GPRC5d antibody-containing chimeric antigen receptor and use thereof

Abstract

Provided are a chimeric antigen receptor, and a GPRC5D antibody-containing chimeric antigen receptor and a use thereof. The chimeric antigen receptor comprises: an extracellular domain, wherein the extracellular domain is capable of binding to a tumor antigen; a transmembrane domain, wherein the transmembrane domain is connected to the extracellular domain, and the transmembrane domain is derived from an I-type transmembrane protein or an artificially synthesized transmembrane protein having a hydrophobic helix; and an intracellular domain, wherein the intracellular domain is connected to the transmembrane domain, and the tumor antigen is a tumor-specific antigen or a tumor-associated antigen. The amino acid sequence of the transmembrane domain is optimized, thereby providing a GPRC5D antibody-containing chimeric antigen receptor and engineered immune cells; and the engineered immune cells obtained on the basis of the GPRC5D antibody have specific killing performance on multiple myeloma.

Description

Chimeric antigen receptor containing GPRC5D antibody and its application

[0001] Priority information

[0002] This application claims priority to Chinese patent application number 202311010359.2, filed with the Chinese Patent Office on August 10, 2023, entitled “Chimeric Antigen Receptor Containing GPRC5D Antibody and Its Application,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] 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

[0004] Multiple myeloma (MM) is a malignant disease characterized by the abnormal proliferation of clonal plasma cells. In many countries, it is the second most common malignant tumor of the blood system, often occurring in the elderly, and is currently incurable. In my country, the incidence of multiple myeloma has exceeded that of acute leukemia, at approximately one in 100,000. As the aging population in my country continues to intensify, the number of patients will further increase. Although treatment strategies have 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), MM is still a common disease.

[0005] G protein-coupled receptor family C group 5 member D (GPRC5D) is an orphan receptor and a seven-transmembrane protein that is specifically and highly expressed in multiple myeloma cells, but expressed in low amounts or not at all in normal tissues. Therefore, this target is expected to become a new target for the treatment of multiple myeloma.

[0006] Chimeric antigen receptors (CARs) can activate immune cells to specifically kill tumors. Previous studies have found that the transmembrane domain of CARs is crucial for immune cell activation (https: / / doi.org / 10.1016 / j.stem.2018.06.002). The transmembrane domain, derived from the NKG2D transmembrane region, contributes to the enhanced tumor-killing efficacy of CAR-NK cells. To further enhance this efficacy, optimization of the transmembrane domain and the structure of the chimeric antigen receptor are urgently needed.

[0007] Summary of the Invention

[0008] The present invention aims to at least partially address one of the technical problems in the related art. 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. The engineered immune cell obtained based on the GPRC5D antibody has specific killing properties against multiple myeloma.

[0009] 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:

[0010] An extracellular domain capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0011] 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

[0012] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0013] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0014] SNLFVASWIAVMX8IFX9IGX 10 AX 11 AX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 PS

[0015] wherein X8 is selected from I or V;

[0016] X9 is selected from R or H;

[0017] X 10 Choose from M or L,

[0018] X 11 Selected from V or I;

[0019] X 12 Selected from I or V,

[0020] X 13 Select from S or F;

[0021] X 14 Selected from C, R or H;

[0022] X 15 Selected from C, T or V,

[0023] X 16Selected from V, L or absent;

[0024] X 17 Selected from F or H,

[0025] X 18 Selected from F or Y,

[0026] X 19 Selected from L or absent,

[0027] X 16 and X 19 Cannot exist at the same time, and X 16 and X 19 They cannot exist at the same time, and the tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0028] 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).

[0029] 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.

[0030] 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.

[0031] 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:

[0032] an extracellular domain capable of binding to a tumor antigen;

[0033] 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

[0034] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0035] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0036] SNLFVASWIAVMX1IFX2IGMAX3AIX4X5CX6FFX7PS

[0037] wherein X1 is selected from I or V;

[0038] X2 is selected from R or H;

[0039] X3 is selected from V or I;

[0040] X4 is selected from S or F;

[0041] X5 is selected from C or R;

[0042] X6 is selected from L or is absent;

[0043] X7 is selected from L or is absent,

[0044] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0045] 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).

[0046] 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.

[0047] 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.

[0048] 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:

[0049] An extracellular domain capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0050] 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

[0051] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0052] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0053] SNLFVASWIAVMIIFX 20 IGX 21 AX 22 AX 23 SX 24 X 25 X 26 X 27 X 28 PS

[0054] Among them, X 20 Selected from R or H,

[0055] X 21 Choose from M or L,

[0056] X 22 Selected from V or I,

[0057] X 23 Selected from V or I,

[0058] X 24 is selected from C, R or H,

[0059] X 25 Selected from C, T or V,

[0060] X 26 Selected from L or V,

[0061] X 27 Selected from F or H,

[0062] X 28 Selected from F or Y;

[0063] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0064] 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).

[0065] 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 to 29.

[0066] 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.

[0067] According to an embodiment of the present invention, the chimeric antigen receptor of the first, second, and third aspects may further include at least one of the following additional technical features:

[0068] According to an embodiment of the present invention, the extracellular domain includes an antibody or 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.

[0069] According to an embodiment of the present invention, the extracellular domain further comprises an antibody or an antigen-binding fragment thereof that can specifically recognize pathogen antigens, and the pathogen includes EBV.

[0070] 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.

[0071] According to an embodiment of the present invention, the extracellular domain comprises an antibody or an antigen-binding fragment thereof that can specifically recognize GPRC5D.

[0072] 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:

[0073] Heavy chain variable region CDR sequences: SEQ ID NOs: 1-3;

[0074] The CDR sequence of the light chain variable region is SEQ ID NO: 4, the amino acid sequence SAS, and SEQ ID NO: 5.

[0075] CDR 2 sequence of the light chain variable region: SAS

[0076] The present invention provides a chimeric antigen receptor whose transmembrane domain is optimized and contains a new GPRC5D antibody outside the cell. The antibody 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.

[0077] According to an embodiment of the present invention, the antibody or antigen-binding fragment capable of specifically recognizing GPRC5D comprises:

[0078] The heavy chain variable region CDR1, CDR2, CDR3 sequences are shown 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.

[0079] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D comprises:

[0080] 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.

[0081] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof capable of specifically recognizing GPRC5D comprises:

[0082] The heavy chain variable region CDR1, CDR2, and 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, and 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, 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.

[0083] According to one embodiment of the present invention, a GPRC5D antibody or an antigen-binding fragment thereof has the heavy chain variable region CDR1, CDR2, CDR3 shown in SEQ ID NOs: 1, 2, and 3, the amino acid sequence SAS shown in SEQ ID NO: 4, and the light chain variable region CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 5. The antibody can specifically bind to GPRC5D on the surface of multiple myeloma cells. Engineered immune cells designed based on the antibody can specifically target multiple myeloma cells, kill target cells, and prevent recurrence.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] According to an embodiment of the present invention, the extracellular domain comprises a bispecific binding molecule comprising:

[0088] A first binding region, comprising the antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D; and

[0089] The second binding region has an activity of binding to an antigen specifically expressed on lymphocytes.

[0090] 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.

[0091] According to an embodiment of the present invention, the second binding region comprises a single-chain antibody or a single-domain antibody.

[0092] According to an embodiment of the present invention, the second binding region has BCMA binding activity.

[0093] 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.

[0094] According to an embodiment of the present invention, the second binding region has an amino acid sequence as shown in SEQ ID NO:9.

[0095] According to an embodiment of the present invention, the first binding region and the second binding region are connected via a connecting peptide.

[0096] According to an embodiment of the present invention, the bispecific binding molecule has the amino acid sequence shown in SEQ ID NO: 10.

[0097] Existing BCMA-targeted bispecific antibodies or CAR-T cells have achieved some efficacy in treating multiple myeloma (MM). However, MM patients still cannot avoid relapse due to BCMA antigen escape. The inventors have discovered that the bispecific antibody of the present invention, consisting of the GPRC5D antibody and the BCMA antibody, and engineered immune cells based on this bispecific antibody, can specifically target multiple myeloma cells, killing target cells while preventing relapse, thus resolving the BCMA antigen escape issue.

[0098] According to an embodiment of the present invention, the extracellular domain is connected to the transmembrane domain via a hinge domain.

[0099] 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:

[0100] 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.

