Chimeric antigen receptor targeting BCMA and use thereof

Abstract

Provided is a chimeric antigen receptor targeting BCMA. The receptor comprises a humanized scFv structure targeting BCMA, wherein the heavy-chain variable region scFv-VH and the light-chain variable region scFv-VL of scFv are connected via one or more G4S sequences. A new second-generation CAR-T cell targeting BCMA is successfully constructed by means of transduction by using a retroviral vector. In vitro and in vivo experiments show that after stimulation with BCMA positive tumor cells, the BCMA CAR 31-T cells can be effectively activated to secrete cytokines such as IFN-γ and TNF-α, thereby promoting the apoptosis of tumor cells. The BCMA CAR 31-T cells can effectively and specifically kill BCMA positive tumor cells.

Description

Chimeric antigen receptor targeting BCMA and its application Technical Field

[0001] The present invention relates to the field of cellular immunotherapy for tumors, and particularly to targeting BCMA chimeric antigen receptors. Background Art

[0002] Multiple myeloma (MM) is a malignant tumor characterized by the abnormal proliferation of terminally differentiated plasma cells and is the second most common hematologic malignancy. In recent years, with the development of new therapeutic agents such as proteasome inhibitors and immunomodulators, patient prognosis has improved significantly. However, MM remains an incurable disease, and almost all patients ultimately face the risk of relapse and drug resistance. Therefore, new treatment options are crucial for these patients. Immunotherapy, represented by chimeric antigen receptor modified T cells (CAR-T), has shown promising efficacy in hematologic malignancies and is expected to become a new and effective treatment for MM.

[0003] Unlike traditional T cell activation pathways, CAR-T cell activation does not rely on MHC presentation. CAR-T cells can recognize and bind to surface antigens on target tumor cells through an extracellular single-chain variable fragment (ScFv). In the original design, the extracellular antigen-binding domain and the intracellular signaling domain were connected by a transmembrane domain (first-generation CARs), directly inducing T cell activation after antigen recognition and binding. The antigen-binding domain binds to a ligand on the cell surface, providing a primary signal. Once bound to the ligand, intracellular costimulatory molecules are activated, providing a secondary signal. These primary and secondary signals are then transmitted to the intracellular activation domain, activating the CAR-T cell and exerting its anti-tumor activity. Studies have found that first-generation CAR structures have little tumor clearance and no proliferative activity. Therefore, researchers have constructed second-generation CARs containing costimulatory domains such as CD28 or 4-1BB, which have shown stronger anti-tumor activity. CARs that incorporate more than one intracellular costimulatory domain (such as CD28, OX40, 4-1BB, CD27, etc.) are considered third-generation CARs. The fourth generation of CARs is capable of secreting cytokines such as IL-12 and expressing cell surface markers such as co-stimulatory ligands. Currently, the second generation of CAR structures have shown better safety and clinical efficacy, and therefore are more widely used in clinical practice.

[0004] Typical CAR-T cell manufacturing requires drawing peripheral blood from the patient, isolating peripheral blood mononuclear cells (PBMCs), and then stimulating and activating the T cells. Then, through genetic engineering, the cells are engineered to express a CAR on their surface that specifically recognizes the tumor target antigen. Successfully prepared CAR-T cells are then amplified and infused back into the patient to exert their anti-tumor function. The production of CAR-T cells generally takes 10-14 days. Since 2017, five CAR-T products have been approved by the FDA for the treatment of hematological malignancies. Among them, Abecma, which targets BCMA, was approved this year and is the first CAR-T cell product for the treatment of multiple myeloma.

[0005] Identifying tumor-specific antigens is crucial for successful CAR-T cell therapy. First, the antigen must be expressed on the surface of tumor cells. Second, the antigen must be uniformly expressed on tumor cells and, ideally, required for tumor survival. Most importantly, the target antigen must not be expressed in related healthy tissues to avoid potential on-target off-tumor toxicity.

[0006] BCMA, also known as CD269 or TNFRSF17, is a 27-kDa type III transmembrane glycoprotein expressed by mature B lymphocytes, plasma cells, and most patients with multiple myeloma. BCMA binds to multiple ligands, including BAFF (B-cell activating factor) and APRIL (A proliferation-inducing ligand), mediating cell survival through downstream NF-κB and MAPK / JNK signaling pathways. BCMA, a member of the tumor necrosis factor family, plays a crucial role in supporting plasma cell differentiation and survival. It is selectively expressed on normal plasma cells and multiple myeloma cells; BCMA expression is not detected on cells from other tissues. BCMA, along with transmembrane activator, calcium regulator, and cyclophilin ligand interactor (TACI), also functions as a receptor for proliferation-inducing ligand (APRIL). In multiple myeloma, the APRIL / BCMA pathway plays a key role in supporting growth, drug resistance, and immune compromise. Due to its unique expression on plasma cells and its crucial role in multiple myeloma, BCMA has become an ideal target for multiple myeloma treatment.

[0007] Although BCMA CAR-T cells targeting MM have been reported, clinical trials of new CAR-T cells are still needed to advance the treatment of MM.

[0008] Summary of the Invention

[0009] To solve the above problems, the present invention provides a chimeric antigen receptor targeting BCMA, which comprises a human ScFv structure targeting BCMA, wherein the heavy chain variable region ScFv-VH and the light chain variable region ScFv-VL of the ScFv are connected by one or more G4S sequences; the amino acid sequence of the heavy chain variable region ScFv-VH has at least 90% homology, preferably at least 95% homology, and more preferably at least 98% homology to the following SEQ ID NO. 8: SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSS;

[0010] The amino acid sequence of the light chain variable region ScFv-VL has at least 90% homology, preferably at least 95% homology, and more preferably at least 98% homology to the following SEQ ID NO.9: QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG.

[0011] In one embodiment, the receptor comprises a human ScFv targeting BCMA, the structure of which is ScFv-VH-(G4S)n-ScFv-VL, wherein n is an integer greater than or equal to 1, preferably 3 or 4.

[0012] In one embodiment, the receptor includes a human BCMA-targeting ScFv structure of ScFv-VH-(G4S)3-ScFv-VL, the amino acid sequence of the heavy chain variable region ScFv-VH is SEQ ID NO.8; the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.9.

[0013] In one embodiment, the receptor comprises a human ScFv targeting BCMA structure of ScFv-VH-(G4S)n-ScFv-VL-(G4S)n-ScFv-VL-(G4S)n-ScFv-VH, wherein n is an integer greater than or equal to 1, preferably 3 or 4.

[0014] In one embodiment, the receptor includes a human BCMA-targeting ScFv structure of ScFv-VH-(G4S)3-ScFv-VL-(G4S)4-ScFv-VL-(G4S)3-ScFv-VH, the amino acid sequence of the heavy chain variable region ScFv-VH is SEQ ID NO.8; the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.9.

[0015] In one embodiment, the receptor includes an upstream signal peptide and a myc tag for detection connected in series; a human BCMA-targeting ScFv structure comprising a heavy chain variable region and a light chain variable region; a CD8 hinge-transmembrane domain; a CD28 or 4-1BB co-activation domain and a CD3ζ intracellular signaling domain.

[0016] In one embodiment, the present invention provides a chimeric antigen receptor T cell targeting BCMA, which expresses the above-mentioned chimeric antigen receptor.

[0017] In one embodiment, the present invention provides a drug for treating tumors, comprising the chimeric antigen receptor T cell described above.

[0018] In one embodiment, the present invention provides the use of the above-mentioned chimeric antigen receptor in preparing chimeric antigen receptor T cells and in treating tumors.

[0019] In one embodiment, the tumor is a surface BCMA-positive tumor.

[0020] In one embodiment, the tumor is multiple myeloma.

[0021] In one embodiment, the present invention provides an application of the above-mentioned chimeric antigen receptor, wherein a gene fragment encoding the chimeric antigen receptor is inserted into a viral expression vector, packaged into viral vector particles, and used to infect human T cells to prepare chimeric antigen receptor T cells for the treatment of surface BCMA-positive tumors.

[0022] In the present invention, we successfully constructed pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19, and pMFG-BCMA CAR20 plasmids and transiently transfected Phoenix-ECO cells with transfection efficiencies exceeding 50%. The transiently transfected retroviral vector particles were used to transduce the PG13 cell line with transduction efficiencies exceeding 95%. pPCR detection showed that the retroviral vector particles had high viral titers, with the highest titers exceeding 1×10 7Copies / mL indicate the successful generation of a PG13 cell line stably expressing a BCMA CAR retroviral vector. Human primary T cells were transduced with the successfully generated amphotropic retroviral vectors BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19, and BCMA CAR20, and CAR expression was detected, demonstrating the successful generation of BCMA CAR-T cells.

[0023] In an in vitro tumor killing experiment, the present invention used flow cytometry to detect target cell apoptosis and luciferase bioluminescence to detect target cell survival. BCMA CAR16-T showed relatively better anti-tumor ability. However, since the killing effect was inferior to that of the positive control group, it indicates that the BCMA CAR16 we screened can be further optimized to improve its binding ability to the target antigen.

