluripotent stem cells include CD34 + Cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells (HSPC), hematopoietic multipotent progenitor cells, T cell progenitor cells, T cells, NKT cells. The source cells can also be T cells, T cell progenitor cells, hematopoietic multipotent progenitor cells, CD34 isolated from peripheral blood, umbilical cord blood, or other tissues (including tumor tissues) or organs. +Cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells.

[0357] In some embodiments, the genetically modified immune cells or cells derived therefrom produced using the methods provided herein, wherein the immune cells are macrophages, including M1 macrophages and M2 macrophages. The sources thereof include macrophages from various tissues, such as bone marrow, lymph nodes, liver, spleen, lungs, and macrophages from the circulatory system, microglia, etc.; also include macrophages derived from pluripotent stem cells or hematopoietic stem cells derived therefrom, in particular, wherein the pluripotent stem cells are iPSCs, cloned iPSCs or iPSC cell lines, and the derived cells of pluripotent stem cells include CD34 + Cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells (HSPC), hematopoietic multipotent progenitor cells, macrophage progenitor cells, macrophages. The source cells can also be macrophages, macrophage progenitor cells, hematopoietic multipotent progenitor cells, CD34 isolated from peripheral blood, umbilical cord blood, or other tissues (including tumor tissues) or organs. + Cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells.

[0358] In some embodiments, the immune cells of the present invention are embryonic stem cells, induced pluripotent cells (iPSCs), cloned iPSCs or iPSC cell lines, or derived cells obtained by differentiating any of the above embryonic stem cells or iPSCs.

[0359] Any of the above cells encodes at least a polypeptide of a chimeric antigen receptor (anti-GPRC5D CAR or anti-GPRC5D CAR) that can recognize GPRC5D.

[0360] In some embodiments of the derivative cells obtained by differentiation of iPSCs, the derivative cells have an enhanced killing effect on tumor cells expressing GPRC5D compared to corresponding primary cells obtained from peripheral blood, umbilical cord blood or any other tissue or organ.

[0361] In some embodiments in which the iPSC and its derived cells contain at least a chimeric antigen receptor that can recognize GPRC5D, the cells may further contain one or more of the following genome editing: (i) B2M and CIITA knockout or low expression; (ii) introduction of CD47, or HLA-E, or HLA-G (including non-cleavable forms), or expression of any combination thereof; (iii) introduction of expression of high-affinity non-cleavable CD16 (hnCD16) or its variants; (iv) introduction of expression of a second or third CAR; (v) introduction of a second or third sc (vi) TGFBR2, PVR, NKG2A, TIM3, TIGIT, CD38, TCR are missing or their expression is reduced; and (vii) the expression of at least one of 4-1BBL, NKG2D, NKp44, NKp46, CD3, CD47, CD113, CD131, CD137, CD80, CD86, CD68, CD226, PDL1, A2AR, CAR, TCR, Fc receptor, adapter and surface triggering receptor for coupling with universal adapter is introduced or increased.

[0362] In some embodiments of the cells or their populations, one or more cells comprising anti-GPRC5D CAR are primary NK cells or NK cells differentiated from pluripotent stem cells, hematopoietic stem cells and progenitor cells, and NK cell progenitor cells, or their populations, and these cells have at least one of the following characteristics compared to similar cells that have not been genetically modified: they have a stronger ability to kill tumor cells expressing GPRC5D.

[0363] The present invention provides a composition for therapeutic use, which comprises immune cells, preferably NK cells, and other pharmaceutically acceptable excipients. In some embodiments, the subject suitable for cell therapy suffers from at least the following diseases: multiple myeloma, and / or tumor cells expressing GPRC5D in their bodies. The composition may also include clinically acceptable polypeptides, cytokines, growth factors, feeder cells or their replacement factors, immune checkpoint inhibitors, antibody drugs (such as anti-CD38 antibodies), etc. In some embodiments of the composition for therapeutic use, the antibodies used with the provided cells may include daratumumab, and / or at least one of the following drugs: rituximab, veltuzumab, ofatumumab, utoximab, okatumumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, dinutuximab, avelumab, daratumumab, isatuximab, etozinomab.

[0364] The present invention also provides a method for obtaining or producing the above-mentioned cells, which comprises directing differentiation of one or more iPSCs containing a chimeric antigen receptor that can recognize GPRC5D.

[0365] The present application further provides a method for obtaining one or more iPSCs containing anti-GPRC5D CARs as described above. It includes using any of the following gene editing methods or a combination thereof to introduce a DNA sequence encoding an anti-GPRC5D CAR polypeptide into an iPSC: 1) using a homologous recombination method to insert an exogenous DNA fragment into a target site, and targeting the target site can be accomplished by setting homologous sequences on both sides of the exogenous DNA fragment; 2) using a non-homologous recombination method to insert an exogenous DNA fragment into a site of double-strand break in the genome; 3) using tools such as viruses, transposons, and plasmids to randomly integrate exogenous DNA fragments into the host genome.

[0366] The present invention further provides a method for obtaining one or more iPSCs comprising the above-mentioned chimeric antigen receptor polypeptide that can recognize GPRC5D. It includes further performing at least any of the following gene modifications, or a combination thereof: 1) introducing high-affinity non-cleavable CD16 or its variant; 2) knocking out CD38; 3) knocking out B2M and CIITA, and / or PVR; and introducing CD47, and / or HLA-G or non-cleavable HLA-G, and / or HLA-E; 4) membrane-bound IL15 (mbIL15). The above selected gene modifications can be completed at one time or in steps. The introduced exogenous gene expression frame or exogenous polypeptide can be expressed independently or co-expressed in a bicistronic construct; an exogenous promoter can be used to drive expression, or the promoter of an endogenous gene can be used for stable expression or tissue-specific expression. The exogenous promoter can be any of the following promoters or a combination thereof: CAG, CMV, EF1α, PGK, UBC or other constitutive, inducible, time-, tissue- or cell type-specific promoters. In some embodiments, targeted gene modification can be performed using engineered nucleases (such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), homing endonucleases (Meganuclease)), homologous recombination, or any other functional variations of these methods.

[0367] The present invention further provides a method for piggyBac-mediated expression of anti-GPRC5D CAR, so as to obtain the above-mentioned method for one or more iPSCs comprising anti-GPRC5D CAR polypeptides. In some embodiments mediated by piggyBac, the DNA sequence encoding anti-GPRC5D CAR is located in the transposed region of piggyBac (the region between the reverse terminal repeats at both ends). Subsequently, with the assistance of transposase, piggybac randomly integrates the DNA sequence encoding anti-GPRC5D CAR into the host genome TTAA site.

[0368] Figure 2 Figure A in the middle lists several expression cassettes containing anti-GPRC5D CAR polypeptides, which can be cloned into the piggyBac plasmid and randomly integrated into the host genome with the help of PBase. Figure 2 Figure B shows an example of CAG-anti-GPRC5D CAR-BGHpA inserted into the piggyBac transposition region to obtain the plasmid anti-GPRC5D CAR.