[0101] According to an embodiment of the present invention, the hinge domain is derived from the hinge region of CD8;

[0102] According to an embodiment of the present invention, the hinge domain has the amino acid sequence shown in SEQ ID NO: 11.

[0103] According to an embodiment of the present invention, the intracellular domain includes a costimulatory signaling domain and an intracellular signaling domain, and the costimulatory signaling domain and the intracellular signaling domain are connected by a connecting peptide.

[0104] 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:

[0105] 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.

[0106] According to an embodiment of the present invention, the costimulatory signaling domain is derived from the intracellular costimulatory signaling domain of 2B4.

[0107] According to an embodiment of the present invention, the costimulatory signal domain has the amino acid sequence shown in SEQ ID NO: 12.

[0108] According to an embodiment of the present invention, the intracellular signaling domain includes a domain containing at least one ITAM motif.

[0109] According to an embodiment of the present invention, the intracellular signaling domain comprises an intracellular signaling domain derived from at least one protein selected from the following group;

[0110] CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, DAP10, DAP-12.

[0111] According to an embodiment of the present invention, the intracellular signaling domain is an intracellular signaling domain derived from CD3ζ.

[0112] According to an embodiment of the present invention, the intracellular signaling domain has the amino acid sequence shown in SEQ ID NO: 13.

[0113] 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.

[0114] According to an embodiment of the present invention, the signal peptide is a signal peptide derived from CD8 protein.

[0115] According to an embodiment of the present invention, the signal peptide has the amino acid sequence shown in SEQ ID NO:14.

[0116] 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. In the present invention, the transmembrane domain of the chimeric antigen receptor can use the transmembrane domains shown in SEQ ID NOs: 18 to 23 according to different experimental needs, with SEQ ID NO: 18 being the amino acid sequence of the unmutated transmembrane domain.

[0117] In a fourth aspect, 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.

[0118] In a fifth aspect, 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.

[0119] In a sixth aspect, the present invention provides a cell. According to an embodiment of the present invention, the cell carries the polynucleotide of the fourth aspect or the expression vector of the fifth aspect, or is capable of expressing the chimeric antigen receptor of the first, second, or third aspect.

[0120] According to an embodiment of the present invention, the cells include immune cells.

[0121] 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.

[0122] 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.

[0123] According to an embodiment of the present invention, the immune cells include at least one selected from NK cells, T cells, and macrophages.

[0124] In a 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:

[0125] The chimeric antigen receptor of the first, second or third aspect;

[0126] The polynucleotide according to the fourth aspect;

[0127] The expression vector according to the fifth aspect; and

[0128] The cell described in the sixth aspect.

[0129] 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.

[0130] According to an embodiment of the present invention, the disease is a hematological tumor or solid tumor disease, an autoimmune disease or a viral infectious disease.

[0131] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0132] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0133] In a ninth aspect, the present invention provides a kit for detecting GPRC5D. According to an embodiment of the present invention, the kit comprises the chimeric antigen receptor according to the first, second or third aspect.

[0134] In a tenth aspect, the present invention provides use of the antibody or antigen-binding fragment thereof of the first, second, or third aspect, the polynucleotide of the fourth aspect, or the expression vector of the fifth aspect in preparing a kit. According to an embodiment of the present invention, the kit is used for detecting GPRC5D or diagnosing a GPRC5D-related disease.

[0135] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0136] 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 treating individual hematological tumors or solid tumor diseases, autoimmune diseases or viral infectious diseases.

[0137] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0138] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0139] In a twelfth aspect, the present invention provides a method for preventing and / or treating a blood or solid tumor disease, an autoimmune disease, or a viral infectious disease in a subject. According to an embodiment of the present invention, the method comprises administering to the subject at least one of the following:

[0140] The chimeric antigen receptor of the first, second or third aspect;

[0141] The polynucleotide according to the fourth aspect;

[0142] The expression vector according to the fifth aspect;

[0143] The cell of the sixth aspect; and

[0144] The composition described in the seventh aspect.

[0145] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0146] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0147] 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 has a GPRC5D-related disease.

[0148] According to an embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0149] 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 using at least one of the following:

[0150] The chimeric antigen receptor of the first, second or third aspect;

[0151] The polynucleotide according to the fourth aspect;

[0152] The expression vector described in the fifth aspect.

[0153] The method for detecting GPRC5D according to an embodiment of the present invention can be used in scientific research, and the sample includes but is not limited to tissue, cells, blood, serum, plasma, urine, feces, saliva or sweat.

[0154] 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.

[0155] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0156] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0157] FIG1 is an expression cassette encoding an anti-GPRC5D CAR polypeptide according to an embodiment of the present invention, wherein:

[0158] 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;

[0159] FIG2 shows the piggyBac-mediated anti-GPRC5D CAR expression according to an embodiment of the present invention, wherein:

[0160] A shows several examples of anti-GPRC5D CAR polypeptide expression cassettes.

[0161] B shows a map of the piggyBac plasmid containing the anti-GPRC5D CAR expression cassette;

[0162] FIG3 is a schematic diagram of site-directed integration of a DNA template containing anti-GPRC5D CAR by homologous recombination according to an embodiment of the present invention, wherein:

[0163] A shows a schematic diagram of several common forms of expression cassettes encoding anti-GPRC5D CAR polypeptides, where the dotted line represents an optional portion that may or may not be present;

[0164] B shows examples of several DNA template components containing anti-GPRC5D CAR polypeptide expression cassettes.

[0165] 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;

[0166] Figure 4 shows a general process for obtaining gene-edited iPSC-derived anti-GPRC5D CAR-iNK cells according to an embodiment of the present invention, wherein "*" indicates that the DNA encoding the anti-GPRC5D CAR can be introduced using different tools, such as donor plasmids, piggyBac, dsDNA, ssDNA, various integrating or non-integrating viruses, etc. Depending on the choice of different introduction tools, PBase can be selected to assist piggyBac introduction, and nucleases can be selected to form single-strand or double-strand breaks to assist other non-viral introduction methods;

[0167] FIG5 is a graph showing the results of FACS detection of the positive rate of CAR molecules in iPSCs and iNK cells differentiated from them that were introduced with PiggyBac (PB)-mediated anti-GPCR5D CAR according to an embodiment of the present invention, wherein the expression ratio of CAR molecules in the tested cell population is detected using an anti-GPRC5D scFV antibody, the left peak is the isotype control, and the right peak is the detection marker;

[0168] FIG6 is a graph showing the results of FACS detection of iPSC-derived anti-GPRC5D CAR-iNK CD56 and CD45 expression levels according to an embodiment of the present invention, wherein the left peak is the isotype control and the right peak is the detection marker;

[0169] FIG7 is a graph showing the results of FACS detection of NKG2D expression levels of iPSC-derived anti-GPRC5D CAR-iNK cells according to an embodiment of the present invention, wherein the left peak is an isotype control and the right peak is a detection marker;

[0170] FIG8 is a graph showing the results of FACS detection of TCRαβ and TCRγ expression levels of iPSC-derived anti-GPRC5D CAR-iNK cells according to an embodiment of the present invention, wherein the left peak is an isotype control and the right peak is a detection marker;

[0171] FIG9 is a graph showing the results of the cytotoxicity test of iPSC-derived anti-GPRC5D or anti-BCMACAR-iNK cells against K562-Luc cells according to an embodiment of the present invention, wherein:

[0172] A shows the results of the cytotoxicity test of different NK cells against K562-Luc under the conditions of 4 hours incubation time and 4:1 effector-target ratio.

[0173] B. Graph showing the test results of the effects of WT-iNK and anti-GPRC5D or anti-BCMACAR-iNK on the killing activity of K562-Luc at different effector-target ratios;

[0174] FIG10 shows the killing activity of iPSC-derived anti-GPRC5D cells against H929-Luc2 cells according to an embodiment of the present invention, wherein,

[0175] The left figure shows the results of the test of the effect 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. The right figure shows the results of the test of the effect of WT-iNK and anti-GPRC5D or anti-BCMACAR-iNK on the killing activity of H929-Luc2 at different effector-target ratios.