[0024] Because BCMA has a trimer structure and a small protein molecular weight of 32.3kDa, we concatenated the ScFv structure of BCMA CAR16 to design BCMA CAR31, BCMA CAR32, and BCMA CAR33. This increases the flexibility of the ScFv region of the extracellular antigen-binding domain, better captures antigens, and enhances the ability to kill tumors.

[0025] BCMA CAR31, BCMA CAR32, and BCMA CAR33 were prepared as amphotropic retroviral vectors and transduced into primary human T cells. The transduction efficiency exceeded 50%, and qPCR detection showed successful integration into the T cell genome.

[0026] In vitro tumor killing experiments, incucyte real-time dynamic live cell imaging screened BCMA CAR31-T cells with the best killing effect, which was further compared with BCMA CAR16-T cells. CD69 is a marker of T cell activation. We found that in the absence of tumor cell stimulation, neither BCMA CAR31-T nor BCMA CAR16-T cells showed obvious self-activation. However, BCMA CAR-T cells were effectively activated by tumor cell stimulation and exerted their anti-tumor effects.

[0027] To determine the in vitro anti-tumor ability of BCMA CAR31-T cells, we selected RPMI-GFP-luc cells expressing human BCMA-positive tumor cells. Luciferase bioluminescence assays for target cell survival revealed that, compared with the BCMA CAR16-T group, BCMA CAR31-T cells demonstrated enhanced cytotoxicity against two different BCMA-positive tumor cell lines at varying effector-target ratios, indicating that the structurally optimized BCMA CAR31-T cells have enhanced tumor cell cytotoxicity.

[0028] To verify the antigen specificity of BCMA CAR31-T cells' anti-tumor effects, BCMA CAR31-T cells were co-incubated with different tumor cell lines: K562-hBCMA-gfp, PMI-gfp-luc, K562-cBCMA, and K562. The results showed that compared with the BCMA CAR16-T group, at different effector-target ratios, BCMA CAR31-T cells had stronger killing abilities against both BCMA-positive tumor cells. However, BCMA CAR31-T cells showed no difference in killing abilities compared to Pan-T cells against non-human BCMA-expressing K562-cBCMA and BCMA-negative K562 cells. This result indicates that BCMA CAR31-T cells have BCMA antigen specificity in killing tumor cells.

[0029] When CAR-T cells bind to tumor antigens, they recruit other immune cells and release a large amount of cytokines, generating an anti-tumor immune response. In cytokine secretion experiments, compared with BCMA CAR16-T, BCMA CAR31-T cells secrete more pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6, IL-17A, and aFasL under the stimulation of tumor cells, thereby promoting tumor cell apoptosis.

[0030] Generating a strong and lasting anti-tumor immune response requires not only eliciting cytotoxicity and cytokine production but also stimulating the proliferation of CAR-T cells. Using CFSE assays, we found that BCMA CAR31-T cells proliferated faster than BCMA CAR16-T cells.

[0031] In addition, the present invention established an NPG mouse xenograft tumor model to confirm the anti-tumor ability of BCMA CAR31-T cells in vivo. Two days after the second tail vein injection of BCMA CAR31-T, the secretion of IFN-γ cytokines was significantly enhanced compared to BCMA CAR16-T, indicating that BCMA CAR31-T cells were better activated; the mice were generally in good condition and did not show symptoms of cytokine release syndrome such as fever, nausea, and vomiting. During the continuous observation of mouse tumor changes, the tumor signal in the BCMA CAR31-T group was significantly weakened compared to BCMA CAR16-T, showing more effective anti-tumor ability; flow cytometry was used to continuously detect the T cell content in the peripheral blood of mice. The results showed that until the 37th day after tumor inoculation, the content of BCMA CAR31-T cells was higher than that of BCMA CAR16-T cells, indicating that BCMA CAR31-T cells continued to survive longer in the mouse body and had more lasting anti-tumor ability.

[0032] Therefore, in the present invention, a new second-generation BCMA-targeted CAR-T cell was successfully constructed through retroviral vector transduction. In vitro experiments showed that BCMA-positive tumor cells can effectively activate BCMA CAR31-T cells after stimulation, secreting cytokines such as IFN-γ and TNF-α, and promoting tumor cell apoptosis. BCMA CAR31-T cells can effectively and specifically kill BCMA-positive tumor cells. BCMA CAR31-T cells have good proliferation capacity in vitro. Further in vivo anti-tumor experiments showed that BCMA CAR31-T cells can be rapidly activated to secrete IFN-γ and have a certain tumor effect. Therefore, the new second-generation CAR-T cell targeting BCMA has efficient and specific anti-tumor activity and may become a new therapy for the clinical treatment of MM. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0034] FIG1 is an experimental scheme diagram of the present invention;

[0035] FIG2 is a schematic structural diagram of the BCMA CAR of the present invention;

[0036] FIG3 is a diagram of the preparation scheme of the BCMA CAR retroviral vector plasmid;

[0037] Figure 4 is a roadmap of BCMA CAR retroviral vector packaging technology;

[0038] Figure 5 is a technical roadmap for the preparation of BCMA CAR-T cells;

[0039] Figure 6 is a technical roadmap for BCMA CAR-T's in vitro anti-tumor function;

[0040] FIG7 is a flow cytometry analysis of the efficiency of BCMA CAR-T cells in killing K562-hBCMA-gfp cells;

[0041] Figure 8 Detection of BCMA CAR-T cell killing efficiency of RPMI-gfp-luc cells (n=3) (compared with bb2121 CAR-T cells, * P < 0.05) result graph;

[0042] FIG9 is a schematic diagram of the structure of an optimized BCMA CAR;

[0043] Figure 10 Gel electrophoresis of enzyme digestion identification, wherein A: pMFG-BCMA CAR31 and pMFG-BCMA CAR32 enzyme digestion identification fragment; B: pMFG-BCMA CAR33 enzyme digestion identification fragment;

[0044] FIG11 is a graph showing the transduction efficiency of optimized BCMA CAR amphotropic retroviral vector particles in transducing primary human T cells (n=3) (ns: no statistical difference);

[0045] FIG12 is a diagram of an optimized BCMA CAR-T cell killing experimental scheme in vitro;

[0046] FIG13 is a graph showing the real-time fluorescence monitoring results of the optimized BCMA CAR-T cells killing K562-hBCMA-gfp in vitro (n=3);

[0047] Figure 14 shows the results of CD69 expression on CAR-T cells (n=3), wherein A: flow cytometry of CD69 expression on the surface of BCMA CAR-T cells; B: histogram of CD69 expression on the surface of BCMA CAR-T cells without tumor cell stimulation; C: histogram of CD69 expression on the surface of BCMA CAR-T cells stimulated by BCMA+ tumor cells (compared with BCMA CAR16-T cells, *P<0.05, ns: no statistical difference);

[0048] Figure 15 is a graph showing the results of luciferase bioluminescence assay to detect the in vitro killing ability of BCMA CAR-T cells (n=3) (compared with BCMA CAR16-T cells, *P<0.05, **P<0.01, ***P<0.001);

[0049] FIG16 is a graph showing the results of BCMA CAR-T cell killing efficiency test for different tumor cells (n=3) (compared with BCMA CAR16-T cells, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: no statistical difference);

[0050] Figure 17 is a graph showing the secretion levels of BCMA CAR-T cells (n=3) (compared with BCMA CAR16-T cells, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001);

[0051] FIG18 is a graph showing the results of BCMA CAR-T cell proliferation ability detection;

[0052] FIG19 is a diagram of the BCMA CAR-T in vivo anti-tumor experimental scheme;

[0053] FIG20 shows the results of in vivo imaging of a mouse xenograft tumor model (n=6), A: in vivo imaging of mice; B: statistical graph of the average signal intensity of mouse tumors (ns: no statistical difference);

[0054] FIG21 shows the results of in vivo imaging of a mouse xenograft tumor model (n=6), A: in vivo imaging of mice; B: statistical graph of the average signal intensity of mouse tumors (*P<0.05, **P<0.01, ***P<0.001 compared with BCMA CAR16-T cells); DETAILED DESCRIPTION

[0055] In order to enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of this application.

[0056] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0057] Using phage display, we screened six novel humanized BCMA-targeting ScFv constructs. The specific experimental scheme is shown in Figure 1. To verify whether these six BCMA-targeting ScFvs could effectively and specifically recognize BCMA-positive tumor cells, we designed and constructed second-generation humanized BCMA CAR constructs using CD8 as the transmembrane domain and CD28 and CD3ζ as intracellular stimulatory domains (see Figure 2). BCMA CAR-T cells were then generated using retroviral vectors. In vitro tumor cell cytotoxicity experiments identified the BCMA CAR16-T cell with the strongest tumor cell cytotoxicity. We further concatenated the BCMA CAR16 ScFvs to further enhance their antigen-binding capacity for BCMA. In in vitro experiments, tumor cells expressing human BCMA antigens were used as target cells to detect the activation of BCMA CAR-T after optimization, verify the killing specificity and effectiveness of BCMA CAR-T cells on target cells, and detect the proliferation ability and cytokine secretion levels of BCMA CAR-T. Further verification was carried out in an in vivo xenograft tumor model to detect cytokine secretion and anti-tumor ability, as well as the maintenance of CAR-T cells in vivo after treatment.