[0369] The DNA encoding anti-GPRC5D CAR can be introduced using different tools, such as donor plasmid, piggyBac, dsDNA, ssDNA, various integrative or non-integrative viruses, etc. Depending on the choice of different introduction tools, PBase can be selected to assist piggyBac introduction, and nuclease can be selected to form single-strand or double-strand breaks to assist other non-viral methods of introduction.

[0370] Figure 3 Figure A in the middle shows a schematic diagram of a homologous recombination template containing an anti-GPRC5D CAR. Figure 3 Figure B provides several examples of DNA templates for homologous recombination containing anti-GPRC5D CAR expression cassettes. These DNA templates can be in the form of single-stranded DNA, double-stranded DNA, or plasmids. Figure 3 Figure C in the middle provides an example of a DNA template with an anti-GPRC5D CAR and hnCD16 expression cassette targeting any site in the human genome, as well as the location of the expression cassette in the genome after homologous recombination.

[0371] In the present invention Figure 1-3 The construct forms in are all included in the protection scope of the construct protected by the present invention.

[0372] The present invention further provides CRISPR-mediated gene editing to obtain the above-mentioned method of constructing one or more immune cells or iPSCs containing a chimeric antigen receptor polypeptide that can recognize GPRC5D. In some CRISPR-mediated embodiments, CRISPR binds to the targeting sequence and produces a double-strand break, and further inserts a DNA sequence encoding an anti-GPRC5DCAR at the targeting sequence by non-homologous end joining or homologous recombination. The targeting sequence may be any of the following sites: AAVS1 site, or any of CISH, SOCS1, SOCS2, SOCS4, SOCS5, SOCS6, SOCS7, NKG2A, B2M, CIITA, CD38, TGFBR2, PVR, TIM3, TIGIT, TCR, etc.

[0373] In some CRISPR-mediated embodiments, the editing further comprises expressing any of the following genes at the same site as above, by tandem expression or parallel expression with the anti-GPRC5D CAR polypeptide: hnCD16 or its variants, mbIL15, CD47, HLA-E, HLA-G or non-cleavable HLA-G, tEGFR or an inducible suicide gene (iCaspase9).

[0374] In some CRISPR-mediated embodiments, the editing further includes expressing at least one of the following genes or polypeptides at at least any of the above-mentioned different sites, such as the AAVS1 site, CISH, SOCS1, SOCS2, SOCS4, SOCS5, SOCS6, SOCS7, NKG2A, B2M, CIITA, CD38, TGFBR2, PVR, TIM3, TIGIT, TCR: hnCD16 or its variants, mbIL15, CD47, HLA-E, HLA-G or non-cleavable HLA-G, tEGFR or iCas9.

[0375] In addition, the present invention provides a donor plasmid or donor DNA fragment for engineering cells to obtain genome-edited cells, which comprises: 1) one or more nucleic acid sequences encoding one or more exogenous polypeptides and, 2) left and right homologous arm sequences targeting a target site. Wherein the genome-edited cells are induced pluripotent cells (iPSCs), cloned iPSCs or iPSC cell lines; wherein the genome-edited cells comprise targeted integration of the one or more nucleic acid sequences encoding one or more exogenous polypeptides at the target site; wherein the genome-edited cells are capable of differentiating into CD34 +iPSCs of cells. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous proteins are linked in-frame to and driven by an endogenous promoter of the target locus after integration; or wherein the selected position in the target locus is in an exon of the target gene. In some embodiments, the one or more nucleic acid sequences encoding one or more exogenous polypeptides are driven by an exogenous promoter after integration; wherein the integration position in the target locus is in an exon of the target gene.

[0376] In some embodiments, the present invention provides a piggybac plasmid for engineering cells to obtain genome-edited cells, comprising: one or more nucleic acid sequences encoding one or more exogenous polypeptides. Wherein the exogenous polypeptides include anti-GPRC5D CAR, and / or at least one of hnCD16 or its variants, CD47, HLA-E, HLA-G or non-cleavable HLA-G, tEGFR, iCas9, mbIL15. The genome-edited cells are induced pluripotent cells (iPSC), cloned iPSC or iPSC cell lines; wherein the genome-edited cells contain the random integration of the one or more nucleic acid sequences encoding one or more exogenous polypeptides in the host cell genome; wherein the genome-edited cells are capable of differentiating into CD34 + iPSC cells.

[0377] In some embodiments using donor plasmids, donor DNA fragments, or piggyBac, the gene-edited iPSC-derived blood cells comprise derived CD34 + Cells, derived hematopoietic stem cells and progenitor cells, derived hematopoietic multipotent progenitor cells, derived T cell progenitor cells, derived NK cell progenitor cells, derived T cells, derived NKT cells, derived NK cells or derived B cells. In one embodiment, the hematopoietic cells derived from the genome-edited iPSC obtained by differentiation include derived NK cells or derived T cells, and compared with their native corresponding cells obtained from peripheral blood, umbilical cord blood or any other donor tissue, have at least one of the following characteristics, the characteristics at least including: (1) specific killing of tumor cells expressing GPRC5D; (2) acquisition or enhancement of ADCC effect.

[0378] The present invention further provides a composition for making gene-edited iPSC or differentiated hematopoietic cells derived therefrom, and the composition comprises a donor plasmid, or a donor DNA, or a piggyBac plasmid as described herein. In addition, a kit is provided herein, and the kit comprises a donor plasmid, or a donor DNA fragment, or a piggyBac plasmid as described herein, and one or more culture media for iPSC culture, maintenance or differentiation. Further provided are donor plasmids, or donor DNA, or piggyBac plasmids and host cells for use in drugs.

[0379] The present invention provides a method for producing genome-edited iPSCs or hematopoietic cells derived therefrom using the donor plasmid, or donor DNA fragment, or piggyBac plasmid as described, the method comprising: 1) introducing the donor plasmid, or donor DNA fragment, or piggyBac plasmid into iPSC, and 2) culturing the iPSC for a sufficient time to obtain genome-edited iPSCs. In some embodiments, the method may further comprise guiding the resulting genome-edited iPSCs to differentiate into hematopoietic cells, the hematopoietic cells comprising the same genotype as the genome-edited iPSCs. In some embodiments of the method, the method further comprises introducing a CRISPR system, ZFN, TALEN, homing nuclease, or any other functional variant thereof.

[0380] The anti-tumor ability of effector immune cells (such as NK cells, T cells, macrophages, etc.) in adoptive cell therapy is still relatively limited in vitro and in vivo, and it is necessary to enhance cell function through engineering methods, so as to benefit tumor patients. One of the purposes of the present invention is to prepare engineered immune cells based on GPRC5D antibodies or bispecific binding molecules containing GPRC5D antibodies as extracellular regions. These immune cells can be separated from peripheral blood, umbilical cord blood or specific tissues or organs (including tumor tissues or other organs) of patients or donors, or differentiated from pluripotent stem cells (such as induced pluripotent stem cells (iPSC) or embryonic stem cells (embryonic stem cells within 14 days of fertilization without in vivo development)), hematopoietic stem cells, or various immune cell progenitor cells. The engineering is mainly completed by genetic modification. The engineering can be completed at the stem cell stage or the progenitor cell stage, or at the immune cell stage. The engineering includes one or more genetic modifications, including DNA insertion, deletion, and replacement. These genetic modifications can be completed at one time or in several steps in different orders. Genetic modifications completed at the stem cell or progenitor cell stage are retained and maintain the corresponding functions in the derived cells produced during subsequent differentiation, expansion, passaging or transplantation.