[0176] FIG11 is a graph showing the results of cytotoxicity assays against H929-Luc2 cells using iPSC-derived anti-GPRC5D / BCMACAR-iNK, anti-GPRC5D CAR-iNK, and anti-BCMACAR-iNK 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;

[0177] FIG12 is a graph showing the comparative detection results of the killing ability of iPSC-derived WT iNK and anti-GPRC5D CAR-iNK against K562-Luc cells and H929-Luc2 cells after cryopreservation and resuscitation according to an embodiment of the present invention;

[0178] FIG13 is a graph showing the results of detecting changes in the killing ability of fresh (Fresh) and cryopreserved (Cryopreserved) anti-GPRC5D CAR-iNK against K562-Luc and H929-Luc2 according to an embodiment of the present invention;

[0179] FIG14 is a graph showing the results of the detection of the killing activity of anti-GPRC5D CAR-iNK and WT iNK at different effector-target ratios against MM.1S-Luc cells and OPM-2Luc2 cells after cryopreservation and thawing according to an embodiment of the present invention;

[0180] Figure 15 is a graph showing the results of detecting the proportion of cells expressing CAR molecules using anti-GPRC5D scFV antibodies after CAR molecules (TM control) and variants thereof (TM1 to TM14) loaded with anti-GPRC5D scFV were transfected into 293T cells according to an embodiment of the present invention. CAR molecule expression was detected using anti-anti-GPRC5D scFV antibodies;

[0181] Figure 16 shows the results of flow cytometry analysis of CD34 and CD43 expression levels on day 10 of differentiation of iPSCs loaded with anti-GPRC5D scFV CAR molecules (TM control) and their variants (TM1 to TM8) into iHSPCs according to an embodiment of the present invention, wherein:

[0182] The left and right figures respectively show the expression percentages of CD34 and CD43 in the iHSPCs obtained by differentiation of the groups in this example;

[0183] Figure 17 shows the expression levels of CD56 and CD314 on day 23 of iPSCs differentiated into CAR-iNK cells loaded with anti-GPRC5D scFV CAR molecules (TM control) and their variants (TM 1-TM8) according to an embodiment of the present invention, wherein,

[0184] The left and right figures respectively show the expression percentages of CD56 and CD314 in the CAR-iNK cells obtained by differentiation of the groups in this example;

[0185] FIG18 is a graph showing the expansion factor of the CD56+CAR-iNK cell population relative to the iPSC cell population at the start of differentiation at day 23 during differentiation of iPSCs loaded with anti-GPRC5D scFV CAR molecules (TM control) or variants thereof (TM1 to TM8) into CAR-iNK cells according to an embodiment of the present invention;

[0186] FIG19 is a graph showing the expression of CD45, CD56, and anti-GPRC5D scFV in cells obtained after further expansion of CAR-iNK cells loaded with anti-GPRC5D scFV CAR molecules (TM control) and their variants (TM 1 to TM 8) according to an embodiment of the present invention for 13 days (i.e., Day 36 CAR-iNK).

[0187] Figures 19A, 19B, and 19C respectively show the results of flow cytometry detection of the expression percentages of each marker (CD45, CD56, anti-GPRC5D scFV) in this example. The peak on the left in the figure is the detection result of the isotype control, and the peak on the right is the detection result of each marker (CD45, CD56, anti-GPRC5D scFV).

[0188] Figure 19D shows the statistical results of flow cytometry detection of the expression percentage of each marker (CD45, CD56, anti-GPRC5D scFV) in this example;

[0189] Figure 20 is a graph showing the detection results of 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 by Dunnett's test (GraphPad Prism 9.5.0 (525)). DETAILED DESCRIPTION

[0190] 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.

[0191] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0192] The endpoints of the ranges and any values ​​disclosed herein 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 endpoints of each range, the endpoints of each range and 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 to be specifically disclosed herein.

[0193] 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 meaning commonly understood by those skilled in the art to which the present invention belongs.

[0194] 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.

[0195] 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.

[0196] the term

[0197] "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.

[0198] "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 antibodies (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 functional antibody fragments thereof.

[0199] "Antibody fragment" refers to a molecule other than an intact antibody that comprises 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.

[0200] As used herein, "heavy chain variable region" refers to the region of an antibody molecule that comprises 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.

[0201] As used herein, the term "single-domain antibody (sdAb)" refers to a heavy chain antibody naturally found in camel / alpaca blood that lacks a light chain and consists solely of the heavier heavy chain (H chain). The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies greatly and is called the variable region (V region), while the carboxyl terminus (C-terminus) is relatively stable and exhibits minimal variation, known as the constant region (C region). The V region of the H chain is called the VH. Within the variable region, certain regions exhibit a higher degree of variation in amino acid composition and sequence, known as the hypervariable region (HVR). The HVR is the site of antigen-antibody binding and is therefore also known as the complementarity-determining region (CDR). The heavy chain variable region has three CDRs. CDR1 and CDR3 are slightly longer than those in humans, and CDR3 protrudes outward from the tertiary structure. Therefore, single-domain antibodies are hypothesized to 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 sequences (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).

[0202] "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 the 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 the heavy chain variable domain (as described by Chothia et al., J. Mol. Biol. 196:901-917 (1987)).

[0203] "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 typically highly specific and are directed against a single antigenic site or epitope. In contrast, polyclonal antibody preparations typically include a mixture of antibodies having different amino acid sequences directed against the same or different determinants (epitopes). In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized in homogenous cultures and are not contaminated by other immunoglobulins having different specificities and characteristics.

[0204] As used herein, the term "hinge" refers to the region between the CH1 and CH2 domains of an antibody heavy chain. This region includes inter-H chain disulfide bonds, is rich in proline, does not form an α-helix, and is prone to stretching and some degree of distortion, which facilitates complementary binding between the antibody's antigen-binding site and the antigen epitope. The antigen-binding domain in the present invention is connected to the transmembrane domain via the hinge domain.

[0205] In the present invention, "antigen" specifically refers to a marker on the surface of tumor cells.

[0206] Chimeric antigen receptor, abbreviated as CAR, is an artificial receptor molecule manufactured using genetic engineering technology. It includes an antigen-binding domain (usually derived from a single-chain antibody variable region gene fragment, scFV, or from a single-domain antibody), a hinge domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

[0207] As used herein, "reduced expression" of a gene refers to the elimination or reduction of expression of one or more gene products encoded by a gene in a cell, compared to normal levels of mRNA or protein expression of the gene. In some cases, this effect on gene expression is temporary or reversible, such as through the inhibition of gene expression by small molecule compounds, nucleic acids such as RNAi, siRNA, or shRNA, or specific proteins. In some cases, this effect on gene expression is permanent, such as through gene editing to introduce base insertions or deletions into the coding region of a target gene, resulting in frameshift mutations, or through homologous recombination to insert a DNA sequence into the coding or noncoding region of a target gene, resulting in frameshift mutations, premature termination of transcription and / or translation, or translation of an entirely different polypeptide, thereby permanently inactivating the gene. The proportion of gene expression reduction achieved by different methods can range from 100%, greater than 90%, greater than 80%, greater than 70%, greater than 60%, greater than 50%, greater than 40%, greater than 30%, or greater than 20%.

[0208] "Inactivated" means loss of function, or loss of protein function.

[0209] 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, encompassing nucleic acids containing known nucleotide analogs or modified backbone residues or connections, the nucleic acids being synthetic, naturally occurring, and non-naturally occurring, having binding properties similar to reference nucleic acids, and 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). A nucleic acid encoding a polypeptide or fusion protein refers to one or more nucleic acid molecules encoding a polypeptide or fusion protein, 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 encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as sequences explicitly indicated.

[0210] 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 into which the genetic element is expressed, such as to produce a protein, RNA, or DNA encoded by the genetic element, or to replicate the genetic element. A 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector can contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector can also contain a replication initiation site. A vector can also include components that assist its entry into cells, including, but not limited to, viral particles, liposomes, or protein coats. A vector can be an expression vector or a cloning vector. In some embodiments, the vector provided by the present invention (e.g., an 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.

[0211] As used herein, the term "cell" in the present invention refers to a eukaryotic cell, particularly a mammalian cell, such as a human cell.

[0212] As used herein, the term "immune cell" in the present invention refers to a cell from a human. More specifically, the immune cells in the present invention can be derived from pluripotent stem cells through 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 isolated 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 isolated 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 typically 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 lymphocyte progenitor cells.

[0213] "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, and 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.

[0214] "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, lymphoid or other tissues, or differentiated from pluripotent stem cells.

[0215] "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, megakaryocyte stem cells, precursor B cells, precursor T cells, NK progenitor cells, and macrophage progenitor cells.