[0058] In the present invention, the amino acid sequence is as follows:

[0059] SP amino acid sequence: MEWSWVFLFFLSVTTGVHSDI (SEQ ID NO. 1);

[0060] Myc amino acid sequence: EQKLISEEDL (SEQ ID NO. 2);

[0061] CD8 amino acid sequence:

[0062] CD28 amino acid sequence:

[0063] CD3ζ amino acid sequence:

[0064] BCMA CAR15 ScFv-VH amino acid sequence:

[0065] BCMA CAR15 ScFv-VL amino acid sequence:

[0066] BCMA CAR16 ScFv-VH amino acid sequence:

[0067] BCMA CAR16 ScFv-VL amino acid sequence:

[0068] BCMA CAR17 ScFv-VH amino acid sequence:

[0069] BCMA CAR17 ScFv-VL amino acid sequence:

[0070] BCMA CAR18 ScFv-VH amino acid sequence:

[0071] BCMA CAR18 ScFv-VL amino acid sequence:

[0072] BCMA CAR19 ScFv-VH amino acid sequence:

[0073] BCMA CAR19 ScFv-VL amino acid sequence:

[0074] BCMA CAR20 ScFv-VH amino acid sequence:

[0075] BCMA CAR20 ScFv-VL amino acid sequence:

[0076] BCMA CAR31 ScFv amino acid sequence:

[0077] BCMA CAR32 ScFv amino acid sequence:

[0078] SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSI YHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG(SEQ ID NO.19), where the underlined characters in the sequence are (G4S)n;

[0079] BCMA CAR33 ScFv amino acid sequence:

[0080] SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLGGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSSQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG (SEQ ID NO.20), wherein the underscores in the sequence are (G4S)n sequences.

[0081] Construction of the BCMA CAR retroviral vector plasmid in Example 1

[0082] Six ScFv sequences targeting BCMA were screened and synthesized by General Biotech Co., Ltd. into the pUC57 vector. Plasmids containing the CD28-CD8-CD3ζ sequence, which can be purchased commercially or prepared in-house, and the retroviral vector pMFG plasmid were used to amplify the ScFv and CD28-CD8-CD3ζ fragments by PCR. The ScFv-CD28-CD8-CD3ζ fragment was then amplified by homologous recombination. The recombinant target fragment and the vector were double-digested with XhoI and NotI and ligated to construct the complete pMFG-BCMA CAR plasmid. The specific experimental scheme is shown in Figure 3.

[0083] 1. Experimental Procedure

[0084] 1. Extraction of vector plasmid and target plasmid

[0085] The pUC57-BCMA CAR15, pUC57-BCMA CAR16, pUC57-BCMA CAR17, pUC57-BCMA CAR18, pUC57-BCMA CAR19, pUC57-BCMA CAR20, pMFG-CD8-CD28-CD3ζ and vector plasmid pMFG bacteria stored at -80°C were taken out, 50 μL of each was added into 25 mL of LB liquid medium (final concentration of Amp was 50 μg / mL), and the culture was shaken at 37°C and 200 rpm for 12-16 h until 3-4×10 9 / mL cell density. Harvest the bacterial pellet, centrifuge, suspend, filter column, add 4mL of isopropanol to the eluate and mix well. Immediately centrifuge at 15000g for 30min at 4°C and carefully discard the supernatant. Add 2mL of 75% ethanol to wash the DNA pellet, centrifuge at 15000g for 10min at room temperature and carefully discard the supernatant. Dry the pellet for 5-10min, add an appropriate volume of nuclease-free water, and gently flick to mix. Measure the DNA concentration and store the plasmid at -20°C for later use.

[0086] 2. PCR amplification of target fragments

[0087] The pUC57-BCMA CAR15, pUC57-BCMA CAR16, pUC57-BCMA CAR17, pUC57-BCMA CAR18, pUC57-BCMA CAR19, and pUC57-BCMA CAR20 plasmids obtained above were used to amplify target bands by PCR, with each target fragment being approximately 800 bp. The pMFG-CD8-CD28-CD3ζ plasmid obtained above was also used to amplify target bands by PCR, with a fragment of approximately 800 bp.

[0088] Table 1 Primer sequence list

[0089] The obtained target fragments were named CAR15-ScFv, CAR16-ScFv, CAR17-ScFv, CAR18-ScFv, CAR19-ScFv, CAR20-ScFv, and CD8-CD28-CD3ζ. The obtained CAR15-ScFv, CAR16-ScFv, CAR17-ScFv, CAR18-ScFv, CAR19-ScFv, and CAR20-ScFv were subjected to homologous recombination PCR with CD8-CD28-CD3ζ to obtain complete ScFv-CD8-CD28-CD3ζ fragments, each approximately 1600 bp. The vector plasmid and homologous recombinant target fragments were digested with enzymes.

[0090] The purified CAR15-ScFv-CD8-CD28-CD3ζ, CAR16-ScFv-CD8-CD28-CD3ζ, CAR17-ScFv-CD8-CD28-CD3ζ, CAR18-ScFv-CD8-CD28-CD3ζ, CAR19-ScFv-CD8-CD28-CD3ζ, and CAR20-ScFv-CD8-CD28-CD3ζ were double-digested with XhoI and NotI restriction endonucleases, and the pMFG vector fragment was recovered by electrophoresis.

[0091] The target fragment was connected to the vector fragment. The XhoI and NotI double-digested target fragments CAR15-ScFv-CD8-CD28-CD3ζ, CAR16-ScFv-CD8-CD28-CD3ζ, CAR17-ScFv-CD8-CD28-CD3ζ, CAR18-ScFv-CD8-CD28-CD3ζ, CAR19-ScFv-CD8-CD28-CD3ζ, and CAR20-ScFv-CD8-CD28-CD3ζ recovered from the gel were connected to the pMFG vector enzyme-digested fragment. The molar ratio of the target fragment to the vector fragment was 3:1-5:1.

[0092] The ligation product was transformed into DH5α competent cells, followed by plasmid DNA extraction and enzyme digestion. The extracted constructed plasmid DNA was double-digested with XhoI and NotI restriction endonucleases to verify the correct bands. The correct plasmid DNA was identified by enzyme digestion. The purified plasmid DNA was sent to Qingke Bio for sequencing. The sequencing primers are shown in the table below:

[0093] Table 2 Sequencing primers

[0094] 2. Experimental results

[0095] 1. Schematic diagram of the BCMA CAR structure

[0096] The ScFv region of the BCMA CAR is a humanized monoclonal antibody sequence targeting BCMA. The SP gene and the myc gene for expression efficiency testing are added before the ScFv gene sequence. The transmembrane region utilizes the CD8 hinge-transmembrane domain. The intracellular costimulatory molecule uses the CD28 molecule as a co-stimulatory domain, and the intracellular signaling molecule uses CD3ζ. The six selected BCMA CAR expression plasmids are shown in Figure 2.

[0097] The results of gel electrophoresis of target fragment amplification showed that the gene-synthesized pUC-BCMA CAR15, pUC-BCMA CAR16, pUC-BCMA CAR17, pUC-BCMA CAR18, pUC-BCMA CAR19, and pUC-BCMA CAR20 plasmids containing ScFv fragments were amplified by PCR, and the sizes were all approximately 800 bp; the plasmid containing the CD8-CD28-CD3ζ fragment was amplified by PCR, and the fragment size was approximately 830 bp. Gel electrophoresis results showed that the target band sizes were all correct.

[0098] 2. Target fragment homologous recombination gel electrophoresis results

[0099] CAR15-ScFv, CAR16-ScFv, CAR17-ScFv, CAR18-ScFv, CAR19-ScFv, and CAR20-ScFv were recombined into CD8-CD28-CD3ζ using homologous recombination PCR. The sizes of the recombinant fragments were all approximately 1.6 kb, and gel electrophoresis showed that the band sizes were all correct.

[0100] 3. Gel electrophoresis results of enzyme-digested vector fragments

[0101] The plasmid containing the pMFG vector was double-digested with XhoI and NotI, and the fragment size was approximately 7 kb. Gel electrophoresis results showed that the fragment size was correct and could be used for ligation experiments with the target band.

[0102] 4. Enzyme digestion identification results

[0103] The constructed plasmids were digested with XhoI and NotI, revealing fragment sizes of 7 kb and 1.6 kb. Gel electrophoresis results showed that the band sizes of the constructed pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19, and pMFG-BCMA CAR20 plasmids were all correct and could be sent for sequencing for further verification.

[0104] 5. Successful construction of BCMA CAR plasmid

[0105] The plasmids identified by enzyme digestion were purified and sent for sequencing. Sequencing results showed that the sequences of pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19, and pMFG-BCMA CAR20 were completely correct.