[0381] Kit for detecting GPRC5D

[0382] According to a specific embodiment of the present invention, the present invention provides a kit for detecting GPRC5D, the kit comprising the above-mentioned antibody or antigen-binding fragment thereof and / or the above-mentioned chimeric antigen receptor. The antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D or a bispecific binding molecule containing the GPRC5D antibody can bind to GPRC5D in the sample to be tested, thereby achieving the purpose of in vitro detection of GPRC5D levels.

[0383] Uses and methods

[0384] According to a specific embodiment of the present invention, the present invention proposes the use of the chimeric antigen receptor, polynucleotide, expression vector, cell and / or composition described above in the treatment of individual blood tumors or solid tumor diseases, autoimmune diseases or viral infectious diseases. According to an embodiment of the present invention, through an antibody or antigen binding fragment thereof that can specifically recognize GPRC5D or a bispecific binding molecule containing a GPRC5D antibody, the chimeric antigen receptor can bind to GPRC5D and the second antibody in the bispecific binding molecule, thereby effectively treating blood tumors or solid tumor diseases, autoimmune diseases or viral infectious diseases.

[0385] According to an embodiment of the present invention, the blood tumor or solid tumor disease is a GPRC5D-related disease. The chimeric antigen receptor can effectively treat GPRC5D-mediated related diseases.

[0386] According to a specific embodiment of the present invention, the GPRC5D-related diseases include but are not limited to multiple myeloma. According to a specific embodiment of the present invention, the chimeric antigen receptor can effectively treat multiple myeloma.

[0387] According to an embodiment of the present invention, the autoimmune disease includes but is not limited to at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, arthritis syndrome, autoimmune anemia, rheumatoid heart disease, Sjögren's syndrome, systemic vasculitis, pemphigus, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, Hashimoto's thyroiditis, type 1 diabetes and multiple sclerosis.

[0388] According to an embodiment of the present invention, the viral infectious disease includes but is not limited to EBV infectious disease.

[0389] According to a specific embodiment of the present invention, the present invention provides a method for preventing and / or treating an individual's blood tumor or solid tumor disease, or an autoimmune disease or a viral infectious disease, the method comprising administering to the individual at least one of the following: the chimeric antigen receptor, polynucleotide, expression vector, cell and composition described above. As described above, through an antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D or a bispecific binding molecule containing a GPRC5D antibody, the chimeric antigen receptor can bind to GPRC5D and the second antibody in the bispecific antibody, thereby effectively treating the blood tumor or solid tumor disease, autoimmune disease or viral infectious disease. The effect of preventing and / or treating blood tumors or solid tumors, autoimmune diseases or viral infectious diseases will not be repeated here.

[0390] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0391] According to an embodiment of the present invention, the autoimmune disease includes but is not limited to at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, arthritis syndrome, autoimmune anemia, rheumatoid heart disease, Sjögren's syndrome, systemic vasculitis, pemphigus, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, Hashimoto's thyroiditis, type 1 diabetes and multiple sclerosis.

[0392] According to an embodiment of the present invention, the viral infectious disease includes but is not limited to EBV infectious disease.

[0393] According to a specific embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0394] According to a specific embodiment of the present invention, the present invention proposes the use of the chimeric antigen receptor, polynucleotide, and expression vector described above in detecting GPRC5D or diagnosing whether an individual suffers from a GPRC5D-related disease. As described above, the chimeric antigen receptor of the embodiment of the present invention can effectively bind to GPRC5D, so the chimeric antigen receptor can be used to detect GPRC5D, and GPRC5D mediates a variety of diseases. Therefore, based on the level of GPRC5D contained in a biological sample from an individual, it can be determined whether the individual suffers from a GPRC5D-mediated disease.

[0395] According to a specific embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0396] According to a specific embodiment of the present invention, the present invention provides a method for detecting GPRC5D, the method comprising using at least one of the following to detect GPRC5D in a sample to be tested: the chimeric antigen receptor, polynucleotide and expression vector described above. As described above, the chimeric antigen receptor of the embodiment of the present invention can effectively bind to GPRC5D, and therefore, the chimeric antigen receptor can be used to detect GPRC5D.

[0397] The method for detecting GPRC5D according to the embodiment of the present invention can be used in scientific research, and the sample includes but is not limited to tissue, cell, blood, serum, plasma, urine, feces, saliva or sweat.

[0398] According to a specific embodiment of the present invention, the present invention proposes a method for diagnosing whether an individual suffers from a GPRC5D-related disease, the method comprising detecting GPRC5D in a sample derived from the individual to be tested using at least one of the following: the chimeric antigen receptor, polynucleotide and expression vector described above. As described above, the chimeric antigen receptor of the embodiment of the present invention can effectively bind to GPRC5D, and therefore, the chimeric antigen receptor can be used to detect GPRC5D. GPRC5D mediates a variety of diseases, and therefore, it can be determined whether the individual suffers from a GPRC5D-mediated related disease based on the level of the GPRC5D contained in the biological sample from the individual. In addition, the above-mentioned substance can also be used to monitor the content of GPRC5D in the sample to be tested from the individual, that is, the above-mentioned substance can also be used to stage the disease of individuals suffering from GPRC5D-mediated related diseases, and can also be used for the prognosis evaluation of GPRC5D-mediated related diseases.

[0399] According to a specific embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma. According to a specific embodiment of the present invention, the above method can effectively diagnose, stage or evaluate the prognosis of whether an individual suffers from a GPRC5D-related disease.

[0400] According to a specific embodiment of the present invention, the sample includes but is not limited to tissue, cell, blood, serum, plasma, urine, feces, saliva or sweat.

[0401] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will appreciate that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. Where the manufacturers of reagents or instruments are not indicated, they are all conventional products that can be obtained commercially.

[0402] Example 1 Anti-GPRC5D CAR polypeptide structure

[0403] Through hybridoma cell screening technology, an anti-GPRC5D antibody that specifically binds to GPRC5D was obtained. Based on the amino acid sequence of the anti-GPRC5D antibody, the inventors obtained two anti-GPRC5D CAR polypeptides, as follows (1) and (2), respectively.

[0404] (1) Figure 1 As shown in Figure A, the anti-GPRC5D CAR structure includes the antigen recognition domain (anti-GPRC5D scFV), hinge region, transmembrane domain, co-stimulatory signaling domain, and intracellular signaling domain from the N-terminus to the C-terminus of the polypeptide.

[0405] The amino acid sequence of the anti-GPRC5D CAR is shown in SEQ ID NO: 15:

[0406] MALPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLV KPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIFCCFFLPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDV YSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 15).

[0407] The transmembrane domain is underlined herein. In the present invention, the transmembrane domain of the chimeric antigen receptor can use the transmembrane domain shown in any one of SEQ ID NOs: 18 to 23 according to different experimental requirements.

