Chimeric antigen receptor simultaneously targeting GPRC5D and CS1 and application thereof

Abstract

The invention discloses a chimeric antigen receptor simultaneously targeting GPRC5D and CS1 and application thereof, and relates to the technical field of biological medicines. Spatial epitopes of a GPRC5D antigen and a CS1 antigen are taken as target spots, an scfv region of a GPRC5D antibody and an scfv region of a CS1 antibody are connected in series through Linker, and an optimal structure VH1-VL1-VH2-VL2 is obtained through screening, VH1 and VL1 are anti-GPRC5D scfv, and VH2 and VL2 are anti-CS1scfv. The chimeric antigen receptor constructed by connecting the optimal structure VH1-VL1-VH2-VL2 with a transmembrane region of CD8, a CD137 intracellular signal region and a CD3zeta intracellular signal region in series is used for treating the multiple myeloma, so that the problems of low remission rate and high relapse rate of a CAR-T cell therapy in treatment of the multiple myeloma are solved, and the chimeric antigen receptor is still effective for the multiple myeloma with BCMA negative or low BCMA antigen expression and can be used for treating the multiple myeloma. And a new strategy is provided for clinical treatment of multiple myeloma patients with negative BCMA expression or recurrence after BCMA CAR-T treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chimeric antigen receptor simultaneously targeting GPRC5D and CS1 and an application thereof. Background Art

[0002] Multiple myeloma (MM) is the second largest malignant tumor in hematology. It originates from the malignant proliferation of plasma cells in the hematopoietic tissue of the bone marrow, which eventually leads to organ or tissue damage. The main clinical manifestations include damage to the skeletal system, kidney damage and blood system damage. In the past decade, the treatment of multiple myeloma has been mainly based on comprehensive treatment, including chemotherapy, radiotherapy, autologous stem cell transplantation, etc. ① Chemotherapy, including traditional chemotherapy drugs (such as melphalan, doxorubicin and cyclophosphamide, etc.), glucocorticoids (such as dexamethasone, prednisone, etc.), immunomodulators (such as thalidomide, lenalidomide, pomalidomide, etc.) and protease inhibitors (such as bortezomib, carfilzomib, ixazomib, panobinostat, etc.); ② Radiotherapy, used for patients with limited myeloma, local bone pain and spinal cord compression symptoms; ③ Autologous stem cell transplantation, by destroying the tumor microenvironment, directly eliminates myeloma cells and promotes T cell-mediated anti-myeloma response. Although the patient's condition has improved, the disease is still inevitably recurring. Due to the disease characteristics of MM that it is difficult to cure and prone to drug resistance and recurrence, the current treatment methods are far from meeting the current medical needs. How to change the poor survival status of patients with refractory relapsed multiple myeloma and improve their quality of life remains a difficult problem that needs to be solved urgently.

[0003] In March 2021, Bristol-Myers Squibb's (BMS) BCMA-targeted CAR-T cell therapy Abecma (idecabtagenevicleucel, ide-cel) was approved by the U.S. FDA, becoming the first BCMA CART cell therapy approved worldwide: for the treatment of adult patients with R / RMM who have previously received 4 or more therapies (including immunomodulators, proteasome inhibitors, and anti-CD38 antibodies). Data from the key Phase 2 KarMMa study showed an overall response rate (ORR) of 72%, a strict complete response rate (sCR) of 28%, a median duration of response (DoR) of 11 months, and a median DoR of 19 months for sCR patients.

[0004] Although many CARTs for MM have been approved for marketing, the remission rate is limited and the patient relapse rate is also high. This may be because the amount of BCMA antigen expressed by MM cells decreases after CAR-T treatment, or the antigen epitope changes under selection pressure and is no longer recognized by CAR-T cells, as well as the heterogeneity of tumor cells. Therefore, in order to improve the survival period of MM patients, especially to provide an effective treatment for patients with multiple myeloma who relapse after BCMA CAR-T treatment, the present invention is proposed. Summary of the invention

[0005] To solve the above technical problems, the present invention uses GPRC5D and CS1 as dual targets, simultaneously targets two surface markers of multiple myeloma, and optimizes the CAR structure, which can not only effectively prevent tumor recurrence caused by the loss of a single target, but also performs excellently in targeted killing of multiple myeloma cells. The effect is also better than the combination of GPRC5D-CART and CS1-CART, can effectively treat tumors and reduce the tumor recurrence rate after treatment, thereby prolonging the patient's survival period.