[0106] Example 2. BCMA CAR retroviral vector packaging

[0107] After constructing the pMFG-BCMA CAR plasmid, the BCMA CAR retroviral vector was packaged and a cell line stably producing the retroviral vector was established. The specific experimental process is shown in Figure 4: First, the pMFG-BCMA CAR plasmid was transiently transfected into Phoenix ECO cells, and the BCMA CAR ecotropic retroviral vector supernatant particles were collected. The collected BCMA CAR ecotropic retroviral vector particles were then transduced into the PG13 cell line to produce amphotropic retroviral vector supernatant particles. A PG13 cell line stably producing BCMA CAR amphotropic retroviral vector supernatant particles was then established. The viral vector titer was measured by qPCR to verify the successful production of BCMA CAR amphotropic retroviral vector supernatant particles.

[0108] 1. Materials

[0109] The human eosinophil packaging cell line Pheonix-ECO and the gibbon ape leukemia virus packaging cell line PG13 were purchased from the American type culture collection (ATCC).

[0110] DMEM complete medium: Working in a biosafety cabinet, add 56 mL of FBS (10%) and 5.6 mL of penicillin-streptomycin (1%) to every 500 mL of DMEM (89%). Vacuum filter the prepared DMEM complete medium using a 0.22 μm 500 mL filter. Label the bottle and store in a 4°C refrigerator until ready for use.

[0111] Cell freezing solution: Prepare FBS and DMSO in a biological safety cabinet at a ratio of FBS:DMSO = 9:1; filter the prepared cell freezing solution through a 0.22 μm filter membrane and set aside.

[0112] Staining buffer: Prepare PBS (1×) and FBS at a ratio of PBS:FBS = 9:1 and store at 4°C until use.

[0113] 2. Experimental Methods

[0114] 1. Phoenix ECO and PG13 cell culture

[0115] 1.1. Cell recovery

[0116] Take out the cells to be revived from the liquid nitrogen tank and resuspend them, then resuspend the cell pellet with DMEM complete medium, transfer the cell suspension to an appropriate culture flask, and culture it in a 37°C, 5% CO2 incubator.

[0117] 1.2 Cell passaging and medium replacement

[0118] Observe the cell attachment and growth status under a microscope. If the cell density is <80% but the culture medium turns yellow, the cells need to be replaced with a culture medium. If the cell density is >80%, the cells need to be passaged. Use an appropriate volume of 1x PBS and shake the culture flask several times to wash the cells, removing any cells that have not successfully attached. Gently discard the PBS used for washing and add an appropriate volume of trypsin to digest the cells. Gently shake the culture flask to ensure that the trypsin is fully accessible to the cells to accelerate digestion. Alternatively, place the flask in a 37°C incubator to accelerate digestion. Observe the cells under a microscope to see if they have detached. When approximately 90% of the cells have detached, add three times the volume of DMEM complete medium to terminate digestion. Transfer the cell suspension to a centrifuge tube and centrifuge at 300g for 5 minutes. Carefully remove the supernatant with a pipette. Resuspend the cell pellet in an appropriate volume of fresh DMEM complete medium. Transfer the cell suspension to an appropriate culture flask and incubate in a 37°C, 5% CO2 incubator. Observe the cell growth status under an inverted microscope every 24 hours.

[0119] 1.3 Cell cryopreservation

[0120] Add an appropriate volume of trypsin to digest the cells, digest at 37°C for about 5 minutes, and take out the cells every 1 minute to observe the digestion status under a microscope. When about 90% of the cells are digested and detached, add 3 times the volume of trypsin to stop the digestion. Use a pipette to gently blow the cell suspension into a single-cell suspension, take out 20 μL of the single-cell suspension and mix it with an equal volume of trypan blue solution. Add it dropwise to the cell counting plate and count it with a cell counter. Transfer the cell suspension to a centrifuge tube and centrifuge at 300g for 5 minutes. Use a pipette to aspirate the supernatant and discard it. Be careful not to touch the cell pellet, and add cell freezing solution to resuspend the cells (the general cell freezing concentration is 5×10 6 -1×10 7 The cell suspension was added to a 2 mL cryovial, placed in a program incubator, and immediately transferred to a -80°C freezer overnight.

[0121] 2. Preparation of Ecotropic Retroviral Vector Supernatant

[0122] 2.1 Plating Phoenix-ECO cells: Discard the old culture medium of Phoenix-ECO cells that are growing well. Add trypsin to digest the cells and incubate at 37°C for approximately 5 minutes to accelerate digestion. Add three times the volume of trypsin to terminate digestion with DMEM complete medium. Centrifuge at 300g for 5 minutes. Discard the solution and resuspend the cells in DMEM complete medium. Take 20 μL of the single-cell suspension and add it to an equal volume of trypan blue solution for cell counting.

[0123] Press 1×10 6 Calculate and prepare the single cell suspension required for inoculation into a six-well cell culture plate, 2.5 mL / well. Dilute to 1×10 6 Add a single-cell suspension of 100 cells / well to a six-well culture plate. Gently rock the plate back and forth or side to side to evenly distribute the cells. Place the plate in a 37°C incubator with 5% CO2 and incubate overnight.

[0124] 2.2 Phoenix-ECO cell transfection

[0125] 24 hours after cell seeding, remove the six-well culture plate and observe under an inverted microscope. Transfection should be performed when cells are growing well and the density reaches approximately 80%. Calculate the required volumes of plasmids pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19, and pMFG-BCMA CAR20, as well as the volume of Fugene HD.

[0126] Change the medium in the six-well plate and add 2.3 mL of complete DMEM to each well. Gently add 200 μL of the mixed solution dropwise to each well to a total volume of 2.5 mL. Gently shake the plate to mix well. Place the six-well plate in a 37°C incubator with 5% CO2.

[0127] 2.3 Harvesting of Ecotropic Retroviral Vector Supernatant

[0128] 24 hours after transfection, remove the six-well plate from the incubator, carefully remove the culture supernatant, and slowly add fresh DMEM complete medium at 2.5 mL / well. Transfer the six-well plate to a 32°C incubator with 5% CO2 and incubate. 48 hours after transfection, carefully collect the supernatant into a centrifuge tube. Slowly add fresh DMEM complete medium at 2.5 mL / well. Transfer the six-well plate to a 32°C incubator with 5% CO2 and incubate. Filter the collected culture supernatant through a 0.45 μm low-adhesion virus filter membrane, aliquot, and store at -80°C until needed. 72 hours after transfection, remove the six-well plate and place it in a biosafety cabinet. Carefully collect the supernatant into a centrifuge tube. Filter the collected culture supernatant through a 0.45 μm low-adhesion virus filter membrane, aliquot, and store at -80°C until needed.

[0129] 2.4 Phoenix-ECO cell transfection efficiency detection

[0130] 72 hours after transfection, trypsinize and terminate the transfection of Phoenix-ECO cells. Collect the cell suspension after centrifugation at 300g for 5 minutes, discard the supernatant, collect the cell pellet, and wash the cells with PBS. Centrifuge at 300g for 5 minutes at room temperature and discard the supernatant. Resuspend the cells in 50 μL of staining buffer and stain with anti-hc-Myc PE antibody. Untransfected Phoenix-ECO cells serve as a negative control. After staining at 4°C in the dark for 1 hour, wash the cells with 900 μL of PBS. Centrifuge at 300g for 5 minutes, discard the supernatant, and resuspend the cell pellet in staining buffer. Analyze transfection efficiency by flow cytometry.

[0131] 3. Preparation of Amphotropic Retroviral Vectors

[0132] 3.1. PG13 cell transduction. Non-treated 12-well culture plates were pretreated with RetroNectin 1 day before transduction. RetroNectin was diluted with PBS to a final concentration of 10 μg / mL, and 1 mL was added to each well. Grouping: PG13 cell control group, BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19 and BCMA CAR20 experimental groups. The supernatant of the ecotropic retroviral vector collected 72 hours after transfection with Phoenix-ECO was taken out from -80°C, re-thawed at room temperature, and 1 mL was added to each well of the 12-well culture plate. The 12-well culture plate with the ecotropic retroviral vector supernatant added was centrifuged at 30°C for 1 hour. The PG13 cells were removed from the 37°C incubator, the cell status was observed under a microscope, trypsinized and terminated, and pipetted into a single cell suspension. Cell count, 1×10 6 The cells were transferred to a centrifuge tube and centrifuged at 300 g for 5 min.

[0133] 48 hours after transduction, a portion of the cells was digested and tested for transduction efficiency. The remaining PG13 cells were subcultured and expanded into T75 flasks. When the cell density reached 80%, the amphotropic retroviral vector supernatant was harvested and designated H0. Fresh DMEM complete medium was added, and the culture plate was transferred to a 32°C, 5% CO2 incubator. The viral vector supernatant was removed and stored at -80°C. The viral vector supernatant was harvested for four consecutive days and designated H1-H4. The harvested viral vector supernatant was stored at -80°C until further use. To test the transduction efficiency of PG13 cells, a small amount of PG13 cells was collected 48 hours after transduction and tested for transduction efficiency. The BCMA CAR retroviral vector titer was determined by qPCR. The viral titer of the amphotropic retroviral vector supernatant produced by the PG13 cell line was determined to verify the successful production of BCMA CAR amphotropic retroviral vector particles.