[0408] For example, the amino acid sequence of another anti-GPRC5D CAR is shown in SEQ ID NO: 25 (wherein the amino acid sequence of the transmembrane domain is SEQ ID NO: 18):

[0409] MALPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKSYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLV KPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLSSVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIFCCFFFPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDV YSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:25).

[0410] (2) Figure 1 As shown in Figure B, the anti-GPRC5D CAR structure includes the antigen recognition domain (anti-GPRC5D scFV), BCMA sdAb, hinge region, transmembrane domain, co-stimulatory signaling domain, and intracellular signaling domain from the N-terminus to the C-terminus of the polypeptide.

[0411] The amino acid sequence of GPRC5D scFV-BCMA sdAb CAR is shown in SEQ ID NO: 16, wherein the transmembrane domain is underlined:

[0412] MALPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYK SYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLS SVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAV IGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIFCCFFLPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDV YSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:16).

[0413] In the present invention, GPRC5D scFV-BCMA sdAb CAR can use the transmembrane domain shown in any one of SEQ ID NOs: 18 to 23, 27 to 35 according to different experimental needs. For example, the amino acid sequence of another GPRC5D scFV-BCMA sdAb CAR is shown in SEQ ID NO: 26 (wherein the transmembrane domain amino acid sequence is SEQ ID NO: 18):

[0414] MALPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGQSPKALIYSASYRDSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQYK SYPLTFGQGTRLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSATYSPSLKSRITISRDTSKNQFSLKLS SVTAADTAVYYCARGGIAGRGRWGAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSQVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAV IGWRDISTSYADSVKGRFTISRDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SNLFVASWIAVMIIFRIGMAVAIFCCFFFPS WRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNPARLSRKELENFDV YSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:26).

[0415] Example 2 Anti-GPRC5D CAR molecule introduction

[0416] Will Figure 2 The expression cassette shown in a of Figure A was cloned into the piggyBac vector carrying Puromycin resistance to obtain the plasmid anti-GPRC5D CAR (see Figure 2 B). The transmembrane domain sequence in the CAR molecule of the present invention may have multiple variants. As an example, the transmembrane domain sequence of SEQ ID NO: 18 is used, and the plasmid sequence is shown in SEQ ID NO: 17. Subsequently, the plasmid antiGPRC5D CAR and the plasmid expressing the transposase (PBase) were transfected by a nuclear transfection instrument (Amaxa, II), using a nuclear transfection kit (LONZA, CAT#VPH5022), co-transfected into an iPSC from a healthy person (WT iPSC, Sanqi Bio's own cells). The transfection steps can refer to the instructions for use of the kit. After 2-7 days of screening with 0.5 μg / ml Puromycin, the iPSC expressing anti-GPRC5D CAR molecules (anti-GPRC5D CAR-iPSC) was obtained by amplification and culture. Mouse anti-anti-GPRC5D scFV antibody (primary antibody, Kangyuan Bochuang) and PE Goat anti-mouse IgG (secondary antibody, Biolegend) were used to identify the proportion of iPSCs expressing anti-GPRC5D CAR molecules using FACS (see Figure 5 Figure A in the middle shows that iPSC expressing anti-GPRC5D CAR molecules were obtained. Using the same method, iPSC expressing anti-GPRC5D and BCMA dual antigen CAR molecules (anti-GPRC5D / BCMACAR-iPSC) were obtained.

[0417]

[0418] Example 3 Differentiation of iPSCs expressing anti-GPRC5D CAR polypeptide into CAR-iNK

[0419] Figure 4 The general process of obtaining gene-edited iPSC-derived anti-GPRC5D CAR-iNK is shown. The iPSC expressing the anti-GPRC5D CAR polypeptide obtained in Examples 2 and 3 was differentiated into CAR-iNK by the following specific steps.

[0420] 1) When the confluence of iPSC cells reached 75% to 85%, they were washed once with DMEM / F12 medium, digested with Accutase or TrypLE digestion enzymes in a 37°C, 5% CO2 incubator. Then, centrifuged and the supernatant was discarded, and the cells were cultured with mTeSR supplemented with 5 μM Y27632, 50 ng / mL BMP4, and 10 μM CHIR99021. TM 1. Resuspend the cells in culture medium to obtain single-cell iPSCs;

[0421] 2) Transfer an appropriate number of single-cell iPSCs to AggreWell TM In microplates, follow the AggreWell TM Follow the 400 or 800 instructions to ensure that the single cells transferred into each well are evenly distributed throughout the well. Centrifuge the microplate containing cells at 100g for 3 minutes and culture at 37°C, 5% CO2 and 95% humidity for 1 to 2 days. Then, replace with the second differentiation medium, and thereafter replace the medium every 2 days for 4 to 5 days. The components of the second differentiation medium are: StemPro-34 complete medium supplemented with 1% ITS-X, 1μM β-mercaptoethanol, 50ug / mL ascorbic acid, 1x GlutaMAX, 50ng / mL VEGF, 50ng / mL bFGF, 50ng / mL BMP4, 35nM UM171;

[0422] 3) Then, change to the third differentiation medium. Third differentiation medium: StemPro-34 TM SFM complete medium supplemented with 1% ITS-X, 1 μM β-mercaptoethanol, 50 μg / mL ascorbic acid, 1x GlutaMAX, 50 ng / mL VEGF, 50 ng / mL bFGF, 50 ng / mL SCF, 10 ng / mL FLT3L, and 30 ng / mL TPO.

[0423] 4) After the above three stages of culture, generally on the 10th to 14th day after differentiation, depending on the CD34 + Depending on the FACS results, CD34+ When the ratio exceeds 80%, the fourth differentiation medium can be replaced to further differentiate into NK cells. This process generally lasts for 3-5 weeks. Fourth differentiation medium: DMEM / F12+15% human AB serum+5ng / mL IL-3 (used in the first week)+20ng / mL IL-7+10ng / mL IL-15+10ng / mL FLT3L+50ng / mL SCF. In some cases, the above CAR-iNK can be stimulated by irradiated aAPC (K562 cells expressing membrane-bound IL-21 and 4-1BBL) and further expanded in expansion medium to achieve sufficient cell mass. Expansion medium formula: 5% FBS and 1000IU / mL IL-2, 10ng / mL IL-15 are added to GT-T551 H3 medium.

[0424] Antibodies (including isotype control and secondary antibodies) were purchased from Biolegend except anti-GPRC5D scFV antibody from Kangyuan Broad. The FACS detection steps can refer to the commonly used operating specifications of relevant instruments. The results of using FACS to detect the expression levels of CD56, CD45, NKG2D, TCRαβ, and TCRγ of GPRC5D CAR-iNK are shown in the figure. Figures 6 to 8 As shown, the results showed that the expression of anti-GPRC5D CAR had no adverse effects on the differentiation and expansion of anti-GPRC5DCAR-iNK, and the expression levels of NK-related markers of the two were similar, among which the expression level of NKG2D (NK cell activation receptor) of anti-GPRC5D CAR-iNK was better than that of WT iNK.