[0216] As used herein, "NK cells" refer to natural killer cells, generally CD56 + CD45 + Double positive, simultaneously expressing multiple activating receptors, such as NKG2D, NKp44, NKp46, etc. NK cells can be differentiated from pluripotent stem cells (such as iPSCs), isolated from peripheral blood, differentiated from CD34+ cells (such as umbilical cord blood or placental blood), or obtained from tumor cell lines (such as NK92).

[0217] 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 cells can be 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. "CBNK" refers to NK cells derived from cord blood, and "WT iNK" 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.

[0218] As used herein, the term "composition" is in a form that permits the biological activity of the active ingredient to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0219] As used herein, the term "treat," ...

[0220] As used herein, the term "diagnosis" refers to the identification, disclosure, identification, and / or definition of the pathogenic process of a pathological state, disease, or condition. In some embodiments, the pharmaceutical compositions of the present invention, when administered to a subject or contacted with a sample from a subject, aid in the diagnosis of cancer, neoplasia, or condition.

[0221] As used herein, an autoimmune disease is a disease caused by the immune system mistakenly attacking the body's own tissues. There are currently over 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.

[0222] In this article, viral infectious diseases refer to diseases caused by viral infection, which 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.

[0223] 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 human suspected of having cancer, an autoimmune disease or condition, and / or an infection.

[0224] As used herein, the term "tumor antigen" is a general term for proteins and polypeptide molecules that appear and are highly expressed 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-associate 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 tumors induced by chemical and physical carcinogens, a protein expressed by tumors induced by viruses, an abnormal protein encoded by oncogenes and mutant tumor suppressor genes, or a protein expressed after the activation of dormant genes. 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; ISBN 978-7-03-053131-5; p339).

[0225] Chimeric antigen receptor

[0226] The present invention provides a chimeric antigen receptor, comprising:

[0227] An extracellular domain capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0228] 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

[0229] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0230] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0231] SNLFVASWIAVMX8IFX9IGX 10 AX 11 AX 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 PS

[0232] wherein X8 is selected from I or V;

[0233] X9 is selected from R or H;

[0234] X 10 Choose from M or L,

[0235] X 11 Selected from V or I;

[0236] X 12 Selected from I or V,

[0237] X 13 Select from S or F;

[0238] X 14 Selected from C, R or H;

[0239] X 15 Selected from C, T or V,

[0240] X 16 Selected from V, L or absent;

[0241] X 17 Selected from F or H,

[0242] X 18 Selected from F or Y,

[0243] X 19 Selected from L or absent,

[0244] X 16 and X 19 Cannot exist at the same time, and X 16 and X 19 Can't exist at the same time.

[0245] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0246] 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).

[0247] 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.

[0248] The present invention provides a chimeric antigen receptor (CAR), comprising:

[0249] an extracellular domain capable of binding to a tumor antigen;

[0250] 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

[0251] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0252] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0253] SNLFVASWIAVMX1IFX2IGMAX3AIX4X5CX6FFX7PS

[0254] wherein X1 is selected from I or V;

[0255] X2 is selected from R or H;

[0256] X3 is selected from V or I;

[0257] X4 is selected from S or F;

[0258] X5 is selected from C or R;

[0259] X6 is selected from L or is absent;

[0260] X7 is selected from L or is absent,

[0261] The tumor antigen may be a tumor-specific antigen or a tumor-associated antigen.

[0262] 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.

[0263] The present invention provides a chimeric antigen receptor, comprising:

[0264] An extracellular domain capable of binding to a tumor antigen, an inflammatory factor, or a pathogen antigen;

[0265] 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

[0266] an intracellular domain, the intracellular domain being connected to the transmembrane domain,

[0267] Wherein, the amino acid sequence of the transmembrane domain has the following sequence:

[0268] SNLFVASWIAVMIIFX 20 IGX 21 AX 22 AX 23 SX 24 X 25 X 26 X 27 X 28 PS

[0269] Among them, X 20 Selected from R or H,

[0270] X 21 Choose from M or L,

[0271] X 22 Selected from V or I,

[0272] X 23 Selected from V or I,

[0273] X 24 is selected from C, R or H,

[0274] X 25 Selected from C, T or V,

[0275] X 26 Selected from L or V,

[0276] X 27 Selected from F or H,

[0277] X 28 Selected from F or Y;

[0278] The tumor antigen is a tumor-specific antigen or a tumor-associated antigen.

[0279] 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).

[0280] 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 to 29.

[0281] According to an embodiment of the present invention, the chimeric antigen receptor proposed above may further include at least one of the following additional technical features:

[0282] 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.

[0283] According to an embodiment of the present invention, the extracellular domain includes an antibody or 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.

[0284] According to an embodiment of the present invention, the extracellular domain further comprises an antibody or an antigen-binding fragment thereof that can specifically recognize pathogen antigens, and the pathogen includes EBV.

[0285] 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.

[0286] According to an embodiment of the present invention, the extracellular domain comprises an antibody or an antigen-binding fragment thereof that can specifically recognize GPRC5D.

[0287] 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:

[0288] Heavy chain variable region CDR sequences: SEQ ID NOs: 1-3;

[0289] The CDR sequence of the light chain variable region is SEQ ID NO: 4, the amino acid sequence SAS, and SEQ ID NO: 5.

[0290] 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:

[0291] The heavy chain variable region CDR1, CDR2, and 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, and 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, 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.

[0292] It should be noted that any antibody or antibody fragment having the above-mentioned heavy chain variable region CDR1, CDR2, CDR3 sequences and light chain variable region CDR1, CDR2, CDR3 sequences is included in the protection scope of the present invention, such as IgG, scFv, Fab, Fab', etc.

[0293] 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.

[0294] 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.

[0295] According to an embodiment of the present invention, the extracellular domain comprises a bispecific binding molecule comprising:

[0296] A first binding region, comprising the antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D; and

[0297] The second binding region has an activity of binding to an antigen specifically expressed on lymphocytes.

[0298] According to an embodiment of the present invention, antibodies that bind to antigens specifically expressed on lymphocytes are all included within the scope of the second binding region of the bispecific binding molecules of the present invention.

[0299] 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. The aforementioned antibodies or antigen-binding fragments thereof that can specifically recognize GPRC5D and bispecific binding molecules that have the activity of binding to antigens specifically expressed on lymphocytes are all included in the scope of protection of the present invention. Preferably, the second binding region can be a whole antibody, a single-chain antibody, or a single-domain antibody.

[0300] 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.

[0301] 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.

[0302] According to a preferred embodiment of the present invention, the bispecific binding molecule comprises an antibody or antigen-binding fragment thereof that specifically recognizes GPRC5D and a single-chain antibody or single-domain antibody that specifically recognizes BCMA. The GPRC5D single-chain antibody and the BCMA single-chain antibody or single-domain antibody are linked by a linker peptide, enabling targeted killing of multiple myeloma cells. Preferably, the bispecific binding molecule has the amino acid sequence set forth in SEQ ID NO: 10.

[0303] 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.

[0304] 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 can 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 can be an intracellular costimulatory signal region derived from 2B4. For example, the costimulatory signal domain has the amino acid sequence shown in SEQ ID NO: 12.

[0305] It should be noted that there is no particular limitation on the selection of the "connector peptide" in the present invention, and it can be, for example, (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 scope of protection of the present invention and can be used as the connecting peptide in the present invention.

[0306] In the present invention, the intracellular signaling domain comprises a domain with 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 the amino acid sequence shown in SEQ ID NO: 13.

[0307] 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 the CD8 protein. For example, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 14.

[0308] 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.

[0309] 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 an anti-GPRC5D single-chain variable region fragment (scFV) or sdAb; b) a hinge region; c) a transmembrane domain; d) a costimulatory signaling domain; and (d) an intracellular signaling domain.

[0310] The polypeptide encoding the anti-GPRC5D CAR, wherein the amino acid sequence of the anti-GPRC5D scFV is shown in SEQ ID NO: 8.

[0311] According to a specific embodiment of the present invention, from N-terminus to 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, and the intracellular signaling domain (as shown in Figure 1A).

[0312] 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 costimulatory signaling domain derived from 2B4, and an intracellular signaling domain derived from CD3ζ.

[0313] 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 co-stimulatory signaling domain, and the intracellular signaling domain, as shown in Figure 1 B. The anti-antigen 2 scFV or sdAb may be an scFV or sdAb that targets an antigen specifically expressed on lymphocytes.

