Chimeric antigen receptors targeting bcma and t cells and uses thereof

Abstract

The application discloses a targeting BCMA chimeric antigen receptor and T cells and application thereof, the targeting BCMA chimeric antigen receptor of the application includes two human-derived target BCMA tandem ScFv heavy chains VH, does not include ScFv light chain VL;Two heavy chains VH are connected by one or more G4S sequences between sequences;The amino acid sequences of the two tandem ScFv heavy chains VH are at least 90% homologous with SEQ ID No.1 and SEQ ID No.2 respectively.The VH developed by the application is a humanized antibody heavy chain variable region, which has lower immunogenicity than traditional murine or chimeric ScFv, multiple VH tandem can cover multiple antigen epitopes on the tumor surface, reduce immune escape;The optimal heavy chain recombinant structure screened by the application has better killing effect compared with the BCMA CAR T cell product CT103A on the market.

Description

Technical Field

[0001] This invention relates to the field of cell immunotherapy for tumors, specifically to targeting the BCMA chimeric antigen receptor and its T cells and their applications. Background Technology

[0002] BCMA (B-cell maturation antigen) is a highly specific biomarker on the surface of plasma cells and myeloma cells. It is almost never expressed on normal hematopoietic stem cells or most tissues, making it a gold standard target for the treatment of recurrent myeloma (RRMM). Traditional treatments, including proteasome inhibitors, immunomodulatory agents, or monoclonal antibodies, have poor prognoses, high rates of relapse and refractory disease, and short survival rates.

[0003] BCMA-targeted therapy has become a core breakthrough in the treatment of multiple myeloma in recent years, but existing regimens still face problems such as antigen escape, immunogenicity, and insufficient affinity. Traditional ScFv consists of a heavy chain (VH) and a light chain (VL). Clinically, murine or chimeric ScFv is commonly used, which easily induces anti-CAR antibodies (ACA), leading to CAR T cell clearance and decreased efficacy. The VH-VL interface is hydrophobic, and when expressed alone, it is prone to aggregation and degradation, has a short half-life in vivo, and is functionally unstable. A single ScFv can only recognize a single epitope, and tumors can easily achieve immune escape through BCMA downregulation or mutation.

[0004] In its earlier application CN116535521A, the applicant disclosed a chimeric receptor targeting BCMA, which includes a human BCMA-targeting ScFv structure, wherein the heavy chain variable region ScFv-VH and the light chain variable region ScFv-VL of ScFv are linked by one or more G4S sequences. Based on this, the applicant further improved the chimeric receptor targeting BCMA, enhancing its target cell killing effect, particularly its killing efficiency under low target-to-cell ratio conditions. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a BCMA-targeting chimeric antigen receptor comprising two tandem ScFv heavy chains (VH) targeting BCMA, excluding the ScFV light chain (VL); the sequences of the two heavy chains (VH) are linked by one or more G4S sequences; the amino acid sequences of the two tandem ScFv heavy chains (VH) have at least 90% homology with SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0006] In one embodiment, the two tandem ScFv heavy chain VH structures of the human-derived BCMA-targeting chain are SEQ ID No. 1 and SEQ ID No. 2, which are connected by one or more G4S sequences.

[0007] In one embodiment, the receptor includes an upstream signal peptide and a myc tag for detection, tandemly connected; SEQ ID No. 1 and SEQ ID No. 2, which are linked by one or more G4S sequences; a CD8 hinge-transmembrane domain; a 4-1BB coactivation domain and a CD3ζ intracellular signal transduction domain.

[0008] In one embodiment, the CD3ζ intracellular signal transduction domain is followed by a membrane-bound IL15.

[0009] In one embodiment, a chimeric antigen receptor T cell that targets BCMA is provided, which expresses the aforementioned chimeric antigen receptor.

[0010] In one embodiment, a drug for treating tumors is provided, which contains the chimeric antigen receptor T cells described above.

[0011] In one embodiment, the chimeric antigen receptor described above is provided for the preparation of chimeric antigen receptor T cells and their application in tumor treatment.

[0012] In one embodiment, the tumor is a BCMA-positive tumor on its surface.

[0013] In one embodiment, the tumor is multiple myeloma.

[0014] In one embodiment, the present invention provides the application of the chimeric antigen receptor described above, wherein 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 BCMA-positive tumors.

[0015] In this application, the killing efficiency of six humanized BCMA-ScFv fragments (numbered 15, 16, 17, 18, 19, and 20) was compared. Fragment 16 was found to have the best killing effect and has been granted a patent (CN116535521A). Secondly, to verify the specific binding activity of the heavy chain (VH) and light chain (VL) of these six BCMA-ScFv fragments to the BCMA antigen, we compared the binding activity of the S15-S20 VH / VL fragments to the BCMA antigen using an indirect ELISA method. The results showed that fragments 16, 19, and 20 exhibited the highest VH affinity. The VHs in the three high-affinity ScFvs selected in the early stage were recombined. Three single VHs were used as negative controls, and 16VH-19VH (174) and 16VH-20VH (175) were used as experimental groups. The killing effect was compared with the original BCMA-ScFv (16). We found that the effect of 16VH-19VH (174) was better than that of BCMA-ScFv (16). In particular, CAR174 showed a much higher killing efficiency than other CARs at a smaller effective-to-target ratio. That is, a small amount of CAR174 can show a very high killing efficiency. CAR174 achieved a killing efficiency of 35% at a low effective-to-target ratio of 1:8, while the other 6 CARs were significantly lower than 35%.