[0425] Example 4. Determination of the killing ability of NK cells against tumor cells by luciferase method

[0426] Tumor cells K562-Luc were used as target cells, anti-GPRC5D CAR-iNK, CBNK, WT iNK and anti-BCMACAR-iNK cells were used as effector cells, and the killing ability of NK cells on target cells was detected at an effector-target ratio of 4:1. The specific experimental operation was as follows:

[0427] Prepare K562-Luc target cell suspension (target cell viability is not less than 90%), count and plate cells to the wells of a 96-well plate according to the specified effector-target ratio (fixed effector-target ratio 4:1), and arrange 3 replicates for each effector cell for each effector-target ratio. Adjust the medium of each well to 100 μL, and then add the required number of effector cells anti-GPRC5D CAR-iNK, CBNK, WT iNK and anti-BCMA CAR-iNK according to the specified effector-target ratio. Place the 96-well plate in a 37°C, 5% carbon dioxide incubator and incubate for the specified time (such as 4h). Take the fluorescein substrate and dilute the substrate 100 times with cell culture medium. Add 50 μl of fluorescein substrate diluent to each well and react at room temperature for 10 minutes away from light. Set the light source of the microplate reader to Lum, the time is 1000ms, and read the reading on the machine. Killing efficiency % = (control group-killing group) / cell control group*100. Among them, WT iNK is an iNK cell differentiated from WT iPSC, and the preparation process of anti-BCMACAR-iNK or anti-GPRC5D / BCMACAR-iNK is similar to the above-mentioned anti-GPRC5D CAR-iNK preparation process, the only difference is that the GPRC5D antibody in the anti-GPRC5D CAR polypeptide is replaced with a BCMA antibody (the amino acid sequence of the BCMA antibody (BCMAsdAb) is shown in SEQ ID NO: 24), or a GPRC5D / BCMA bispecific antibody (the amino acid sequence of the GPRC5D / BCMA bispecific antibody is shown in SEQ ID NO: 10).

[0428] QVKLEESGGGLVQAGRSLRLSCAASEHTFSSHVMGWFRQAPGKERESVAVIGWRDISTSYADSVKGRFTISRDDNAKKTLYLQMNSLKPEDTAVYYCAARRIDAADFDSWGQGTQVTVSSGGGGSGGGG SGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFTMGWFRQAPGKEREFVAAISLSPTLAYYAESVKGRFTISRDNAKNTVVLQMNSLKPEDTALYYCAADRKSVMSIRPDYWGQGTQVTVSS(SEQ ID NO: 24).

[0429] The results are as follows Fig. 9 Figure A in the middle shows that different effector cells have similar killing abilities against K562-Luc. Since K562-Luc does not express GPRC5D or BCMA, Fig. 9 Center A shows that these effector cells have similar basal killing abilities against target cells.

[0430] Example 5. Determination of the basic killing activity of effector cells on target cells

[0431] Tumor cells K562-Luc were used as target cells, and anti-GPRC5D CAR-iNK, WT iNK and anti-BCMACAR-iNK cells were used as effector cells. They were mixed at different effector-target ratios (4:1, 2:1, 1:1), and the killing ability of NK cells against K562-Luc cells was determined according to the method in Example 4. Fig. 9 Middle B shows that the basal killing ability of WT iNK, anti-GPRC5D CAR-iNK and anti-BCMACAR-iNK on target cells K562-Luc was dose-dependent.

[0432] Example 6. Anti-GPRC5D CAR molecules enhance iNK cell proliferation and proliferation to GPRC5D + Tumor cell killing activity

[0433] The effector cells (CBNK, WT iNK, GPRC5D CAR-iNK, anti-BCMACAR-iNK, anti-GPRC5D / BCMACAR-iNK) and the target cells NCI-H929-Luc2 (hereinafter referred to as H929-Luc2) highly expressing GPRC5D and BCMA were mixed at a fixed effector-target ratio of 4:1 and different effector-target ratios (8:1, 4:1, 1:1), and unless otherwise specified, they were co-cultured at 37°C and 5% CO2 for 4 hours, and the killing of the target cells H929-Luc2 by iNK cells of different groups was determined using the method provided in Example 4.

[0434] The results are as follows Fig.10 Middle A shows that under the condition of an effector-target ratio of 4:1 and a total incubation of 4 hours, CBNK is insensitive to H929-Luc2 and has basically no killing effect; WT iNK can kill about 10% of H929-Luc2 cells; and anti-GPRC5D CAR-iNK can kill about 90% of H929-Luc cells. Fig.10 Middle B shows that the killing effect of WT iNK and anti-GPRC5D CAR-iNK on H929-Luc2 is dose-dependent and decreases as the effector-target ratio decreases.

[0435] The killing effects of anti-GPRC5D CAR-iNK, anti-BCMACAR-iNK and anti-GPRC5D / BCMACAR-iNK on H929-Luc2 were further compared. The experimental method was also referred to the above Example 4. The results are as follows: Fig.11 The results showed that at the same target-efficiency ratio, the killing effect of CAR-iNK carrying anti-GPRC5D was stronger than that of CAR-iNK carrying anti-BCMACAR. In addition, anti-GPRC5D / BCMACAR-iNK showed better killing effect at a low target-efficiency ratio.

[0436] In summary, the expression of anti-GPRC5D CAR molecules can make NK cells have a much better ability to resist GPRC5D than WT iNK cells or CBNK cells. + The killing ability of CAR-iNK cells was also improved, and the expression of anti-GPRC5D CAR molecules had a better killing effect on the MM tumor cell line H929-Luc2 than the iNK expressing anti-BCMA CAR molecules. After adding the BCMA antigen recognition domain, the killing effect of anti-GPRC5D CAR molecules on the MM tumor cell line H929-Luc2 can be further improved. In addition, after loading anti-GPRC5D or anti-BCMA chimeric antigen receptors at the iPSC stage, these iPSC cells further differentiated into anti-GPRC5D or anti-BCMA CAR-iNK cells, which can specifically kill target cells expressing GPRC5D or BCMA.

[0437] Example 7. Anti-GPRC5D CAR molecules enhance the activity of iNK cells after cryopreservation and resuscitation against GPRC5D + Tumor cell killing activity

[0438] The effector cells of this example are the iNK cells (anti-GPRC5D CAR-iNK and WT iNK) differentiated in Example 3, the target cells are K562-Luc, and the killing activity of the iNK cells against tumor cells after resuscitation is detected.

[0439] First, the iNK cells (anti-GPRC5D CAR-iNK and WT iNK) differentiated in Example 3 were revived. The specific experimental operation was to open the water bath / metal bath in advance and wait for the temperature to rise to 37°C and stabilize. Take out the pre-cooled iNK cell culture medium and pipette 9mL into a new centrifuge tube. Take out the cryopreservation tube from liquid nitrogen, rub your hands a few times, and then quickly transfer it to a 37°C water bath or metal bath, shaking continuously until only a small piece of ice remains in the cryopreservation tube. After passing through the transfer window, put it in a safety cabinet after alcohol disinfection. Gently open the cell cryopreservation tube, and after pipetting with a pipette, gently transfer the cells to a centrifuge tube containing the culture medium and mix well. Mix the cell suspension and centrifuge (300g*5min). Remove the supernatant, add 10mL of NK culture medium again, resuspend and mix, and count. Centrifuge and remove the supernatant. According to the cell number, add NK medium at a density of 1 million cells / mL, mix well, and inoculate into culture flasks / culture plates, or use immediately for cell killing experiments.