[0134] 3. Experimental results

[0135] 1. Successfully prepared BCMA CAR ecotropic retroviral vector

[0136] Phoenix-ECO cells were transfected with pMFG-BCMA CAR15, pMFG-BCMA CAR16, pMFG-BCMA CAR17, pMFG-BCMA CAR18, pMFG-BCMA CAR19 and pMFG-BCMA CAR20 plasmids. The transfection efficiency was detected by flow cytometry 72 hours later. The transfection efficiency of pMFG-BCMA CAR15 was 56.70%, pMFG-BCMA CAR16 was 64.91%, pMFG-BCMA CAR17 was 59.11%, pMFG-BCMA CAR18 was 57.88%, pMFG-BCMA CAR19 was 59.85%, and pMFG-BCMA CAR20 was 57.95%. All six plasmids were successfully transfected into Phoenix-ECO cells. The supernatant of the ecotropic retroviral vector collected 72 hours after transfection can be used to transduce PG13 cells.

[0137] 2. Successfully constructed a cell line that stably produces the BCMA CAR amphotropic retroviral vector

[0138] The supernatant of the successfully prepared amphotropic retroviral vectors for BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19, and BCMA CAR20 was used to transduce PG13 cells, and the transduction efficiency was determined by flow cytometry 48 hours later. The transduction efficiency of BCMA CAR15 was 96.91%, BCMA CAR16 was 97.53%, BCMA CAR17 was 98.50%, BCMA CAR18 was 98.69%, BCMA CAR19 was 97.82%, and BCMA CAR20 was 98.62%. The results demonstrated the successful preparation of amphotropic retroviral vector particles and the successful establishment of a PG13 cell line capable of stably producing amphotropic retroviral vector particles.

[0139] 3. Successful preparation of BCMA CAR dual-tropic retroviral vector

[0140] The PG13 cell line transduced with the BCMA CAR ecotropic retroviral vector was expanded and cultured in a T75 culture flask. The amphotropic retroviral vector particles were harvested for 5 consecutive days for qPCR detection. The results showed that the highest titer of the BCMA CAR amphotropic retroviral vector was BCMA CAR15H2: 1.40×10 7 ±1.13×10 6 copies / mL, BCMA CAR16H1: 1.33×10 7 ±7.07×10 4 Copies / mL, BCMA CAR17H2: 3.09×10 7 ±1.70×10 6 Copies / mL, BCMA CAR18H2: 2.44×10 7 ±2.55×10 6 Copies / mL, BCMA CAR19H2: 1.95×10 7 ±2.36×10 4 Copies / mL, BCMA CAR20H2: 2.68×10 7 ±4.24×10 6 The results indicate that we have successfully prepared amphotropic retroviral vectors for BCMA CAR15, BCMA CAR16, BCMA CAR17, BCMA CAR18, BCMA CAR19, and BCMA CAR20. The amphotropic retroviral vector particles with the highest titer for each BCMA CAR were selected for human primary T cell transduction experiments.

[0141] Example 3 Preparation of BCMA CAR-T cells

[0142] Based on the preparation of BCMA CAR amphotropic retroviral vector particles, BCMA CAR amphotropic retroviral vector particles are transduced into human primary T cells to construct BCMA CAR-T cells. The technical route is shown in Figure 5: First, human peripheral blood mononuclear cells (PBMCs) are separated by density gradient centrifugation. Primary T cells are activated by stimulation with CD3 monoclonal antibody and cytokine IL-2. The activated T cells are then transduced with BCMA CAR by centrifugation to construct BCMA CAR-T cells. Flow cytometry is used to detect the expression of the cell surface Myc tag to determine whether BCMA CAR is expressed on the T cell surface.

[0143] 1. Experimental Methods

[0144] 1. Construction of BCMA CAR-T cells

[0145] One day before transduction, non-treated 12-well culture plates were pretreated with RetroNectin. RetroNectin was diluted with PBS to a final concentration of 10 μg / mL, and 1 mL was added to each well.

[0146] Groups were divided into a non-T cell transduction control group (Pan-T group), BCMA CAR15-T group, BCMA CAR16-T group, BCMA CAR17-T group, BCMA CAR18-T group, BCMA CAR19-T group, BCMA CAR20-T group, and bb2121 CAR-T (a currently marketed BCMA CAR, with the same structure as our other BCMA CARs except for the ScFv). The experiment was repeated using T cells isolated from different volunteers.

[0147] Aspirate the RetroNectin solution, add 1 mL of PBS to the 12-well culture plate for washing, and discard the liquid. Take out the supernatant of the amphotropic retroviral vector collected from the PG13 cell line from -80°C, rethaw at room temperature, and add 1 mL to each well of the 12-well culture plate. Centrifuge the 12-well culture plate with the amphotropic retroviral vector supernatant at 30°C for 1 hour. Remove the T cells from the 37°C incubator, observe the cell status under a microscope, and pipette into a single cell suspension. Count the cells and take 1×10 6Transfer the cell suspension to a centrifuge tube and centrifuge at 300g for 5 minutes. Discard the supernatant and resuspend the cells in 1 mL of amphotropic retroviral vector supernatant per well. For the control group, resuspend the cells in AIM-V complete medium. Disperse the cells into a single-cell suspension and slowly add the suspension dropwise to a 12-well culture plate. Centrifuge the 12-well culture plate with the cells suspended in amphotropic retroviral vector supernatant at 30°C for 1 hour. Incubate the 12-well culture plate in a 37°C, 5% CO2 incubator for at least 1 hour. Remove the 12-well culture plate, harvest the cells, centrifuge at 300g for 5 minutes, and carefully remove the supernatant. Slowly add 1 mL of fresh amphotropic retroviral vector supernatant per well. For the control group, add fresh AIM-V complete medium. Harvest the cells, centrifuge at 300g for 5 minutes, and carefully remove the supernatant. Resuspend the cells in fresh AIM-V complete medium. Incubate the 12-well culture plate in a 37°C, 5% CO2 incubator.

[0148] 2. T cell transduction efficiency detection

[0149] 48 hours after transduction, mix the primary human T cells with a pipette and gently disperse them into a single-cell suspension. Transfer 300 μL of the cell suspension to a 1.5 mL centrifuge tube. Centrifuge at 300 g for 5 minutes, collect the cell pellet, and wash the cells with PBS. Centrifuge at 300 g for 5 minutes at room temperature and discard the supernatant. Resuspend the cells in 50 μL of staining buffer and stain with APC anti-human CD3 antibody and anti-hc-Myc PE antibody. Untransduced T cells serve as a negative control. After staining at 4°C in the dark for 1 hour, wash the cells with 900 μL of PBS. Centrifuge at 300 g for 5 minutes, discard the supernatant, and resuspend the cell pellet in staining buffer. Analyze the transduction efficiency by flow cytometry. The percentage of CD3-positive and Myc-positive cells is the transduction efficiency of the BCMA CAR-transduced T cells.

[0150] 2. Experimental results

[0151] The BCMA CAR retroviral vector successfully transduced primary human T cells. The BCMA CAR amphotropic retroviral vector was transduced into primary human T cells, and the transduction efficiency was measured 48 hours later. The transduction efficiency of bb2121 CAR-T cells was 55.30%, BCMA CAR15-T cells was 43.18%, BCMA CAR16-T cells was 55.35%, BCMA CAR17-T cells was 65.87%, BCMA CAR18-T cells was 62.66%, BCMA CAR19-T cells was 55.42%, and BCMA CAR20-T cells was 54.59%. These results demonstrate that BCMA CAR-T cells were successfully constructed and can be used in subsequent experiments to verify their ability to kill tumor cells.

[0152] Example 4 Verification of BCMA CAR-T Anti-tumor Function in Vitro

[0153] After successfully constructing BCMA CAR-T cells, their ability to kill tumor cells was verified. BCMA CAR-T cells were co-incubated with target cells, and target cell apoptosis was detected using luciferase flow cytometry to verify the BCMA CAR-T cell's ability to kill tumor cells. The technical route is shown in Figure 6.

[0154] 1. Luciferase bioluminescence assay to detect the anti-tumor ability of BCMA CAR-T in vitro

[0155] Firefly luciferase is a monomeric protein with a size of approximately 61 kDa. Its substrate is ATP-Mg 2+ It catalyzes the oxidation of luciferin, converting chemical energy into electron transitions to generate light energy, forming the product molecule oxidized luciferin. RPMI-gfp-luc cells stably expressing the luciferase reporter gene in the laboratory can generate chemical signals under the catalytic action of the substrate, which can be used to monitor tumor cell survival.

[0156] 1. Groups: Pan-T group, BCMA CAR15-T group, BCMA CAR16-T group, BCMA CAR17-T group, BCMA CAR18-T group, BCMA CAR19-T group, and BCMA CAR20-T group.

[0157] 2. Plating: Blow RPMI-gfp-luc into a single cell suspension and count the cells; when the cells are growing well, dilute the cells to 4×10 4 / 50μL, take 50μL / well and inoculate into 96-well all-white culture plate.