[0006] The first object of the present invention is to provide a chimeric antigen receptor that simultaneously targets GPRC5D and CS1 for use in preparing a drug for treating multiple myeloma, wherein the chimeric antigen receptor that simultaneously targets GPRC5D and CS1 comprises an extracellular antigen binding region, wherein the extracellular antigen binding region is a GPRC5D antibody heavy chain, a GPRC5D antibody light chain, a CS1 antibody heavy chain, and a CS1 antibody light chain that are sequentially connected in series via a flexible linker;

[0007] The sequence of the GPRC5D antibody heavy chain is shown in SEQ ID NO.1, the sequence of the GPRC5D antibody light chain is shown in SEQ ID NO.2, the sequence of the CS1 antibody heavy chain is shown in SEQ ID NO.3, and the sequence of the CS1 antibody light chain is shown in SEQ ID NO.4.

[0008] GPRC5D is a G protein-coupled orphan receptor. Under normal circumstances, its expression is limited to hair follicles. In multiple myeloma cells, it is specifically highly expressed in malignant plasma cells, so it is a potential excellent target for the treatment of multiple myeloma. However, GPRC5D is not expressed or expressed at a low level on normal B cells and plasma cells, so GPRC5D CAR-T cells will not attack normal B cells and plasma cells, which is better than BCMCAR-T, which is also highly expressed on normal B cells and plasma cells. CS1 (also known as CD319, SLAMF7) is a member of the signaling lymphocyte activation molecule family 7 and is a surface receptor protein that regulates the immune function of NK cells. CS1 is highly expressed in multiple myeloma (MM) cell lines, but is not found in healthy tissues, primary tumor tissues, or hematological and non-hematological cancer cell lines, and is an excellent target for the treatment of multiple myeloma. However, there are no reports of CAR-T therapy combining these two targets, let alone optimization of their structures to ensure synergy. Moreover, it is well known that the challenges faced by tandem CAR-T cell therapy strategies include design complexity, possible structural optimization requirements, and factors affecting CAR efficacy (such as the order of scFv, the arrangement of VH and VL, and the length and rigidity of the linker), which may require complex structural optimization and empirical testing.

[0009] In the present invention, the optimal structure is obtained by screening the extracellular structure of the chimeric antigen receptor, specifically: the extracellular antigen binding regions VH1-VL1-VH2-VL2, VL1-VH1-VH2-VL2, VH1-VL1-VL2-VH2, VL1-VH1-VL2-VH, VH2-VL2-VH1-VL1, VH2-VL2-VL1-VH1, VL2-VH2-VH1-VL1, and VL2-VH2-VL1-VH1 are designed respectively, wherein 1 is anti-GPRC5D scfv and 2 is anti-CS1 scfv. The effects of target cell lysis are compared under the same conditions, and it is found that the lysis effect is optimal only when the extracellular structure is VH1-VL1-VH2-VL2.

[0010] Furthermore, the flexible linker is (GGGGS)n, where n is an integer selected from 1-5.

[0011] Furthermore, the amino acid sequence of the extracellular antigen binding region is shown in SEQ NO.5.

[0012] Furthermore, the chimeric antigen receptor also contains an extracellular signal peptide region, a hinge region, a transmembrane region and an intracellular signal region, and the chimeric antigen receptor contains an extracellular signal peptide region, an extracellular antigen binding region, a hinge region, a transmembrane region and an intracellular signal region connected in series.

[0013] Furthermore, the extracellular signal peptide may be a CD8a signal peptide.

[0014] Preferably, the chimeric antigen receptor contains a CD8a signal peptide, an extracellular antigen binding region, a CD8 hinge region, a CD8 transmembrane region, a CD137 intracellular signal region, and a CD3zeta intracellular signal region connected in series.

[0015] Furthermore, the amino acid sequences of the CD8a signal peptide, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signal region, and CD3zeta intracellular signal region are shown in SEQ ID NOs.6-10, respectively.

[0016] Furthermore, the amino acid sequence of the chimeric antigen receptor that simultaneously targets GPRC5D and CS1 is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12.