[0440] iNK cells were obtained within 72 hours after resuscitation, and the killing activity of the above NK cells against the target cells K562-Luc was determined using the method in Example 4. The results are as follows Fig.12As shown in Figure A, the basal killing ability of WT iNK and anti-GPRC5D CAR-iNK cells after resuscitation showed a dose effect, and the basal killing ability of anti-GPRC5D CAR-iNK was slightly higher than that of WT iNK. Fig.12 Figure B in the middle shows that the resuscitated WT iNK completely lost its ability to kill H929-Luc2, while the resuscitated anti-GPRC5DCAR-iNK still had a good ability to kill H929-Luc2.

[0441] Comparison of the basic killing ability (against K562-Luc) and specific killing ability (against H929-Luc2) of anti-GPRC5D CAR-iNK before and after cryopreservation and thawing showed that the cryopreservation and thawing process had a greater impact on the basic killing ability of anti-GPRC5D CAR-iNK, but had a smaller impact on its specific killing ability (see Fig.13 Figures A and B in the figure).

[0442] We further tested the killing ability of cryopreserved WT iNK and anti-GPRC5D CAR-iNK against other multiple myeloma tumor cell lines, such as Fig.14 The results showed that regardless of whether the target cells were cells expressing GPRC5D in high abundance (MM.1S-Luc2) or cells expressing GPRC5D in low abundance (OPM-2-Luc2), the anti-GPRC5D CAR-iNK after cryopreservation and resuscitation had a high efficiency in killing them, while WT iNK only showed killing ability against MM.1S-Luc2 at a high efficiency target ratio (Effector: Target = 8:1, about 40% killing; Effector: Target = 4:1, about 20% killing).

[0443] The above shows that the cryopreservation and thawing process has little effect on the specific killing ability of anti-GPRC5D CAR-iNK, and anti-GPRC5D CAR-iNK has good killing activity against both target cells expressing GPRC5D in high abundance and low abundance.

[0444] Example 8. Anti-GPRC5D CAR molecule optimization

[0445] In NK cells, the NKG2D transmembrane sequence plays an important role in the specific recognition function of CAR molecules (https: / / doi.org / 10.1016 / j.stem.2018.06.002). In order to further enhance the function of CAR-iNK, the inventors mutated some sites based on the NKG2D transmembrane sequence (SEQ ID NO: 18; TM control) and designed the following NKG2D transmembrane sequence variants (see Table 1, the mutated amino acid sites are underlined) for use as the transmembrane domain of the CAR molecule. The transmembrane domain (SEQ ID NO: 18) in the amino acid sequence SEQ ID NO: 15 of the anti-GPRC5D CAR was replaced with the transmembrane sequence shown in Table 1 to obtain 14 anti-GPRC5D CAR molecule variants (CAR-TM1~14; TM1, TM2, TM3, TM4, TM4, TM5..., TM14 are used below). Using the method in Example 2, a transposon plasmid expressing the above 14 transmembrane sequence variants of the anti-GPRC5D CAR molecule was obtained. Use DNA transfection reagent was used to transfect 2.5 μg of the transposon plasmid prepared above into 293T cells, and the expression of anti-GPRC5D scFV (anti-GPRC5D scFV) in the CAR molecule was detected by flow cytometry 24 hours later. The antibody information is shown in Example 2. The results are shown in Fig.15 As shown in the figure, the expression level of CAR molecules containing TM1 is similar to that of the control CAR molecule (TM control); the expression level of CAR molecules containing TM4, TM5, TM6, and TM8 is slightly higher than that of the control CAR molecule (TM control). The effects of TM9 to TM14 will not be tested in subsequent experiments.

[0446] Further, using the method in Example 2, the transposon plasmid and PBase plasmid containing CAR molecules of TM control and TM1 to TM8 were introduced into iPSC to obtain iPSC expressing CAR molecules and their variants. Using the method in Example 3, the above iPSC expressing exogenous anti-GPRC5D CAR was differentiated into NK cells. iHSPCs were obtained on the 10th day of differentiation. At this time, the CD34 expression level of iHSPC containing TM6 CAR molecules was slightly lower, and the CD34 expression levels of other iHSPCs were all above 70% ( Fig.16 ). The CD43 expression levels in all iHSPCs were similar, all above 97% ( Fig.16We further differentiated the above iHSPCs to day 23 and obtained NK cells expressing CD314 and CD56 (Day 23CAR-iNK). Interestingly, the expression levels of CD314 and CD56 in all CAR-iNKs containing transmembrane sequence variants were higher than those in the control group (TM control) ( Fig.17 ), which shows that the use of CAR molecules containing transmembrane sequence variants not only does not affect the differentiation of iPSC into NK cells, but may have different degrees of enhancement of its differentiation. At the same time, at the iHSPC stage, the CD34 expression level of CAR carrying TM6 was low (50.18%), which did not affect its further differentiation into NK cells ( Fig.17 ). Later, we further counted the number of Day 23CD56 + The proliferation of CAR-iNK cells relative to iPSC cells at the initial differentiation stage showed that CD56 containing TM variants + CAR-iNK cells have similar or higher proliferation rates than control cells. The proliferation rate of TM5 group cells is 100 times higher than that of the control group, the proliferation rate of TM4, TM7, and TM8 groups cells is 10 times higher than that of the control group, and the proliferation rate of TM6 group cells is 7 times higher than that of the control group ( Fig.18 ).

[0447] Furthermore, the inventors further expanded Day 23 CAR-iNK cells to 36 days to make them mature CAR-iNK cells. At this time, the CD45 expression levels of all groups of cells were above 96% (except for the TM2 group, the others were above 99.6%), the CD56 expression levels were above 90% (including TM5 and TM8 groups above 99%), and the CAR molecule expression levels were above 95% (measured by anti-GPRC5D scFV expression level; Figure 19, that is, Fig.19A , 19B , 19C, 19D). They showed a high-purity mature NK cell phenotype, and the CAR molecule expression levels of the TM variant group and the TM control group were similar.