[0158] 3. 50 μL / well of Pan-T, BCMA CAR15-T, BCMA CAR16-T, BCMA CAR17-T, BCMA CAR18-T, BCMA CAR19-T, and BCMA CAR20-T cells were mixed with target cells at different effector-target ratios (1:4, 1:2, 1:1, 2:1, and 4:1). Tumor cells were used as blank controls. Culture the cells in a 37°C, 5% CO2 incubator for 12 h.

[0159] 4. Add ONE-Glo equal to the volume of culture medium to each well TM Luciferase assay reagent and mix thoroughly.

[0160] 5. The following formula was used for analysis: Cell lysis rate = 1 - (lysis of experimental group - lysis of blank group) / (lysis of maximum release pore - lysis of blank group) × 100%. Each experiment was repeated three times.

[0161] 6. Data processing: GraphPad Prism 8 software was used for statistical analysis. The measured data were expressed as (x±s). The t-test was used to compare the two groups. When P < 0.05, the difference was considered statistically significant.

[0162] 2. Flow cytometry detection of BCMA CAR-T anti-tumor ability in vitro

[0163] Annexin V is a phospholipid-binding protein with a high affinity for phosphatidylserine (PS). It can specifically bind to the cell membrane of early apoptotic cells through PS exposed on the extracellular side of the cells. Therefore, Annexin V is a sensitive indicator for detecting early cell apoptosis.

[0164] 1. Groups: Pan-T group, BCMA CAR15-T group, BCMA CAR16-T group, BCMA CAR17-T group, BCMA CAR18-T group, BCMA CAR19-T group, and BCMA CAR20-T group.

[0165] 2. Plating: Spread the K562-hBCMA-gfp cells into a single cell suspension and count them; when the cells are growing well, dilute the cells to 4×10 4 / 100μL, take 100μL / well and inoculate into 96-well plate.

[0166] 3. The effector cells were mixed with target cells at different effector-target ratios (1:4, 1:2, 1:1, 2:1, 4:1) (the tumor cell group was set as a blank control) and cultured in a 37°C, 5% CO2 incubator for 12 hours.

[0167] 4. Add 130 μL staining buffer to each well to wash the cells, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0168] 5. Add BV421 anti-human CD3 antibody to each well and stain at 4°C in the dark for 40 minutes.

[0169] 6. Add 150 μL of staining buffer to each well to terminate staining, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0170] 7. Add Annexin V-Alexa Fluor 647 to each well to detect tumor cell apoptosis and stain for 40 minutes at 4°C in the dark.

[0171] 8. Add 150 μL of staining buffer to each well to terminate staining, centrifuge at 300 g for 5 minutes, and discard the supernatant.

[0172] 9. Resuspend cells in 200 μL staining buffer per well.

[0173] 10. Data were collected by flow cytometry and analyzed using FlowJo. Tumor cell apoptosis was calculated as the percentage of CD3-negative and Annexin V-positive cells relative to total cells. The experiment was repeated three times, with blood drawn from different volunteers. The BCMA CAR-T cells with the best killing effect were selected.

[0174] 3. Flow cytometry further validates BCMA CAR-T's in vitro anti-tumor ability

[0175] 1. Grouping: Pan-T group, positive control group (bb2121 CAR-T group), and BCMA CAR16-T group.

[0176] 2. Plating: Blow RPMI-gfp-luc into a single cell suspension and count the cells; when the cells are growing well, dilute the cells to 4×10 4 / 100μL, take 100μL / well and inoculate into 96-well plate.

[0177] 3. Pan-T, bb2121 CAR-T, and BCMA CAR16-T cells were mixed with target cells at different effector-target ratios (1:4, 1:2, 1:1, and 2:1) (the tumor cell group was set as a blank control) and cultured in a 37°C, 5% CO2 incubator for 12 h.

[0178] 4. The flow cytometry method and data processing are the same as above.

[0179] 4. Experimental Results

[0180] 1. BCMA expression detection results on tumor cell surface

[0181] K562-hBCMA-gfp, RPMI-gfp-luc, K562-cBCMA, and K562 tumor cells were stained with BV421 anti-human BCMA antibody, and surface BCMA expression was detected by flow cytometry. The results showed that BCMA was highly expressed on the surface of K562-hBCMA-gfp and RPMI-gfp-luc cells to varying degrees, with BCMA expression on the surface of K562-hBCMA-gfp and RPMI-gfp-luc cells at 85.5% and 58.5%, respectively. These cells can be used as target cells in this study. K562-cBCMA and K562 cells basically do not express BCMA on their surface and can be used as negative control cells.

[0182] 2. In vitro tumor killing activity of BCMA CAR-T cells detected by luciferase bioluminescence assay

[0183] To validate the in vitro cytotoxicity of BCMA CAR-T cells, we incubated BCMA CAR15-T, BCMA CAR16-T, BCMA CAR17-T, BCMA CAR18-T, BCMA CAR19-T, and BCMA CAR20-T cells with target cells at varying effector-to-target ratios. Luciferase bioluminescence assay was used to measure target cell apoptosis 12 hours later. Results showed that compared with the Pan-T group, the BCMA CAR16-T and BCMA CAR17-T groups exhibited enhanced cytotoxicity against tumor cells, positively correlated with the effector-to-target ratio. BCMA CAR16-T exhibited the strongest cytotoxicity against tumor cells. Three independent replicates demonstrated consistent results.

[0184] 3. In vitro tumor killing activity of BCMA CAR-T cells detected by flow cytometry

[0185] To replicate the ability of BCMA CAR-T cells to kill tumor cells in vitro, we incubated BCMA CAR15-T, BCMA CAR16-T, BCMA CAR17-T, BCMA CAR18-T, BCMA CAR19-T, and BCMA CAR20-T cells with K562-hBCMA-GFP target cells at varying effector-to-target ratios. Target cell apoptosis was assessed 12 hours later by flow cytometry. The results are shown in the figure: Compared with the Pan-T group, the BCMA CAR16-T and BCMA CAR17-T groups showed enhanced tumor cell killing ability, and this killing ability was positively correlated with the effector-to-target ratio. BCMA CAR16-T exhibited the strongest tumor cell killing effect. Three independent replicates demonstrated consistent results, consistent with those detected by firefly bioluminescence assays (Figure 7). The BCMA CAR16-T cell with the highest killing effect was compared with a positive BCMA CAR (bb2121 CAR-T cell).

[0186] 4. Further verification of BCMA CAR-T's in vitro anti-tumor ability

[0187] The BCMA CAR16-T cells, which showed the most significant tumor cell killing effect, were screened and compared with the positive bb2121 CAR-T cells. The results are shown in Figure 8 below: While the BCMA CAR16-T cells showed a more pronounced tumor cell killing effect than the Pan-T group, the effect was not as significant as the positive control group, bb2121 CAR0-T. Further optimization is needed based on the selected BCMA CAR16 cells to improve their tumor cell killing efficiency.

[0188] Example 5 Optimization of BCMA CAR Retroviral Vector Plasmid Construction and BCMA CAR Retroviral Vector Packaging

[0189] 1. Optimizing the Construction of BCMA CAR Retroviral Vector Plasmid

[0190] We successfully screened BCMA CAR16-T cells with the strongest tumor cell-killing ability. However, compared to the marketed bb2121 CAR-T cells, their tumor cell-killing ability needs to be enhanced. Because BCMA CAR16-T and bb2121 CAR-T cells are identical except for the ScFv, we designed a tandem design of two or three BCMA CAR16-T ScFvs to increase antigen affinity and, in the hope of enhancing anti-tumor efficacy.

[0191] 1. Schematic diagram of the BCMA CAR31, BCMA CAR32, and BCMA CAR33 structures

[0192] The ScFv region of BCMA CAR uses the human BCMA-targeting monoclonal antibody sequence BCMA CAR16 screened by phage display in the previous laboratory. The three optimized BCMA CAR structure ScFv regions are two ScFv connected, and three ScFv regions connected, while the rest of the structure remains unchanged. The newly constructed BCMA CAR expression plasmid map is shown in Figure 9

[0193] 2. Gel electrophoresis results of target fragment and vector fragment

[0194] After double digestion with XhoI and NgoMIV, the vector pMFG fragment was approximately 7 kb in size, the target fragment BCMA CAR31 was approximately 1.7 kb in size, the BCMA CAR32 was approximately 1.7 kb in size, and the BCMA CAR33 was approximately 2.5 kb in size. Electrophoresis results showed that the vector fragment and the target fragment bands were of the correct size.

[0195] 3. pMFG-BCMA CAR enzyme digestion and gel electrophoresis results

[0196] The constructed pMFG-BCMA CAR31, pMFG-BCMA CAR32, and pMFG-BCMA CAR33 plasmids were extracted and identified using double enzyme digestion (XhoI / NgoMIV). The target fragments were approximately 1.7 kb for BCMA CAR31, 1.7 kb for BCMA CAR32, and 2.5 kb for BCMA CAR33. The pMFG vector fragment was approximately 7 kb. The electrophoresis results are shown in Figure 10: pMFG-BCMA CAR31 plasmid enzyme digestion identified ①, ②, ③, ④, and ⑤ as correct; pMFG-BCMA CAR32 plasmid enzyme digestion identified ①, ④, and ⑤ as correct; and pMFG-BCMA CAR33 plasmid enzyme digestion identified ②, ③, and ⑤ as correct. Correctly identified plasmids were sent for sequencing for further verification. Correctly identified plasmids were purified and sent for sequencing. Sequencing results showed that the sequences of pMFG-BCMA CAR31, pMFG-BCMA CAR32, and pMFG-BCMA CAR33 were completely correct.