[0017] Furthermore, the multiple myeloma therapeutic drug is used to treat multiple myeloma, especially multiple myeloma with negative BCMA expression or relapse after BCMA CART-T treatment.

[0018] The second object of the present invention is to provide a method for killing myeloma cells in vitro, comprising the step of co-incubating CAR-T cells with myeloma cells;

[0019] Among them, the CAR-T cell contains a chimeric antigen receptor that simultaneously targets GPRC5D and CS1, and the chimeric antigen receptor that simultaneously targets GPRC5D and CS1 contains an extracellular antigen binding region, and the extracellular antigen binding region is a GPRC5D antibody heavy chain, a GPRC5D antibody light chain, a CS1 antibody heavy chain, and a CS1 antibody light chain that are sequentially connected in series through a flexible connector. The sequence of the GPRC5D antibody heavy chain is shown in SEQ ID NO.1, the sequence of the GPRC5D antibody light chain is shown in SEQ ID NO.2, the sequence of the CS1 antibody heavy chain is shown in SEQ ID NO.3, and the sequence of the CS1 antibody light chain is shown in SEQ ID NO.4.

[0020] Furthermore, during co-incubation, the ratio of the number of CAR-T cells to myeloma cells is greater than or equal to 1:1, such as 1-16:1, and most preferably, 8:1.

[0021] Further, the co-incubation is 1-96 hours, preferably, greater than 6 hours.

[0022] The third object of the present invention is to provide a chimeric antigen receptor that simultaneously targets GPRC5D and CS1, wherein the chimeric antigen receptor contains an extracellular antigen binding region, wherein the extracellular antigen binding region is a GPRC5D antibody heavy chain, a GPRC5D antibody light chain, a CS1 antibody heavy chain, and a CS1 antibody light chain that are sequentially connected in series via a flexible linker;

[0023] The sequence of the GPRC5D antibody heavy chain is shown in SEQ ID NO.1, the sequence of the GPRC5D antibody light chain is shown in SEQ ID NO.2, the sequence of the CS1 antibody heavy chain is shown in SEQ ID NO.3, and the sequence of the CS1 antibody light chain is shown in SEQ ID NO.4.

[0024] Furthermore, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.11.

[0025] The fourth object of the present invention is to provide a nucleic acid molecule encoding the chimeric antigen receptor.

[0026] Furthermore, the nucleic acid sequence is shown as SEQ ID NO.12.

[0027] The fifth object of the present invention is to provide an expression vector containing the nucleic acid molecule.

[0028] The sixth object of the present invention is to provide a recombinant cell containing the chimeric antigen receptor.

[0029] Furthermore, the starting cells of the recombinant cells include but are not limited to immune cells, such as T cells.

[0030] The seventh object of the present invention is to provide a drug for treating multiple myeloma, comprising the chimeric antigen receptor, nucleic acid molecule, expression vector or recombinant cell.

[0031] By means of the above scheme, the present invention has at least the following advantages:

[0032] The present invention combines GPRC5D and CS1 targets, and screens the CAR tandem structure when combined to further enhance the synergistic effect of the dual targets. By comparing the killing effects of CAR-T containing different structures, it is found that anti-GPRC5D scfv and anti-CS1 scfv are connected in series through Linker, and the extracellular structure is optimal when it is VH1-VL1-VH2-VL2. Thus, the present invention provides a CAR molecule for the treatment of multiple myeloma and a CAR-T therapy based thereon. After CAR-T binds to tumor cells, it can lyse tumor cells and achieve anti-tumor function. Moreover, compared with existing CAR-T, the dual-target CAR-T cells provided by the present invention have more significant advantages in lysing tumor cells and stimulating immunity. This cell therapy also brings hope to MM patients with BCMA-negative and relapsed after BCMA-CART treatment.

[0033] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0035] Figure 1 is a schematic diagram of a chimeric antigen receptor in the present invention;

[0036] Figure 2 This is a comparison of the killing ability of GPRC5D-CS1-CAR-T cells on target cells H929;

[0037] Figure 3 This is a comparison of the ability of GPRC5D-CS1-CAR-T cells to secrete IL-2;

[0038] Figure 4 This is a comparison of the ability of GPRC5D-CS1-CAR-T cells to secrete IFN-γ. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] Example 1 Construction of chimeric antigen receptor (CAR) lentiviral expression vector

[0041] The intracellular domain of 4-1BB (also known as CD137) and the ITAM region of CD3Zeta were used as activation signals, fused with single-chain antibodies of GPRC5D and CS1, and a chimeric antigen receptor expression vector was constructed and subcloned into the PLVX-EF1a (purchased from clontech) vector. The combination order of each element in the constructed chimeric antigen receptor lentiviral expression vector (named PLVX-EF1a-CAR) is as follows Figure 1 shown.