[0448] In order to further verify the effect of CAR molecules carrying TM mutants on the antigen-dependent specific killing function of iNK cells, the method of Example 4 was used to use K562-Luc and K562-GPRC5D-Luc (K562-Luc cell lines expressing exogenous GPRC5D antigen; cells from Shenzhen Sanqi Biotechnology Co., Ltd., which were constructed by overexpressing GPRC5D protein in K562 cells by lentivirus) as target cells, and CAR-iNK carrying TM control or TM variant CAR molecules were co-incubated for 4 hours at an effector-target ratio of 1:1 to determine the killing ability of these CAR-iNKs on target cells. Since iNK can exert a broad-spectrum killing ability and has a certain degree of killing on K562-Luc, CAR molecules carrying anti-GPRC5D can further enhance the killing of iNK on GPRC5D. + The target cells exert antigen-dependent specific killing ability. Therefore, this experiment measured the killing ability of each group of CAR-iNK against K562-Luc and K562-GPRC5D-Luc respectively. The killing ability of CAR-iNK against K562-GPRC5D-Luc minus the killing ability of CAR-iNK against K562-Luc is the antigen-dependent specific killing ability of anti-GPRC5D CAR-iNK due to the expression of CAR molecules. The results are shown in Fig. 20 As shown, TM5, TM6, and TM8 groups had significantly improved antigen-dependent specific killing abilities against K562-GPRC5D-Luc compared with TM control group; while TM1, TM4, and TMcontrol groups had similar antigen-dependent specific killing abilities against K562-GPRC5D-Luc, and TM2, TM3, and TM7 groups had no obvious antigen-dependent specific killing abilities against K562-GPRC5D-Luc.

[0449] In summary, compared with CAR molecules carrying the reverse NKG2D transmembrane domain (NKG2D-TM control, or TM control for short), CAR molecules carrying TM5, TM6, and TM8 variants can make NK cells have better antigen-dependent specific killing ability; CAR molecules carrying TM1 and TM4 variants can make NK cells have similar antigen-dependent specific killing ability. At the same time, TM1 to TM8 variants do not affect the differentiation and proliferation of iPSCs into NK cells, and CAR molecules carrying some TM variants (TM1 to TM8) have a promoting effect on the differentiation and proliferation of iPSCs into NK cells, among which TM5 is the most obvious (CD56 + The proliferation rate of NK cells was 100 times higher than that of the control group), followed by TM4, TM7, and TM8 (CD56 +The proliferation rate of NK cells was 10 times higher than that of the control group), and again TM6 (CD56 + The proliferation rate of NK cells was 7 times that of the control group). Finally, TM3, TM2 and TM1, whose CD56 + The proliferation rates of NK cells were 3.7, 3.5 and 1.7 times that of the control group, respectively.

[0450] Table 1: Transmembrane region sequences used in CAR molecules

[0451] name Transmembrane region sequences for CAR molecules Serial number NKG2D-TMControl SNLFVASWIAVMIIFRIGMAVAIFCCFFFPS SEQ ID NO: 18 NKG2D-TM1 SNLFVASWIAVMIIFRIGMAVAIFCCFFLPS SEQ ID NO: 19 NKG2D-TM2 SNLFVASWIAVMVIFRIGMAVAIFRCLFFPS SEQ ID NO: 20 NKG2D-TM3 SNLFVASWIAVMIIFRIGMAVAIFCCLFFPS SEQ ID NO: 21 NKG2D-TM4 SNLFVASWIAVMIIFHIGLAIAISHVLFFPS SEQ ID NO: 22 NKG2D-TM5 SNLFVASWIAVMIIFHIGMAIAISRCLFFPS SEQ ID NO: 23 NKG2D-TM6 SNLFVASWIAVMIIFHIGMAIAISCTVFFPS SEQ ID NO: 27 NKG2D-TM7 SNLFVASWIAVMIIFRIGMAVAVSRCLHFPS SEQ ID NO: 28 NKG2D-TM8 SNLFVASWIAVMIIFHIGLAIAISRCLFYPS SEQ ID NO: 29 NKG2D-TM9 SNLFVASWIAVMVIFRIGMAIAISRALFFPS SEQ ID NO: 30 NKG2D-TM10 SNLFVASWIAVMVIFRVGMAVAISRALFFPS SEQ ID NO: 31 NKG2D-TM11 SNLFVASWIAVMVIFRIAMAVAISRALFFPS SEQ ID NO: 32 NKG2D-TM12 SNLFVASWIAVMVIFRIAMSIAIFRGLFFPS SEQ ID NO:33 NKG2D-TM13 SNLFVASWIAVMVTFRIGMAIAISRALFFPS SEQ ID NO:34 NKG2D-TM14 SNLFVASWIAVMVLFRIAMAIAIIYGLFFPS SEQ ID NO:35

[0452] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", "some implementation schemes" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0453] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that include: An extracellular domain, which is capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen; A transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from a type I transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and an intracellular domain, the intracellular domain being connected to the transmembrane domain, Wherein, the amino acid sequence of the transmembrane domain has the following sequence: SNLFVASWIAVMX8IFX9IGX 10 AX 11 AX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 PS Wherein, X8 is selected from I or V; X9 is selected from R or H; X 10 Select from M or L, X 11 Selected from V or I; X 12 Selected from I or V, X 13 Selected from S or F; X 14 Selected from C, R or H; X 15 Selected from C, T or V, X 16 Selected from V, L or absent; X 17 Select from F or H, X 18 Selected from F or Y, X 19 Selected from L or absent, X 16 and X 19 Cannot exist at the same time, and X 16 and X 19 Can't exist at the same time. The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

2. The chimeric antigen receptor according to claim 1, characterized in that The transmembrane domain has an amino acid sequence as shown in any one of SEQ ID NOs: 19-23, 27-29.

3. The chimeric antigen receptor according to claim 1, characterized in that The extracellular domain includes an antibody or antigen-binding fragment thereof that can specifically recognize at least one of the following: GPRC5D, CD19, BCMA, CD123, CD138, CD20, CD22, CD30, CD33, CD38, CD4, CD7, CLL-1, CD10, CD34, CS1, CD16, CD5, IL-1-RAP, ITGB7, k-IgG, TACI, TRBC1, MUC1, NKG2DL, PD-L1, CD133, CD117, LeY, CD70, ROR1, CD147, CD171, CD267, AXL, B7-H3, CD44v6, CEA, CAIX, CLDN18.2, CLDN6, DLL3, DR5, EGFR, EpCAM, ErbB, FAP, FBP, FRα, GD2, gp100, HER2, IL-13Rα2, LFA1, LMP1, MAGE-A3, MAGE-A1, MAGE-A 4. MART1, MET, MG7, MMP2, MSLN, MUC1, MUC16, MUC16ecto, NECTIN4, NKG2D, PMEL, PSCA, PSMA, ROR1, ROR2, TM4SF1, TnMUC1, VEGFR2, CLD18 and GPC3; Optionally, the extracellular domain further comprises an antibody or an antigen-binding fragment thereof capable of specifically recognizing a pathogen antigen, wherein the pathogen comprises EBV; Optionally, the extracellular domain further comprises an antibody or an antigen-binding fragment thereof that can specifically recognize an inflammatory factor, wherein the inflammatory factor comprises at least one of the following: IL-1, TNF-α, IFN-γ, IL-12, IL-18, GMCSF, IL-6, IL-8, IL-17, CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL16 and CCL2-2; Preferably, the extracellular domain comprises an antibody or an antigen-binding fragment thereof that can specifically recognize GPRC5D.