[0314] 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 orientation, a costimulatory signaling domain derived from 2B4, and an intracellular signaling domain derived from CD3ζ.

[0315] According to a more specific embodiment of the present invention, from N-terminus to C-terminus, the CAR may sequentially comprise a signal peptide, GPRC5D scFV, BCMA scFV or BCMA sdAb, the hinge region, the transmembrane domain, the costimulatory signal region and the intracellular signal region.

[0316] The BCMA sdAb has the amino acid sequence shown in SEQ ID NO:9.

[0317] Patent CN 109153731 B provides an anti-BCMA sdAb sequence and CAR molecule, and assembles them onto T cells to develop CAR-T therapy for multiple myeloma. The present invention compares the performance of anti-BCMA sdAb sequences and anti-GPRC5D scFV sequences and CARs on NK cells after assembly, and finds that the anti-GPRC5D CAR NK 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-GPRC5D CAR-iNK can be further improved.

[0318] Polynucleotides, expression vectors, cells, and compositions

[0319] The present invention provides an isolated polynucleotide encoding the chimeric antigen receptor described above.

[0320] The present invention provides an expression vector carrying the polynucleotide described above.

[0321] The present invention provides a cell carrying the aforementioned polynucleotide or the aforementioned expression vector or 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.

[0322] It should be noted that the "suitable conditions" described in this specification refer to conditions suitable for the expression of the chimeric antigen receptor described herein. Those skilled in the art will readily understand that conditions suitable for chimeric antigen receptor expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cells, suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for chimeric antigen receptor expression based on the specific laboratory environment.

[0323] Nucleic acids for antibodies, antigen-binding fragments thereof, bispecific binding molecules, or chimeric antigen receptors described above can be introduced into cells using various tools, such as donor plasmids, piggyBac, dsDNA, ssDNA, and various integrating or non-integrating viruses. If necessary, depending on the chosen delivery tool, PBase can be used to facilitate piggyBac delivery, while nucleases can be used to create single-strand or double-strand breaks to facilitate other non-viral delivery methods.

[0324] The cells provided by the present invention may be recombinant cells carrying the aforementioned polynucleotides or the aforementioned expression vectors, or may be engineered immune cells capable of expressing the aforementioned chimeric antigen receptors.

[0325] According to an embodiment of the present invention, immune cells with enhanced function can be obtained by using any one of the following methods or a combination thereof:

[0326] 1) Randomly integrate one or more circular or linear exogenous DNA fragments into target cells;

[0327] 2) One or more circular or linear exogenous DNA fragments are site-specifically integrated into the target cell. Site-specific integration can enhance the efficiency of homologous recombination of the exogenous DNA by introducing a nuclease at the target site. Homologous recombination efficiency can also be enhanced by other physical means, such as electroporation or nuclear transfection.

[0328] 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 through non-homologous end joining;

[0329] 4) One or more endonucleases 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. The cells generate base insertions or base deletions at the selected sites through homologous recombination.

[0330] In this case, the target cell can be the immune cell, stem cell, or progenitor cell mentioned above, or a donor cell for reprogramming into iPSCs, or any cell derived from a stem cell or progenitor cell during the process of differentiating into an immune cell. The exogenous DNA fragment may contain a gene expression cassette, i.e., at least components such as a promoter and an open reading frame, or may not contain a gene expression cassette.

[0331] 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, temporal, 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 insertion site of the exogenous DNA fragment can be the AAVS1 site or other site where cell function is not affected by the insertion of the exogenous gene, or a site where the gene at the target site is destroyed after insertion, or the stop codon position of a functional gene.

[0332] The construction of the CAR expression vector can be in various forms, as shown in Figures 1 and 2. It can be inserted into the exon position of the target gene through the 2A or IRES sequence In-frame, and the expression is started by borrowing the promoter of the target gene (Figure C in Figure 1). In some embodiments, the expression cassette may also include a promoter and a polyadenylic acid sequence (Figure D in Figure 1). After integration into the genome, the expression cassette uses the expression elements it carries to start expression. The expression cassette can also express multiple polypeptides at the same time, wherein two or more polypeptide coding sequences can be connected with a 2A or IRES sequence. As an example, an expression cassette comprising a promoter, anti-GPRC5D CAR-2A / IRES-hnCD16, and polyadenylic acid, anti-GPRC5D CAR and hnCD16 can be expressed simultaneously under the drive of an exogenous promoter (Figure E in Figure 1).

[0333] In some embodiments, the genetically modified immune cells produced by the above methods or the cells from which they are derived express genetically engineered antigen receptors, which can be chimeric antigen receptors (CARs), T cell receptors (TCRs), or NK cell receptors (NCRs).

[0334] 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, particularly, 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 isolated from peripheral blood, umbilical cord blood, or other tissues (including tumor tissue) or organs, CD34 + cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells.

[0335] In some embodiments, the genetically modified immune cells produced using the methods provided herein or cells derived therefrom, 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 tissue) or organs. + cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells.

[0336] 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.; and 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 the 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 tissue) or organs. + cells, hemogenic endothelial cells, hematopoietic stem and progenitor cells.

[0337] 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 derivative cells obtained by differentiating any of the above embryonic stem cells or iPSCs.

[0338] Any of the above cells encodes at least a polypeptide that can recognize a chimeric antigen receptor (anti-GPRC5D CAR or anti-GPRC5D CAR) that can recognize GPRC5D.

[0339] In some embodiments, the derived cells obtained by differentiation of iPSCs 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.

[0340] In some embodiments in which the iPSCs and their derivatives contain at least a chimeric antigen receptor that recognizes GPRC5D, the cells may further comprise one or more of the following genome editings: (i) B2M and CIITA knockout or low expression; (ii) introduction of expression of CD47, or HLA-E, or HLA-G (including an indegradable form), or any combination thereof; (iii) introduction of expression of high-affinity indegradable CD16 (hnCD16) or its variants; (iv) introduction of expression of a second or third CAR; (v) introduction of a second or third sc (vi) expression of FV or single domain antibody (sdAb); (vi) deletion or reduced expression of TGFBR2, PVR, NKG2A, TIM3, TIGIT, CD38, TCR; and (vii) introduction or increase of 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 to a universal adapter.

[0341] In some embodiments of the cells or their populations, the one or more cells comprising the anti-GPRC5D CAR are primary NK cells or NK cells differentiated from pluripotent stem cells, hematopoietic stem cells and progenitor cells, or NK cell progenitor cells, or their populations. 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 that express GPRC5D.

[0342] 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 contains tumor cells expressing GPRC5D in their body. 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, okatuzumab, obinutuzumab, trastuzumab, pertuzumab, alemtuzumab, cetuximab, denosumab, avelumab, daratumumab, isatuximab, etozuzumab.

[0343] The present invention also provides a method for obtaining or producing the above-mentioned cells, which comprises directing the differentiation of one or more iPSCs containing a chimeric antigen receptor that recognizes GPRC5D.

[0344] The present application further provides a method for obtaining one or more iPSCs containing the above-mentioned anti-GPRC5D CAR. 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 iPSC: 1) using a homologous recombination method to insert an exogenous DNA fragment into a target site, and targeting the target site can be achieved 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 double-strand break site in the genome; 3) using tools such as viruses, transposons, and plasmids to randomly integrate exogenous DNA fragments into the host genome.

[0345] The present invention further provides a method for obtaining one or more iPSCs containing the aforementioned chimeric antigen receptor polypeptide that recognizes GPRC5D. This method comprises further performing at least one of the following genetic modifications, or a combination thereof: 1) introduction of high-affinity, uncleavable CD16 or its variant; 2) knockout of CD38; 3) knockout of B2M and CIITA, and / or PVR; and introduction of CD47, and / or HLA-G or uncleavable HLA-G, and / or HLA-E; and 4) membrane-bound IL15 (mbIL15). These selected genetic modifications can be performed all at once or in separate steps. The introduced exogenous gene expression cassette or exogenous polypeptide can be expressed independently or co-expressed in a bicistronic construct. Expression can be driven by an exogenous promoter or by leveraging the promoter of an endogenous gene for stable or tissue-specific expression. The exogenous promoter can be any one or a combination of the following: CAG, CMV, EF1α, PGK, UBC, or other constitutive, inducible, temporal, 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.