[0042] The amino acid sequences of each element in the constructed chimeric antigen receptor are as follows:

[0043] VH1(GPRC5D-VH): SEQ ID NO.1

[0044] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSS

[0045] VL1(GPRC5D-VL): SEQ ID NO.2

[0046] DIQMTQSPSSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIK

[0047] VH2 (CS1-VH): SEQ ID NO. 3

[0048] EVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSS

[0049] VL2 (CS1-VL): SEQ ID NO.4

[0050] DIQMTQSPSSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK

[0051] Extracellular antigen-binding region sequence: SEQ ID NO.5

[0052] QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIK

[0053] Signal peptide: SEQ ID NO.6

[0054] MALPVTALLLPLALLLHAARP

[0055] CD8 hinge region: SEQ ID NO.7

[0056] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0057] CD8 transmembrane region: SEQ ID NO.8

[0058] IYIWAPLAGTCGVLLLSLVITLYC

[0059] CD137 (4-1BB) intracellular domain: SEQ ID NO.9

[0060] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCEL

[0061] CD3Zeta: SEQ ID NO.10

[0062] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0063] Full-length amino acid sequence of the CAR molecule: SEQ ID NO.11

[0064] MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQAPGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVATHVGWYQQKPGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCQQYNRYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSSTINYAPSLKDKFIISRDNAKNSLYLQMNSLRAEDTAVYYCARPDGNYWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQDVGIAVAWYQQKPGKVPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQYSSYPYTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYQGLSTATKDTYDALHMQALPPRFull-length nucleotide sequence of the CAR molecule: SEQ ID NO.12

[0065]

[0066] Example 2 Lentivirus Preparation

[0067] The specific experimental steps are as follows:

[0068] S1, prepare a 15cm dish, inoculate 5*10 6 293T cells (purchased from ATCC) were added with complete culture medium (DMEM high glucose, 10% FBS, double antibody), placed in a 37°C, 5% CO2 incubator, and cultured overnight.

[0069] S2. Take out 100μM PEI and lentiviral packaging plasmids (PLVX-EF1a-CAR, pGP, pVSVG) from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS or HBSS buffer and warm to room temperature. Take 2mL PBS to one well of a 6-well plate, add 10μg PLVX-EF1a-CAR, 4μg pGP, and 2μg pVSVG respectively, pipette up and down to mix thoroughly, add 18μL 100μM PEI, immediately pipette up and down to mix, and let stand at room temperature for 10 minutes.

[0070] S3. Add the above DNA / PEI complex dropwise into a 15 cm culture dish, gently shake the dish and mix thoroughly. Place the dish in a 37°C, 5% CO2 incubator, and after 6 to 8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh complete culture medium.

[0071] S4. After continuous culture for 48 hours, collect the supernatant of the culture medium containing the virus in the culture dish, filter it with a 0.45μm filter membrane, add 20% volume of 50% PEG6000 solution, incubate at 4℃ for 2h, transfer to a centrifuge tube, balance it, and centrifuge it at 3000xg at 4℃ for 0.5h. After centrifugation, carefully remove the liquid in the centrifuge tube in the biosafety cabinet, add 500μL PBS buffer to resuspend the precipitate, and store the virus at -80℃.

[0072] Example 3 Isolation of primary T cells

[0073] The specific experimental steps are as follows:

[0074] S1. Invert the lymphocyte separation solution upside down several times to mix the Lymphoprep reagent thoroughly.

[0075] S2. In a biosafety cabinet, add 15 mL of Lymphoprep reagent to a 50 mL centrifuge tube (or a 15 mL centrifuge tube, depending on the volume of the blood sample to be separated) and set aside.

[0076] S3. Dilute the blood sample with an equal volume of PBS + 2% FBS.

[0077] S4. Use a pipette to carefully add the diluted blood sample slowly along the tube wall to the upper layer of the separation reagent to avoid mixing the separation reagent and the blood sample.