[0197] 2. Optimizing BCMA CAR Retroviral Vector Packaging

[0198] Based on the optimized pMFG-BCMA CAR plasmid, the BCMA CAR retroviral vector was packaged and a cell line stably producing the retroviral vector was established. The specific experimental process is shown in Figure 4: First, the optimized pMFG-BCMA CAR plasmid was transiently transfected into Phoenix ECO cells, and the BCMA CAR ecotropic retroviral vector supernatant particles were collected. The collected BCMA CAR ecotropic retroviral vector supernatant particles were then transduced into the PG13 cell line to produce amphotropic retroviral vector supernatant particles. Furthermore, a PG13 cell line stably producing amphotropic retroviral vector supernatant particles was established. The viral vector titer was measured by qPCR to verify the successful production of amphotropic retroviral vector supernatant particles. Using a similar method as previously described, the following results were obtained.

[0199] 1. Successfully prepared BCMA CAR ecotropic retroviral vector

[0200] The pMFG-BCMA CAR31, pMFG-BCMA CAR32, and pMFG-BCMA CAR33 plasmids were transfected into Phoenix-ECO cells. The transfection efficiency was detected by flow cytometry 72 hours later: the transfection efficiency of pMFG-BCMA CAR31 was 59%, the transfection efficiency of pMFG-BCMA CAR32 was 58.07%, and the transfection efficiency of pMFG-BCMA CAR33 was 60.62%. All three plasmids were successfully transfected into Phoenix-ECO cells.

[0201] 2. Successfully constructed a cell line that stably produces the BCMA CAR amphotropic retroviral vector

[0202] The supernatant of the successfully prepared amphotropic retroviral vectors for BCMA CAR31, BCMA CAR32, and BCMA CAR33 was used to transduce PG13 cells, and the transduction efficiency was measured by flow cytometry 48 hours later. The results showed that the transduction efficiency of BCMA CAR31 was 93.66%, the transduction efficiency of BCMA CAR32 was 95.35%, and the transduction efficiency of BCMA CAR33 was 98.24%. These results demonstrate the successful preparation of amphotropic retroviral vector particles and the establishment of a PG13 cell line capable of stably producing amphotropic retroviral vector particles.

[0203] 3. Successful preparation of BCMA CAR dual-tropic retroviral vector

[0204] The PG13 cell lines transduced with BCMA CAR31, BCMA CAR32, and BCMA CAR33 ecotropic retroviral vectors were expanded and cultured in T75 culture flasks. The amphotropic retroviral vector particles were harvested for 5 consecutive days for qPCR detection. The results showed that the highest titer of BCMA CAR31 retroviral vector was BCMA CAR31 H4: 2.29×10 7 ±1.06×10 6 Copies / mL, BCMA CAR32 H3: 1.64×10 7 ±1.12×10 6 Copies / mL, BCMA CAR33 H4: 2.40×10 7 ±4.53×10 6 These results indicate that we have successfully prepared amphotropic retroviral vectors for BCMA CAR31, BCMA CAR32, and BCMA CAR33. The amphotropic retroviral vector particles with the highest titer were selected for transduction of primary human T cells.

[0205] Example 6 Optimization of BCMA CAR-T cell preparation

[0206] Based on the successful preparation of BCMA CAR amphotropic retroviral vector particles, the BCMA CAR amphotropic retroviral vector particles were transduced into primary human T cells to construct BCMA CAR-T cells. The technical route is shown in Figure 5: First, human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. Primary T cells were activated by stimulation with CD3 monoclonal antibody and cytokine IL-2. The activated T cells were then transduced with BCMA CAR by centrifugation to construct BCMA CAR-T cells. Flow cytometry was used to detect the expression of the Myc tag on the cell surface to verify BCMA CAR expression on the T cell surface. Furthermore, qPCR was used to determine the copy number of the BCMA CAR retroviral vector in the genome of each primary human T cell to verify successful integration. Using a similar method as previously described, the following results were obtained.

[0207] 1. BCMA CAR retroviral vector successfully transduced primary human T cells

[0208] The BCMA CAR amphotropic retroviral vector was transduced into human primary T cells, and the transduction efficiency was detected 48 hours later. The results are shown in Figure 11: the transduction efficiency of BCMA CAR16-T was 61.10% ± 3.65%, the transduction efficiency of BCMA CAR31-T was 61.97% ± 2.98%, the transduction efficiency of BCMA CAR32-T was 62.20% ± 1.59%, and the transduction efficiency of BCMA CAR33-T was 57.80% ± 3.02%. The results showed that the transduced human primary T cells successfully expressed BCMA CAR, and there was no statistical difference in the transduction efficiency between the three groups compared with the control group BCMA CAR16-T. We successfully prepared BCMA CAR-T cells.

[0209] BCMA CAR successfully integrated into the genome of primary human T cells. Human primary T cells were transduced with the BCMA CAR amphotropic retroviral vector, and the number of copies integrated into the T cell genome was measured 48 hours later. The results showed that the BCMA CAR16-T cells had an integrated genome copy number of 0.44±0.05 copies / T cell, the BCMA CAR31-T cells had an integrated genome copy number of 0.48±0.02 copies / cell, the BCMA CAR32-T cells had an integrated genome copy number of 0.43±0.04 copies / T cell, and the BCMA CAR33-T cells had an integrated genome copy number of 0.32±0.04 copies / T cell. These results indicate that the BCMA CARs were successfully integrated into the genome of primary human T cells.

[0210] Example 7 Optimization of BCMA CAR-T in vitro anti-tumor function verification

[0211] Based on the successful construction of three optimized BCMA CAR-T cells, their in vitro tumor cell cytotoxicity was first verified. Using different tumor cells expressing the human BCMA antigen as target cells and target cells not expressing the human BCMA antigen as controls, tumor cell survival was monitored using luciferase bioluminescence assays and incucyte real-time dynamic live cell imaging, and tumor cell apoptosis was detected using flow cytometry to verify the specificity and efficacy of BCMA CAR-T cells in killing target cells. Cytokine secretion by BCMA CAR-T cells was measured using CBA assays, and the proliferation capacity of BCMA CAR-T cells was assessed using CFSE assays. The specific experimental scheme is shown in Figure 12. Using similar methods as previously described, the following experimental results were obtained.

[0212] 1. BCMA CAR31-T has significant killing ability in vitro

[0213] BCMA CAR16-T, BCMA CAR31-T, BCMA CAR32-T, and BCMA CAR33-T were co-incubated with K562-hBCMA-gfp cells at a 1:1 effector-target ratio. Real-time fluorescence was used to continuously monitor tumor cell fluorescence changes, recording every 2 hours for 48 hours. The results are shown in Figure 13: BCMA CAR31-T demonstrated a stronger tumor cell-killing effect than the control BCMA CAR16-T. Therefore, in the following experiments, we selected BCMA CAR31-T to continue comparing its tumor killing, cytokine secretion, and proliferation abilities with BCMA CAR16-T.

[0214] 1. BCMA CAR31-T can effectively activate

[0215] Flow cytometry was used to detect CD69 expression on the surface of BCMA CAR-T cells. The results are shown in Figure 14: In the absence of tumor cell stimulation, there was no statistical difference in surface CD69 expression between BCMA CAR31-T and BCMA CAR16-T. Under stimulation of BCMA-expressing K562-hBCMA-gfp cells, BCMA CAR31-T cells expressed higher surface CD69 than BCMA CAR16-T cells, and the difference was statistically significant. This study result shows that the optimized BCMA CAR31-T can be better activated by tumor cells.

[0216] 2. BCMA CAR31-T has highly effective anti-tumor ability in vitro

[0217] To determine the lysis ability of BCMA CAR31-T cells against BCMA-positive tumor cells, BCMA CAR31-T cells, BCMA CAR16-T cells, or Pan-T cells were incubated with RPMI-gfp-luc or cells at different effector-target ratios for 12 hours, and the chemiluminescence signal intensity was detected. The experimental results are shown in Figure 15: Compared with BCMA CAR16-T, BCMA CAR31-T cells showed stronger ability to kill tumor cells in vitro at all effector-target ratios, and the differences were statistically significant.

[0218] BCMA CAR31-T cells demonstrated highly effective and specific anti-tumor activity in vitro. To validate the antigen specificity of BCMA CAR31-T cell anti-tumor activity, BCMA CAR31-T cells were co-incubated with K562-hBCMA-gfp and PMI-gfp-luc cells expressing human BCMA at varying effector-target ratios. K562-cBCMA cells expressing cynomolgus macaque BCMA and K562 cells not expressing BCMA served as negative target cell controls. A BCMA CAR16-T control group was also included. Samples were analyzed by flow cytometry. The results, as shown in Figure 16, show that compared to the BCMA CAR16-T group, BCMA CAR31-T cells demonstrated enhanced cytotoxicity against two different BCMA-positive tumor cell lines at varying effector-target ratios. However, BCMA CAR31-T showed the same killing ability as Pan-T against non-human BCMA-expressing K562-cBCMA and BCMA-negative K562 cells. This result shows that BCMA CAR31-T cells are specific for the BCMA antigen of tumor cells.