[0346] The present invention further provides a method for expressing anti-GPRC5D CAR mediated by piggyBac, so as to obtain the above-mentioned method of 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 piggyBac transposition region (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.

[0347] Figure 2, Panel A, illustrates several expression cassettes containing anti-GPRC5D CAR polypeptides. These expression cassettes can be cloned into piggyBac plasmids and randomly integrated into the host genome with the help of PBase. Figure 2, Panel B, illustrates the insertion of CAG-anti-GPRC5D CAR-BGHpA into the piggyBac transposable region to generate the anti-GPRC5D CAR plasmid.

[0348] The DNA encoding the anti-GPRC5D CAR can be introduced using various tools, such as donor plasmids, piggyBac, dsDNA, ssDNA, and various integrating or non-integrating viruses. Depending on the chosen introduction tool, PBase can be used to facilitate piggyBac introduction, while nucleases can be used to create single-strand or double-strand breaks to facilitate other non-viral introduction methods.

[0349] Figure 3, Panel A, shows a schematic diagram of a homologous recombination template containing an anti-GPRC5D CAR. Figure 3, Panel B, provides several examples of DNA templates for homologous recombination containing an anti-GPRC5D CAR expression cassette. These DNA templates can be in the form of single-stranded DNA, double-stranded DNA, or plasmids. Figure 3, Panel C, provides an example of a DNA template containing an anti-GPRC5D CAR and hnCD16 expression cassette targeting any site in the human genome, as well as the genomic location of the expression cassette after homologous recombination.

[0350] The construct forms in Figures 1-3 of the present invention are all included in the protection scope of the construct protected by the present invention.

[0351] 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 embodiments mediated by CRISPR, CRISPR binds to the targeting sequence and produces a double-strand break, and further inserts a DNA sequence encoding an anti-GPRC5D CAR at the targeting sequence by non-homologous end joining or homologous recombination. The targeting sequence can 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.

[0352] 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).

[0353] In some CRISPR-mediated embodiments, the editing further includes expressing at least one of the following genes or polypeptides at at least any one 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.

[0354] 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 homology 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 +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.

[0355] 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 CAR against GPRC5D, 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 (iPSCs), cloned iPSCs or iPSC cell lines; wherein the genome-edited cells comprise random integration of the one or more nucleic acid sequences encoding one or more exogenous polypeptides into the host cell genome; wherein the genome-edited cells are capable of differentiating into CD34 + iPSC cells.

[0356] 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 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 differentiated genome-edited iPSCs include derived NK cells or derived T cells, and have at least one of the following characteristics compared to their native counterparts obtained from peripheral blood, umbilical cord blood, or any other donor tissue, the characteristics comprising at least: (1) specific killing of tumor cells expressing GPRC5D; (2) acquisition or enhancement of ADCC effect.

[0357] The present invention further provides a composition for making gene-edited iPSCs or hematopoietic cells differentiated therefrom, and the composition comprises a donor plasmid, or donor DNA, or piggyBac plasmid as described herein. In addition, a kit is provided herein, and the kit comprises a donor plasmid, or donor DNA fragment, or 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.

[0358] 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 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.

[0359] 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 to benefit cancer patients. One of the objectives of the present invention is to prepare engineered immune cells based on GPRC5D antibodies or bispecific binding molecules containing GPRC5D antibodies, using them as extracellular regions. These immune cells can be isolated from the patient's or donor's peripheral blood, umbilical cord blood, or specific tissues or organs (including tumor tissue or other organs), or can be differentiated from pluripotent stem cells (such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (embryonic stem cells within 14 days of fertilization that have not undergone in vivo development)), hematopoietic stem cells, or various immune cell progenitor cells. The engineering is mainly achieved through genetic modification. The engineering can be completed at the stem cell stage or progenitor cell stage, or at the immune cell stage. The engineering includes one or more genetic modifications, including DNA insertion, deletion, and substitution. These genetic modifications can be completed in one step 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 derivative cells produced during subsequent differentiation, expansion, passaging or transplantation.

[0360] Kit for detecting GPRC5D

[0361] According to a specific embodiment of the present invention, a kit for detecting GPRC5D is provided, comprising the aforementioned antibody or antigen-binding fragment thereof and / or chimeric antigen receptor. The antibody or antigen-binding fragment thereof, or a bispecific binding molecule comprising the GPRC5D antibody, that specifically recognizes GPRC5D can bind to GPRC5D in a sample to be tested, thereby achieving the purpose of in vitro detection of GPRC5D levels.

[0362] Uses and methods

[0363] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned chimeric antigen receptor, polynucleotide, expression vector, cell and / or composition in treating a blood tumor or solid tumor disease, autoimmune disease or viral infectious disease in an individual. According to an embodiment of the present invention, the chimeric antigen receptor can bind to GPRC5D and the second antibody in the bispecific binding molecule through an antibody or antigen-binding fragment thereof that can specifically recognize GPRC5D, or a bispecific binding molecule containing the GPRC5D antibody, thereby effectively treating the blood tumor or solid tumor disease, autoimmune disease or viral infectious disease.

[0364] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease. The chimeric antigen receptor can effectively treat GPRC5D-mediated related diseases.

[0365] 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.

[0366] 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.

[0367] According to an embodiment of the present invention, the viral infectious disease includes but is not limited to EBV infectious disease.

[0368] According to a specific embodiment of the present invention, a method for preventing and / or treating a hematological or solid tumor disease, or an autoimmune disease, or a viral infectious disease in an individual is provided, 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, the chimeric antigen receptor is capable of binding to GPRC5D and the second antibody in the bispecific antibody through an antibody or antigen-binding fragment thereof that specifically recognizes GPRC5D, or a bispecific binding molecule comprising the GPRC5D antibody, thereby effectively treating the hematological or solid tumor disease, autoimmune disease, or viral infectious disease. The effectiveness of the method for preventing and / or treating hematological or solid tumor diseases, autoimmune diseases, or viral infectious diseases will not be further described herein.

[0369] According to an embodiment of the present invention, the hematological tumor or solid tumor disease is a GPRC5D-related disease.

[0370] 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.

[0371] According to an embodiment of the present invention, the viral infectious disease includes but is not limited to EBV infectious disease.

[0372] According to a specific embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0373] According to a specific embodiment of the present invention, the present invention provides the use of the aforementioned chimeric antigen receptor, polynucleotide, and expression vector for detecting GPRC5D or diagnosing whether an individual suffers from a GPRC5D-related disease. As previously described, the chimeric antigen receptor of the present invention can effectively bind to GPRC5D. Therefore, the chimeric antigen receptor can be used to detect GPRC5D. 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.

[0374] According to a specific embodiment of the present invention, the GPRC5D-related disease includes multiple myeloma.

[0375] According to a specific embodiment of the present invention, a method for detecting GPRC5D is provided, comprising detecting GPRC5D in a sample using at least one of the following: a chimeric antigen receptor, a polynucleotide, and an expression vector as described above. As described above, the chimeric antigen receptor of the embodiments of the present invention can effectively bind to GPRC5D; therefore, the chimeric antigen receptor can be used to detect GPRC5D.

[0376] The method for detecting GPRC5D according to an embodiment of the present invention can be used in scientific research, and the sample includes but is not limited to tissue, cells, blood, serum, plasma, urine, feces, saliva or sweat.

[0377] According to a specific embodiment of the present invention, a method for diagnosing whether an individual suffers from a GPRC5D-related disease is provided. The method comprises detecting GPRC5D in a sample derived from the individual using at least one of the following: the chimeric antigen receptor, polynucleotide, and expression vector described above. As described above, the chimeric antigen receptors of the embodiments of the present invention can effectively bind to GPRC5D, and therefore, the chimeric antigen receptors can be used to detect GPRC5D. GPRC5D mediates a variety of diseases, and therefore, the level of GPRC5D in a biological sample derived from the individual can be used to determine whether the individual suffers from a GPRC5D-related disease. Furthermore, the above-mentioned substances can also be used to monitor the level of GPRC5D in a sample derived from the individual, i.e., the above-mentioned substances can be used to stage the disease in individuals suffering from GPRC5D-related diseases and to assess the prognosis of GPRC5D-related diseases.

[0378] 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.

[0379] According to a specific embodiment of the present invention, the sample includes but is not limited to tissue, cells, blood, serum, plasma, urine, feces, saliva or sweat.