[0078] S5. Set the centrifuge to 800 x g, set the speed reduction rate to the slowest, and centrifuge at room temperature for 20 minutes.

[0079] S6. After centrifugation, collect the upper light yellow serum into another sterile centrifuge tube and store at -80℃.

[0080] S7. Gently aspirate the mononuclear cell layer at the interface of serum and separation reagent into a new centrifuge tube and wash the cells once with culture medium.

[0081] S8. Adjust the cell density to 1*10 8 cells / mL (total volume not to exceed 2.5 mL) and resuspend in a 5 mL round-bottom tube.

[0082] S9. Add 100 μl / mL antibody cocktail, mix thoroughly, and incubate at room temperature for 15 minutes.

[0083] S10. Take out the magnetic beads and mix them thoroughly by pipetting them up and down at least 5 times.

[0084] S11. Pipette 50 μl of magnetic beads / mL into the above sample, mix thoroughly, and incubate at room temperature for 10 minutes.

[0085] S12. Add complete culture medium to a total volume of 2.5 mL in the tube, insert the tube (with the lid open) into the magnet, and let stand at room temperature for 5 minutes.

[0086] S13. After incubation, keep the tube in the magnetic pole and gently invert it to pour out the cells in the tube.

[0087] S14. Resuspend the cells in X-vivo 15 medium and add 10% FBS, 300 U / mL IL-2, 5 ng / mL IL-15 and 10 ng / mL IL-7.

[0088] Example 4 Activation of primary T cells and lentiviral infection

[0089] The specific experimental steps are as follows:

[0090] S1. Adjust the cell density to 1*10 6cells / mL, cytokine and antibody complexes (final concentration of 300U / mL IL-2, 10ng / mL IL-7, 5ng / mL IL-15, 500ng / mL Anti-CD3 (OKT3), 2ug / mL Anti-CD28) were added and cultured for 48 hours.

[0091] S2. Calculate the required amount of virus according to MOI=20. The calculation formula is as follows: Required amount of virus (mL) = (MOI*number of cells) / virus titer.

[0092] S3. After taking out the virus from the -80℃ refrigerator, quickly melt it in a 37℃ water bath. Add the above-calculated amount of virus to the six-well plate, add polybrene with a final concentration of 6μg / mL, mix thoroughly, seal the four sides of the six-well plate with sealing film, and centrifuge at 800xg for 1 hour.

[0093] S4. After centrifugation, remove the sealing film and place the six-well plate in an incubator at 37°C and 5% CO2 for 24 hours.

[0094] S5. Centrifuge at 250 x g for 10 minutes, remove the culture medium supernatant containing the virus, resuspend the cell pellet with fresh culture medium, transfer the cells to a new six-well plate, and continue culturing for 3-6 days for use.

[0095] Example 5 CAR-T cells lyse target cells

[0096] The specific experimental steps are as follows:

[0097] S1. Adjust the target cells to the logarithmic growth phase and pass them twice before the experiment.

[0098] S2. Resuspend the target cells overexpressing luciferase in complete medium and adjust the cell density to 5*10 5 / mL, take a new 96-well plate and inoculate the target cells at 100μL / well. Add 100μL of sterile water to each of the unused wells around the 96-well plate to prevent the water in the middle experimental well from evaporating. Place the plate in a 5% CO2 37℃ incubator and culture overnight.

[0099] S3. Collect the prepared CAR-T cells by centrifugation and resuspend them in X-vivo15 medium without phenol red; remove the 96-well plate from the incubator, completely aspirate the medium in the wells, gently wash the cells with sterile PBS, and then add CAR-T cells according to the above E / T ratio, and make up the final volume to 100 μL / well; the control group and the experimental group were inoculated with the same number of target cells, but without CAR-T cells. Place the well plate in a 5% CO2 37°C incubator and culture for 6 hours.

[0100] S4. Take out the 96-well plate, centrifuge at 500g for 5 minutes, discard 80μL of cell supernatant from each well, add 100μL of Luciferase substrate that has been restored to room temperature in advance, and mix well. After standing for 3 minutes, transfer to a white or black bottom opaque 96-well plate and detect on an ELISA reader.

[0101] The target cell lysis percentage is calculated as follows:

[0102] Target cell killing rate (%) = (value of control well - value of test well) / value of control well × 100%.