[0219] 3. BCMA CAR31-T has strong cytokine secretion ability

[0220] Cytokine production is a hallmark of effective CAR-T cell activation. BCMA CAR31-T cells were co-incubated with K562-hBCMA-gfp cells expressing the human BCMA antigen at a 1:1 effector-target ratio for 12 hours. Cell culture fluid was harvested, and cytokine levels, such as TNF-α, IFN-γ, IL-6, IL-17A, and aFasL, were measured using a CBA kit. The results are shown in Figure 17: Compared to BCMA CAR16-T cells, BCMA CAR31-T cells secreted significantly more cytokines, including TNF-α, IFN-γ, IL-6, IL-17A, and aFasL, upon stimulation with BCMA-positive tumor cells. The release of these cytokines indicates that BCMA CAR31-T cells can be effectively activated, further confirming the more potent anti-tumor activity of BCMA CAR31-T cells.

[0221] BCMA CAR31-T cells have strong in vitro proliferation capacity. To evaluate the in vitro proliferation capacity of BCMA CAR31-T cells, we used a CFSE-based assay to measure proliferation, with the Pan-T group and BCMA CAR16-T group serving as controls. The results are shown in Figure 18: After 72 hours of cell culture, the CFSE green fluorescence signal of BCMA CAR16-T cells was significantly reduced compared to that of the BCMA CAR31-T group, indicating that these BCMA CAR31-T cells proliferated faster.

[0222] Example 8 Optimization of BCMA CAR-T Anti-tumor Function Verification in Vivo

[0223] BCMA CAR31-T cells were found to be highly effective and specific in killing tumor cells, with strong proliferation and cytokine secretion in vitro. The anti-tumor activity of BCMA CAR31-T cells in vivo was further validated using a xenograft tumor model. The experimental protocol is shown in Figure 19.

[0224] 1. Successful establishment of mouse xenograft tumor model

[0225] NPG mice were injected intravenously with 2 × 10 6 RPMI-gfp-luc cells were injected into the mouse body. Ten days later, in vivo imaging of the mice was performed. The results are shown in Figure 20: Tumor signals were visible in all mice, and the tumors were uniformly formed, indicating that a mouse xenograft tumor model was successfully established. The mice were randomly divided into four groups. No statistically significant difference in tumor signals was found between the BCMA CAR31-T group and the BCMA CAR16-T group.

[0226] BCMA CAR-T cells are effective against tumors in vivo

[0227] Starting from the sixth day after the first BCMA CAR-T injection, in vivo imaging of mice was performed once a week to detect changes in mouse tumor signal intensity. The results are shown in Figure 21: After the first injection of BCMA CAR31-T, there was no statistical difference in the mouse tumor signal between the mice and BCMA CAR16-T. After the second injection, the mouse imaging showed that the tumor signal in the BCMA CAR31-T group was significantly weakened compared with the BCMA CAR16-T group, and the difference was statistically significant. This result indicates that BCMA CAR31-T has effective anti-tumor ability in vivo.

[0228] 3. Increased IFN-γ secretion by BCMA CAR-T

[0229] 48 hours after the two BCMA CAR-T cell injections, peripheral blood was collected from mice, serum was separated, and ELISA was used to detect the secretion level of human IFN-γ in the serum. The results are as follows: After the first BCMA CAR-T injection, the IFN-γ secretion level of BCMA CAR31-T was low at 180.29±31.63pg / mL, which was no different from BCMA CAR16-T (157.74±45.51pg / mL). The results were consistent with the above-mentioned in vivo imaging results of mice, indicating no significant anti-tumor effect. After the second BCMA CAR-T injection, the IFN-γ secretion level of BCMA CAR31-T was significantly increased (3778.37±934.68pg / mL) compared with BCMA CAR16-T (1254.03±1523.75pg / mL), and the results were consistent with the above-mentioned in vivo imaging results of mice, indicating a significant anti-tumor effect. The results of this study showed that BCMA CAR31-T was effectively activated after the second CAR-T injection.

[0230] BCMA CAR31-T has a long-lasting anti-tumor effect in vivo

[0231] Blood samples were collected on the 24th, 31st and 37th days after the mice were inoculated with tumor cells to detect the injected CD3 + The results are as follows: 24 days after tumor cell inoculation, the number of CD3 T cells in each 100 μL mouse peripheral blood was + The T cell counts in the model group (-1474.00±3670.66), Pan-T group (-288.33±8110.37), BCMA CAR16-T group (11800.00±7019.97), and BCMA CAR31-T group (29850.00±15939.23) were statistically significant. The BCMA CAR31-T group had a significantly higher T cell count in the peripheral blood of mice than the BCMA CAR16-T group. On the 31st day after tumor cell inoculation, the number of CD3 + The T cell counts in the model group (-4996.00±2067.62), Pan-T group (-983.33±1922.31), BCMA CAR16-T group (8000.00±7186.93), and BCMA CAR31-T group (23800.00±11440.80) were statistically significant. The BCMA CAR31-T group had a significantly higher T cell count in the peripheral blood of mice than the BCMA CAR16-T group. On the 37th day after tumor cell inoculation, the CD3 +The T cell counts in the model group (1783.33±8431.47), the Pan-T group (-1750.00±3573.65), the BCMA CAR16-T group (11400.00±9017.98), and the BCMA CAR31-T group (54866.67±18619.20) were statistically significant. The results showed that compared with the BCMA CAR16-T group, the BCMA CAR31-T group survived longer in vivo, demonstrating more sustained anti-tumor activity.

[0232] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A chimeric antigen receptor targeting BCMA, characterized in that: The receptor includes a human ScFv structure targeting BCMA, wherein the heavy chain variable region ScFv-VH and the light chain variable region ScFv-VL of the ScFv are connected by one or more G4S sequences; the amino acid sequence of the heavy chain variable region ScFv-VH has at least 90% homology with the following SEQ ID NO.8, preferably at least 95% homology, and more preferably at least 98% homology: SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSS; The amino acid sequence of the light chain variable region ScFv-VL has at least 90% homology with the following SEQ ID NO.9, preferably at least 95% homology, and more preferably at least 98% homology: QSALTQPASASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCAIWHSSAWVFGGGTKLTVLG.

2. The BCMA-targeted chimeric antigen receptor according to claim 1, characterized in that The receptor includes a human ScFv targeting BCMA, the structure of which is ScFv-VH-(G4S)n-ScFv-VL, wherein n is an integer greater than or equal to 1, preferably 3 or 4.

3. The chimeric antigen receptor targeting BCMA according to claim 1, characterized in that The receptor includes a human ScFv targeting BCMA, the structure of which is ScFv-VH-(G4S)3-ScFv-VL, the amino acid sequence of the heavy chain variable region ScFv-VH is SEQ ID NO.8, and the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.

9.

4. The chimeric antigen receptor targeting BCMA according to claim 1, characterized in that The receptor includes a human ScFv targeting BCMA, the structure of which is ScFv-VH-(G4S)n-ScFv-VL-(G4S)n-ScFv-VL-(G4S)n-ScFv-VH, wherein n is an integer greater than or equal to 1, preferably 3 or 4.

5. The chimeric antigen receptor targeting BCMA according to claim 1, characterized in that The receptor includes a human ScFv targeting BCMA, the structure of which is ScFv-VH-(G4S)3-ScFv-VL-(G4S)4-ScFv-VL-(G4S)3-ScFv-VH, the amino acid sequence of the heavy chain variable region ScFv-VH is SEQ ID NO.8; the amino acid sequence of the light chain variable region ScFv-VL is SEQ ID NO.

9.

6. The chimeric antigen receptor according to any one of claims 1 to 5, characterized in that The receptor includes an upstream signal peptide and a myc tag for detection connected in series; a human BCMA-targeting ScFv structure comprising a heavy chain variable region and a light chain variable region; a CD8 hinge-transmembrane domain; a CD28 or 4-1BB synergistic activation domain and a CD3ζ intracellular signaling domain.

7. A chimeric antigen receptor T cell targeting BCMA, characterized in that: The chimeric antigen receptor according to any one of claims 1 to 6 is expressed.

8. A drug for treating tumors, characterized in that: It contains the chimeric antigen receptor T cell according to claim 7.

9. Use of the chimeric antigen receptor according to any one of claims 1 to 6 in preparing chimeric antigen receptor T cells and in tumor treatment.

10. The use according to claim 9, wherein the tumor is a surface BCMA-positive tumor.

11. The use according to claim 9, wherein the tumor is multiple myeloma.

12. The use of the chimeric antigen receptor according to any one of claims 1 to 6, characterized in that: The gene fragment encoding the chimeric antigen receptor is inserted into a viral expression vector, packaged into viral vector particles, and used to infect human T cells to prepare chimeric antigen receptor T cells for the treatment of surface BCMA-positive tumors.

Images