4. The chimeric antigen receptor according to claim 3, characterized in that The antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D contains a CDR sequence selected from at least one of the following or an amino acid sequence having at least 90% identity therewith: Heavy chain variable region CDR sequences: SEQ ID NOs: 1-3; Light chain variable region CDR sequence: SEQ ID NO: 4, amino acid sequence SAS and SEQ ID NO: 5; Optionally, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D comprises: The heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences at least 90% identical to SEQ ID NOs: 1, 2 and 3; Optionally, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D comprises: The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO:4, the amino acid sequence SAS, and SEQ ID NO:5, or an amino acid sequence that is at least 90% identical to SEQ ID NO:4, the amino acid sequence SAS, and SEQ ID NO:5, respectively; Optionally, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D comprises: The heavy chain variable region CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NOs: 1, 2, and 3, and the light chain variable region CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 4, the amino acid sequence SAS, and SEQ ID NO: 5, respectively, or amino acid sequences that are at least 90% identical to SEQ ID NO: 4, the amino acid sequence SAS, and SEQ ID NO: 5; Optionally, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 6; Optionally, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D has a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 7; Optionally, the antibody capable of specifically recognizing GPRC5D is a single-chain antibody having an amino acid sequence as shown in SEQ ID NO:

8.

5. The chimeric antigen receptor according to claim 4, characterized in that The extracellular domain comprises a bispecific binding molecule comprising: A first binding region, wherein the first binding region comprises the antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D; and The second binding region has the activity of binding to an antigen specifically expressed on lymphocytes.

6. The chimeric antigen receptor according to claim 5, characterized in that The antigens specifically expressed on the lymphocytes include at least one selected from BCMA, CD38, CD138, CD3E, and FCGR3A; Optionally, the second binding region comprises a single chain antibody or a single domain antibody; Optionally, the second binding region has BCMA binding activity; Optionally, the second binding region comprises a single-chain antibody or a single-domain antibody capable of specifically recognizing BCMA; Optionally, the second binding region has the amino acid sequence shown in SEQ ID NO:

9.

7. The chimeric antigen receptor according to claim 5, characterized in that The first binding region and the second binding region are connected via a connecting peptide; Optionally, the bispecific binding molecule has the amino acid sequence shown in SEQ ID NO:

10.

8. The chimeric antigen receptor according to claim 1, characterized in that The extracellular domain is connected to the transmembrane domain via a hinge domain; Optionally, the hinge domain is derived from the hinge region of at least one protein in the following group: 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 and LIGHT; Optionally, the hinge domain is derived from the hinge region of CD8; Optionally, the hinge domain has the amino acid sequence shown in SEQ ID NO:

11.

9. The chimeric antigen receptor according to claim 7 or 8, characterized in that The intracellular domain includes a co-stimulatory signal domain and an intracellular signal transduction domain, and the co-stimulatory signal domain and the intracellular signal transduction domain are connected by a connecting peptide; Optionally, the costimulatory signal domain comprises an intracellular costimulatory signal domain of at least one protein selected from the following group: 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, ligand of CD83, CD40, and MyD88; Optionally, the costimulatory signaling domain is derived from the intracellular costimulatory signaling domain of 2B4; Optionally, the costimulatory signal domain has an amino acid sequence as shown in SEQ ID NO: 12; Optionally, the intracellular signaling domain comprises a domain comprising at least one ITAM motif; Optionally, the intracellular signaling domain comprises an intracellular signaling domain derived from at least one protein of the following group; CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, DAP10, DAP-12; Optionally, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ; Optionally, the intracellular signaling domain has an amino acid sequence as shown in SEQ ID NO: 13; Optionally, the chimeric antigen receptor further comprises a signal peptide, which is linked to the N-terminus of the extracellular domain of the chimeric antigen receptor; Optionally, the signal peptide is a signal peptide derived from CD8 protein; Optionally, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 14; Optionally, the chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO:15 or SEQ ID NO:

16.

10. An isolated polynucleotide, characterized in that The polynucleotide encodes the chimeric antigen receptor according to any one of claims 1 to 9.

11. An expression vector, characterized in that: Carrying the polynucleotide according to claim 10.

12. A cell, characterized in that The method comprises carrying the polynucleotide according to claim 10 or the expression vector according to claim 11 or being capable of expressing the chimeric antigen receptor according to any one of claims 1 to 9.

13. The cell according to claim 12, characterized in that The cells include immune cells; Optionally, the immune cells are obtained by separation from peripheral blood, umbilical cord blood, tissues, or organs, or the immune cells are obtained by differentiation of stem cells or immune cell progenitor cells; Optionally, the stem cells include induced pluripotent stem cells, hematopoietic stem cells, or embryonic stem cells within 14 days of fertilization that have not undergone in vivo development; Optionally, the immune cells include at least one selected from NK cells, T cells, and macrophages.

14. A composition, characterized in that Contains at least one of the following: The chimeric antigen receptor according to any one of claims 1 to 9; The polynucleotide of claim 10; The expression vector according to claim 11; The cell of claim 12 or 13.

15. Use of the composition according to claim 14 in preparing a drug for treating a disease, wherein the disease is a blood tumor or a solid tumor disease, an autoimmune disease or a viral infectious disease; Optionally, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

16. The use according to claim 15, characterized in that The GPRC5D-related disease includes multiple myeloma.

17. A kit for detecting GPRC5D, characterized in that: The invention comprises the chimeric antigen receptor according to any one of claims 1 to 9.

18. Use of the chimeric antigen receptor according to any one of claims 1 to 9, the polynucleotide according to claim 10, or the expression vector according to claim 11 in preparing a kit, characterized in that: The kit is used for detecting GPRC5D or diagnosing GPRC5D-related diseases.

19. The use according to claim 18, characterized in that The GPRC5D-related disease includes multiple myeloma.

20. Use of the chimeric antigen receptor according to any one of claims 1 to 9, the polynucleotide according to claim 10, the expression vector according to claim 11, the cell according to claim 12 or 13 and / or the composition according to claim 14 in treating individual hematological or solid tumor diseases, autoimmune diseases or viral infectious diseases.

21. A method for preventing and / or treating a blood tumor or solid tumor disease, an autoimmune disease or a viral infectious disease in an individual, characterized in that: comprising administering to the individual at least one of the following: The chimeric antigen receptor according to any one of claims 1 to 9; The polynucleotide of claim 10; The expression vector according to claim 11; The cell of claim 12 or 13; and The composition of claim 14.

22. Use of the chimeric antigen receptor according to any one of claims 1 to 9, the polynucleotide according to claim 10, or the expression vector according to claim 11 in detecting GPRC5D or diagnosing GPRC5D-related diseases.

23. A method for detecting GPRC5D, characterized in that: The method comprises detecting GPRC5D in a sample to be tested using at least one of the following: The chimeric antigen receptor according to any one of claims 1 to 9; The polynucleotide of claim 10; and The expression vector according to claim 11.

24. A method for diagnosing whether an individual suffers from a GPRC5D-related disease, characterized in that: The method comprises detecting GPRC5D in a sample derived from an individual to be tested using at least one of the following: The chimeric antigen receptor according to any one of claims 1 to 9; The polynucleotide of claim 10; and The expression vector according to claim 11.

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