[0380] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand 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 specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0381] Example 1 Anti-GPRC5D CAR polypeptide structure

[0382] Through hybridoma cell screening technology, anti-GPRC5D antibodies that specifically bind to GPRC5D were 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).

[0383] (1) As shown in Figure 1A, the anti-GPRC5D CAR structure includes the antigen recognition domain (anti-GPRC5D scFV), hinge region, transmembrane domain, costimulatory signaling domain, and intracellular signaling domain from the N-terminus to the C-terminus of the polypeptide.

[0384] The amino acid sequence of the anti-GPRC5D CAR is shown in SEQ ID NO: 15:

[0385] 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.

[0386] 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):

[0387] (2) As shown in Figure 1B, the anti-GPRC5D CAR structure includes the antigen recognition domain (anti-GPRC5D scFV), BCMA sdAb, hinge region, transmembrane domain, costimulatory signaling domain, and intracellular signaling domain from the N-terminus to the C-terminus of the polypeptide.

[0388] The amino acid sequence of GPRC5D scFV-BCMA sdAb CAR is shown in SEQ ID NO: 16, wherein the transmembrane domain is underlined:

[0389] In the present invention, the 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):

[0390] Example 2 Anti-GPRC5D CAR molecule introduction

[0391] The expression cassette shown in a of Figure 2A was cloned into the piggyBac vector carrying Puromycin resistance to obtain the plasmid anti-GPRC5D CAR (see Figure 2B). The transmembrane domain sequence in the CAR molecule of the present invention can 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 anti-GPRC5D CAR and the plasmid expressing transposase (PBase) were transfected by a nuclear transfection instrument (Amaxa, II) were co-transfected into a healthy human iPSC (WT iPSC, Sanqi Bio's own cells) using a nucleofection kit (LONZA, CAT#VPH5022). Transfection procedures can be referred to the kit's instructions. After selection with 0.5 μg / ml Puromycin for 2-7 days, iPSCs expressing the anti-GPRC5D CAR molecule (anti-GPRC5D CAR-iPSCs) were expanded and cultured. FACS analysis was performed using a mouse anti-anti-GPRC5D scFV antibody (primary antibody, Kangyuan Broad) and PE goat anti-mouse IgG (secondary antibody, Biolegend) to identify the proportion of iPSCs expressing the anti-GPRC5D CAR molecule (see Figure 5, Panel A), indicating that iPSCs expressing the anti-GPRC5D CAR molecule were obtained. Using the same method, iPSCs expressing a dual-antigen CAR molecule against GPRC5D and BCMA (anti-GPRC5D / BCMA CAR-iPSCs) were obtained.

[0392] Example 3 Differentiation of iPSCs Expressing Anti-GPRC5D CAR Polypeptide into CAR-iNK

[0393] Figure 4 shows the general process for obtaining gene-edited iPSC-derived anti-GPRC5D CAR-iNK cells. The iPSCs expressing the anti-GPRC5D CAR polypeptide obtained in Examples 2 and 3 were differentiated into CAR-iNK cells using the following specific steps.

[0394] 1) When the iPSC cell confluence reaches 75% to 85%, wash once with DMEM / F12 medium, add Accutase digestion enzyme or TrypLE digestion enzyme and place in a 37°C, 5% CO2 incubator for digestion. Then, centrifuge and discard the supernatant. Use mTeSR supplemented with 5μM Y27632, 50ng / mL BMP4 and 10μM CHIR99021. TM1. Resuspend the cells in culture medium to obtain single-cell iPSCs;

[0395] 2) Transfer an appropriate number of single-cell iPSCs to AggreWell TM Microplate, according to 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 incubate at 37°C, 5% CO2 and 95% humidity for 1 to 2 days. Then, replace with the second differentiation medium, and change the medium every 2 days thereafter 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;

[0396] 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.

[0397] 4) After the above three stages of culture, generally on the 10th to 14th day after differentiation, the CD34 + Depending on the FACS results, CD34 + When the ratio exceeds 80%, the fourth differentiation medium can be replaced for further differentiation 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 quantity. Expansion medium formula: GT-T551 H3 medium with 5% FBS, 1000IU / mL IL-2, and 10ng / mL IL-15.

[0398] All antibodies (including isotype controls and secondary antibodies) were purchased from Biolegend, except for the anti-GPRC5D scFV antibody from Kangyuan Broad. For FACS analysis, refer to the commonly used operating procedures for the relevant instruments. The results of FACS analysis of GPRC5D CAR-iNK expression levels of CD56, CD45, NKG2D, TCRαβ, and TCRγ are shown in Figures 6 to 8. These results demonstrate that expression of the anti-GPRC5D CAR has no adverse effects on the differentiation and expansion of anti-GPRC5D CAR-iNK cells. The expression levels of NK-related markers are similar between the two cells, with anti-GPRC5D CAR-iNK cells expressing NKG2D (NK cell activating receptor) at higher levels than WT iNK cells.

[0399] Example 4. Luciferase method to determine the killing ability of NK cells against tumor cells

[0400] Tumor cells K562-Luc were used as target cells, and anti-GPRC5D CAR-iNK, CBNK, WT iNK, and anti-BCMACAR-iNK cells were used as effector cells. The cytotoxicity of NK cells against target cells was tested at an effector-target ratio of 4:1. The specific experimental procedures are as follows:

[0401] Prepare a K562-Luc target cell suspension (target cell viability no less than 90%) and, after counting, plate cells into the wells of a 96-well plate at the specified effector-target ratio (fixed effector-target ratio 4:1). Set three replicates per well for each effector cell at each effector-target ratio. Adjust the volume of culture medium to 100 μL per well, 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% CO2 incubator and incubate for the specified time (e.g., 4 hours). Dispense luciferin substrate and dilute it 100-fold with cell culture medium. Add 50 μL of the luciferin substrate dilution to each well and incubate at room temperature in the dark for 10 minutes. Set the light source to Lum and the timer to 1000 ms on a microplate reader for reading. 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, except that the GPRC5D antibody in the anti-GPRC5D CAR polypeptide is replaced with a BCMA antibody (BCMA antibody (BCMA sdAb) amino acid sequence as shown in SEQ ID NO: 24) or a GPRC5D / BCMA bispecific antibody (GPRC5D / BCMA bispecific antibody amino acid sequence as shown in SEQ ID NO: 10).

[0402] The results are shown in Figure 9A. Different effector cells showed similar cytotoxicity against K562-Luc. Since K562-Luc does not express GPRC5D or BCMA, Figure 9A shows that these effector cells had similar basal cytotoxicity against target cells.

[0403] Example 5. Determination of basal killing activity of effector cells against target cells

[0404] 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, and 1:1), and the killing ability of NK cells against K562-Luc cells was determined according to the method in Example 4. Figure 9B shows that the basal killing ability of WT iNK, anti-GPRC5D CAR-iNK, and anti-BCMACAR-iNK against target cells K562-Luc was dose-dependent.

[0405] Example 6. Anti-GPRC5D CAR molecules enhance iNK cell proliferation and GPRC5D + Tumor cell killing activity

[0406] Effector cells (CBNK, WT iNK, GPRC5D CAR-iNK, anti-BCMACAR-iNK, anti-GPRC5D / BCMA CAR-iNK) and target cells NCI-H929-Luc2 (hereinafter referred to as H929-Luc2) that highly express 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). Unless otherwise specified, they were co-cultured at 37°C and 5% CO2 for 4 hours. The killing of target cells H929-Luc2 by iNK cells of different groups was determined using the method provided in Example 4.

[0407] The results, as shown in Figure 10A, show that at an effector-target ratio of 4:1 and a 4-hour incubation period, CBNK cells were insensitive to H929-Luc2 cells, exhibiting virtually no killing. WT iNK cells killed approximately 10% of H929-Luc2 cells, while anti-GPRC5D CAR-iNK cells killed approximately 90% of H929-Luc cells. Figure 10B demonstrates that the killing effects of WT iNK cells and anti-GPRC5D CAR-iNK cells on H929-Luc2 cells were dose-dependent, decreasing with decreasing effector-target ratio.

[0408] 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 similar to that in Example 4 above. The results, as shown in Figure 11, show that at the same effector-target ratio, the killing effect of the anti-GPRC5D CAR-iNK was stronger than that of the anti-BCMACAR-iNK. In addition, the anti-GPRC5D / BCMACAR-iNK showed a better killing effect at a low effector-target ratio.