[0103] S5. The processed data were plotted using GraphPad 6.0.

[0104] Experimental results:

[0105] Using CAR-T cells as effector cells and myeloma cell line H929-luc cells naturally expressing GPRC5D as target cells, a co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well was 50,000, and different numbers of CAR-T cells were added, respectively. The co-culture system was cultured in serum-free medium. After continuous culture for 6 hours, the well plate was taken out and centrifuged at 500g for 5 minutes at room temperature to allow all suspended cells to precipitate to the bottom of the well plate, and then Luciferase substrate was added, and the fluorescence intensity of luciferase in the target cells was detected by an ELISA instrument to reflect the lysis ability of CAR-T cells on target cells. The results are shown in FIG. Figure 2 As shown, the chimeric antigen receptor targeting GPRC5D / CS1 can efficiently mediate T cell killing of tumor cells or recombinant cells; as the effector-target ratio increases, the killing effect of CAR-T cells on target cells H929 also increases, reaching the highest level when the effector-target ratio is 8:1.

[0106] Example 6 Detection of CAR-T cell factor secretion level

[0107] The specific experimental steps are as follows:

[0108] S1. Adjust the target cells to the logarithmic growth phase and pass them twice before the experiment.

[0109] S2. Digest the adherent target cells with trypsin and resuspend them in complete medium. Adjust the cell density to 5*10 5 / mL, take a new 96-well plate and inoculate the target cells at 100μL / well. Add 100μL of sterile water to each of the unused wells around the 96-well plate to prevent the water in the middle experimental well from evaporating. Place the plate in a 5% CO2 37℃ incubator and culture overnight.

[0110] S3. Collect the prepared CAR-T cells by centrifugation and resuspend them in serum-free 1640 medium; remove the 96-well plate from the incubator, completely aspirate the medium in the wells, gently wash the cells with sterile PBS, and then add CAR-T cells according to the above E / T ratio, and make up the final volume to 100 μL / well; place the well plate in a 5% CO2 37°C incubator and culture for 6 hours. At the same time, set up a control T cell group.

[0111] S4. After the culture is completed, take the well plate out of the incubator, centrifuge the 96-well plate at 1200xg at room temperature for 5 minutes, gently take out the plate, transfer 50 μL of culture medium supernatant from each well, use ELISA kit to detect the expression of IFN-γ and IL-2, and use a microplate reader to read the OD value.

[0112] S5. The above acquired data were plotted using GraphPad 6.0.

[0113] Experimental results:

[0114] Using CAR-T cells as effector cells and myeloma cell line H929 naturally expressing GPRC5D and CS1 as target cells, a co-culture system was established according to different effector-target ratios, that is, in a 96-well plate, the number of fixed target cells in each well was 50,000, and different numbers of CAR-T cells were added, respectively. The co-culture system was cultured in serum-free medium. After continuous culture for 8 hours, the well plate was taken out and centrifuged at 1200×g for 10 minutes at room temperature to allow all suspended cells to precipitate to the bottom of the well plate, and then 30 μl of supernatant was taken from each well, and the expression levels of IFN-γ and IL-2 secreted by CAR-T cells after being activated by tumor cells in the culture medium supernatant were detected by ELISA. The results are shown in FIG. Figure 3 and Figure 4 As shown, after the GPRC5D-CS1-CART chimeric antigen receptor binds to the targeted tumor cells, it can effectively activate primary T cells and cause an increase in the secretion and expression of cytokines; when the effector-target ratio is 1:1, after the CAR-T cells are activated by tumor cells, they can secrete a large amount of IFN-γ and IL-2, which is significantly higher than that of the control T cells; the secretion amount reaches the highest value when the effector-target ratio is 8:1.

[0115] Figure 2-4In the above, the preparation methods of GPRC5D-CART, CS1-CART, and BCMA-CART are the same as above, and the only difference is that the scFv antibody sequences are different. From the above results, it can be seen that the target cells treated with GPRC5D-CS1-CART, GPRC5D-CART, CS1-CART, and BCMA-CART have significantly stronger ability to secrete IFN-γ and IL-2 than the untreated target cell group (T: NCI-H929). Moreover, compared with CAR-T containing a single target (GPRC5D, CS1 or BCMA), CAR-T containing dual targets GPRC5D and CS1 performs best in killing tumor cells and has the strongest ability to stimulate immunity.