[0409] In summary, the expression of anti-GPRC5D CAR molecules can make NK cells have a much better effect on GPRC5D than WT iNK cells or CBNK cells. + The killing ability of CAR-iNK cells was significantly improved, and the expression of anti-GPRC5D CAR molecules had a better killing effect on the MM tumor cell line H929-Luc2 than that of iNK cells 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 was further improved. In addition, after loading anti-GPRC5D or anti-BCMA chimeric antigen receptors at the iPSC stage, these iPSC cells were further differentiated into anti-GPRC5D or anti-BCMA CAR-iNK cells, which could specifically kill target cells expressing GPRC5D or BCMA.

[0410] Example 7. Anti-GPRC5D CAR molecules enhance the activity of iNK cells against GPRC5D after cryopreservation and thawing. + Tumor cell killing activity

[0411] The effector cells of this example are the iNK cells (anti-GPRC5D CAR-iNK and WT iNK) differentiated in Example 3, and the target cells are K562-Luc. The cytotoxicity of the revived iNK cells against tumor cells is detected.

[0412] 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 9 mL into a new centrifuge tube. Take out the cryopreservation tube from liquid nitrogen, rub it with your hands a few times, and then quickly transfer it to a 37°C water bath or metal bath, shaking it continuously until only a small piece of ice remains in the cryopreservation tube. After passing through the transfer window, place it in a safety cabinet after alcohol disinfection. Gently open the cell cryopreservation tube, pipette it, and gently transfer the cells to a centrifuge tube containing the culture medium and mix well. Mix the cell suspension and centrifuge it (300g*5min). Remove the supernatant, add 10 mL of NK culture medium again, resuspend and mix, and count. Centrifuge and remove the supernatant. Depending on 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.

[0413] iNK cells were collected within 72 hours of resuscitation, and the cytotoxic activity of these NK cells against target cells K562-Luc was determined using the method described in Example 4. As shown in Figure 12A, the basal cytotoxicity of both resuscitated WT iNK and anti-GPRC5D CAR-iNK cells showed a dose-dependent effect, with the basal cytotoxicity of anti-GPRC5D CAR-iNK cells slightly higher than that of WT iNK cells. However, Figure 12B shows that the resuscitated WT iNK cells completely lost their cytotoxicity against H929-Luc2, while the resuscitated anti-GPRC5D CAR-iNK cells still had a good cytotoxicity against H929-Luc2.

[0414] Comparison of the basal 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 basal killing ability of anti-GPRC5D CAR-iNK, but had little effect on its specific killing ability (see Figure 13, Panels A and B).

[0415] The killing ability of WT iNK and anti-GPRC5D CAR-iNK after cryopreservation and resuscitation against other multiple myeloma tumor cell lines was further tested. The results in Figure 14 show that regardless of whether the target cells are cells with high abundance of GPRC5D expression (MM.1S-Luc2) or cells with low abundance of GPRC5D expression (OPM-2-Luc2), the anti-GPRC5D CAR-iNK after cryopreservation and resuscitation has a high killing ability against them, while WT iNK only shows 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).

[0416] The above results indicate that the freezing 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 target cells with high and low abundance of GPRC5D expression.

[0417] Example 8. Anti-GPRC5D CAR molecule optimization

[0418] 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 improve 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) to be used 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 sequences shown in Table 1 to obtain 14 anti-GPRC5D CAR molecule variants (CAR-TM1~14; hereinafter represented by TM1, TM2, TM3, TM4, TM4, TM5..., TM14). 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 293T cells with 2.5 μg of the transposon plasmid prepared above. 24 hours later, flow cytometry was used to detect the expression of anti-GPRC5D scFV (anti-GPRC5D scFV) in the CAR molecule. Antibody information is shown in Example 2. The results are shown in Figure 15. The expression level of the CAR molecule containing TM1 was similar to that of the control CAR molecule (TM control); the expression level of the CAR molecule containing TM4, TM5, TM6, and TM8 was slightly higher than that of the control CAR molecule (TM control). The effects of TM9 to TM14 were not further tested in subsequent experiments.

[0419] Furthermore, 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-mentioned iPSC expressing exogenous anti-GPRC5D CAR was differentiated into NK cells. iHSPCs were obtained on day 10 of differentiation. At this time, the CD34 expression level of iHSPCs containing TM6 CAR molecules was slightly lower, while the CD34 expression levels of other iHSPCs were above 70% (Figure 16). The CD43 expression levels in all iHSPCs were similar, all above 97% (Figure 16). We further differentiated the above-mentioned 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-iNK cells containing transmembrane sequence variants were higher than those in the control group (TM control) (Figure 17). This shows that the use of CAR molecules containing transmembrane sequence variants not only did not affect the differentiation of iPSC into NK cells, but may have different degrees of enhancement effects on their differentiation. At the same time, at the iHSPC stage, the expression level of CD34 in CAR carrying TM6 was low (50.18%), which did not affect its further differentiation into NK cells (Figure 17). The Day 23CD56 expression level was further counted later. + The proliferation of CAR-iNK cells relative to iPSC cells at the initial differentiation stage showed that CD56 containing TM variants + CAR-iNK cells had similar or higher proliferation rates than control cells. The proliferation rate of cells in the TM5 group exceeded 100 times that of the control group, the proliferation rate of cells in the TM4, TM7, and TM8 groups exceeded 10 times that of the control group, and the proliferation rate of cells in the TM6 group exceeded 7 times that of the control group (Figure 18).

[0420] Furthermore, the inventors further expanded Day 23 CAR-iNK cells to 36 days to 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% (of which the TM5 and TM8 groups were above 99%), and the CAR molecule expression levels were above 95% (measured by anti-GPRC5D scFV expression levels; Figure 19, i.e., Figures 19A, 19B, 19C and 19D). They showed a highly pure mature NK cell phenotype, and the CAR molecule expression levels of the TM variant group and the TM control group were similar.

[0421] 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, respectively, with K562-Luc and K562-GPRC5D-Luc (K562-Luc cell lines expressing exogenous GPRC5D antigen; cells were from Shenzhen Sanqi Biotechnology Co., Ltd., and their construction method was to overexpress GPRC5D protein in K562 cells by lentivirus) as target cells, with a 1:1 effector-target ratio, and CAR-iNK carrying TM control or TM variant CAR molecules were co-incubated for 4 hours to measure the killing ability of these CAR-iNK on target cells. Since iNK can exert a broad-spectrum killing ability, it has a certain degree of killing on K562-Luc, and the CAR molecules carrying anti-GPRC5D can further enhance the iNK's effect on GPRC5D. + Target cells exert antigen-dependent specific killing ability. Therefore, this experiment measured the killing ability of each CAR-iNK group against K562-Luc and K562-GPRC5D-Luc. The antigen-dependent specific killing ability of the anti-GPRC5D CAR-iNK due to the expression of the CAR molecule was obtained by subtracting the killing ability of the CAR-iNK group against K562-GPRC5D-Luc from the killing ability of the CAR-iNK group against K562-Luc. The results are shown in Figure 20. The TM5, TM6, and TM8 groups showed significantly enhanced antigen-dependent specific killing ability against K562-GPRC5D-Luc compared to the TM control group. While the TM1, TM4, and TM control groups showed similar antigen-dependent specific killing ability against K562-GPRC5D-Luc, the TM2, TM3, and TM7 groups showed no significant antigen-dependent specific killing ability against K562-GPRC5D-Luc.

[0422] In summary, compared with CAR molecules carrying the reverse NKG2D transmembrane domain (NKG2D-TM control, or abbreviated as TM control), CAR molecules carrying TM5, TM6, and TM8 variants can give NK cells better antigen-dependent specific killing ability; CAR molecules carrying TM1 and TM4 variants can give NK cells similar antigen-dependent specific killing ability. At the same time, TM1 to TM8 variants do not affect the differentiation and expansion 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 more than 100 times that of the control group), followed by TM4, TM7, and TM8 (CD56 + NK cell proliferation rate 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 times, 3.5 times and 1.7 times that of the control group, respectively.

[0423] Table 1: Transmembrane region sequences used in CAR molecules

[0424] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify 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, CDR3 sequences as 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, CDR3 sequences as set forth in SEQ ID NO: 4, 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, 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, wherein the signal peptide is connected to the N-terminus of the extracellular domain of the chimeric antigen receptor. even; 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.

Images