[0116] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. Use of a chimeric antigen receptor simultaneously targeting GPRC5D and CS1 in the preparation of a drug for treating multiple myeloma, characterized in that: The chimeric antigen receptor simultaneously targeting GPRC5D and CS1 contains an extracellular antigen binding region, wherein the extracellular antigen binding region is a GPRC5D antibody heavy chain, a GPRC5D antibody light chain, a CS1 antibody heavy chain, and a CS1 antibody light chain connected in series in sequence through a flexible linker; The sequence of the GPRC5D antibody heavy chain is shown in SEQ ID NO.1, the sequence of the GPRC5D antibody light chain is shown in SEQ ID NO.2, the sequence of the CS1 antibody heavy chain is shown in SEQ ID NO.3, and the sequence of the CS1 antibody light chain is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that: The extracellular antigen binding region is a GPRC5D antibody heavy chain, a flexible linker, a GPRC5D antibody light chain, a flexible linker, a CS1 antibody heavy chain, a flexible linker, and a CS1 antibody light chain connected in series in sequence; the flexible linker is (GGGGS)n, where n is an integer selected from 1-5.

3. The use according to claim 1, characterized in that: The chimeric antigen receptor also contains an extracellular signal peptide region, a hinge region, a transmembrane region and an intracellular signal region.

4. The use according to claim 3, characterized in that: The chimeric antigen receptor contains a CD8a signal peptide, an extracellular antigen binding region, a CD8 hinge region, a CD8 transmembrane region, a CD137 intracellular signal region, and a CD3zeta intracellular signal region connected in series; the amino acid sequence of the extracellular antigen binding region is shown in SEQ NO.5, and the amino acid sequences of the CD8a signal peptide, CD8 hinge region, CD8 transmembrane region, CD137 intracellular signal region, and CD3zeta intracellular signal region are shown in SEQ ID NOs.6-10, respectively.

5. The use according to claim 1, characterized in that: The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO.11; the nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID NO.

12.

6. A method for killing myeloma cells in vitro, characterized in that: The method includes the step of co-incubating CAR-T cells with myeloma cells; Among them, the CAR-T cell contains a chimeric antigen receptor that simultaneously targets GPRC5D and CS1, and the chimeric antigen receptor that simultaneously targets GPRC5D and CS1 contains an extracellular antigen binding region, and the extracellular antigen binding region is a GPRC5D antibody heavy chain, a GPRC5D antibody light chain, a CS1 antibody heavy chain, and a CS1 antibody light chain that are sequentially connected in series through a flexible connector. The sequence of the GPRC5D antibody heavy chain is shown in SEQ ID NO.1, the sequence of the GPRC5D antibody light chain is shown in SEQ ID NO.2, the sequence of the CS1 antibody heavy chain is shown in SEQ ID NO.3, and the sequence of the CS1 antibody light chain is shown in SEQ ID NO.

4.

7. The method according to claim 6, characterized in that Preferably, during co-incubation, the ratio of the number of CAR-T cells to myeloma cells is greater than or equal to 1:1; Preferably, the co-incubation is for 1-96 hours.

8. A chimeric antigen receptor that simultaneously targets GPRC5D and CS1, characterized in that: The chimeric antigen receptor contains an extracellular antigen binding region, and the extracellular antigen binding region is a GPRC5D antibody heavy chain, a GPRC5D antibody light chain, a CS1 antibody heavy chain, and a CS1 antibody light chain connected in series in sequence through a flexible linker; The sequence of the GPRC5D antibody heavy chain is shown in SEQ ID NO.1, the sequence of the GPRC5D antibody light chain is shown in SEQ ID NO.2, the sequence of the CS1 antibody heavy chain is shown in SEQ ID NO.3, and the sequence of the CS1 antibody light chain is shown in SEQ ID NO.

4.

9. A nucleic acid molecule encoding the chimeric antigen receptor of claim 8, an expression vector containing the nucleic acid molecule, or a recombinant cell containing the chimeric antigen receptor.

10. A tumor treatment drug, characterized in that: Containing the chimeric antigen receptor according to claim 8, or the nucleic acid molecule, expression vector or recombinant cell according to claim 9.

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