[0016] Based on previous experiments, this application adds membrane-bound IL15 to enhance the anti-tumor activity of CAR T cells to the traditional second-generation CAR, and adds a combination of three VH tandem (340) for comparison. Finally, the best-performing 16VH-19VH is compared with the CT103A (ADI-34860) ScFv already on the market by Reindeer Biotech. It was found that our independently developed heavy chain recombinant CAR T (341) has a higher killing efficiency than CT103A CAR T, and can show better killing effect at a low target-to-efficiency ratio, significantly reducing the amount of effector cells used, while achieving efficient clearance of target cells.

[0017] In this technical solution, firstly, our independently developed VH is a humanized antibody heavy chain variable region, which has lower immunogenicity than traditional mouse-derived or chimeric ScFv; secondly, multiple VH tandemly can cover multiple antigenic epitopes on the tumor surface, reducing immune escape; finally, the optimal heavy chain recombinant structure we screened has better killing effect than the commercially available BCMA CAR T cell product CT103A. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a dose-dependent binding curve of each single-chain antibody fragment to the BCMA antigen; Figure 2 This is a schematic diagram of the VH recombinant CAR structure; Figure 3 This is a curve comparing the killing efficiency of VH recombinant CAR T cells; Figure 4 This is a schematic diagram of a CAR structure with added mbIL15; Figure 5 shows the PCR results of 16VH-19VH CAR-mbIL15 (341) colonies; Figure 6 shows the PCR results of 16VH-20VH CAR-mbIL15 (342) colonies; Figure 7 shows the results of the retroviral vector biological titer detection. Figure 8 This is a comparison chart of CAR-T cell killing efficiency, in which... Figure 8 In the diagram, A represents the lethal RPMI, and B represents the lethal Nalm6-BCMA. Figure 9 shows the CAR-T positivity rate test results; Figure 10 This is a curve comparing the killing efficiency of CAR-T cells. Detailed Implementation

[0020] 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 described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application.

[0021] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0022] Cell source: HEK293T and Phoenix-Ampho cells were both commercially available cell lines purchased from ATCC. Unless otherwise specified, all reagents and raw materials used in this invention are commercially available.

[0023] Sequence information: 16-VH (SEQ ID No. 1): SQVTLRESGPGLVRPSQTLSLTCTVSGGSIDSGGHYWSWIRQHPGKGLEWIGSIYHSGNTYYNPSLKSRVTMSVDTSKNQFSLKLTSVTAADTAIYYCARDIPHYFEPAYWGQGTLVTVSS 19-VH (SEQ ID No. 2): SQVQLVESGGGLVQPGGSLRLSCAASGFTFNTFVLNWVRLAPGKGLEWVSGISGSGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTAVYYCANLWTAAGIDYWGQGTLVTVSS 20-VH (SEQ ID No. 3): SQVQLVESGGGLVQPEGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEVWDVWGKGTIVTVSS mbIL15 (SEQ ID No. 4): MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL CD8 (SEQ ID No. 5): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC 41BB co-stimulatory domain (SEQ ID No. 6): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3ζ activation domain (SEQ ID No. 7): RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 3×G4S (SEQ ID No. 8): GGGGSGGGGSGGGGS.

[0024] Example 1: Comparison of VH and VL affinity Recombinant BCMA antigen was coated onto an enzyme-linked immunosorbent assay (ELISA) plate using an indirect method. Different concentration gradients of the target antibody (15-20 VH / VL fragments) were added, and the plate was then stained with HRP (horseradish peroxidase)-labeled secondary antibody. After HRP catalysis and the development of the substrate TMB (3,3',5,5'-tetramethylbenzidine), the OD value was measured. The OD value was positively correlated with the specific binding strength between the antibody and antigen. Results are shown below. Figure 1 As shown, VH exhibited dose-dependent binding. The OD values ​​of 19VH and 20VH showed a significant increasing trend with increasing BCMA concentration, exhibiting typical antigen-specific binding characteristics. The OD value of 16VH remained at a high plateau of 3.25-3.48, without significant concentration dependence, suggesting the possible presence of nonspecific high signal or saturation. In contrast, the OD values ​​of all VLs and some VHs (15VH, 17VH, 18VH) remained at the baseline level (<0.2), showing no concentration-dependent change, indicating nonspecific BCMA binding. Figure 1 Table 1 shows the dose-dependent binding curves of each single-chain antibody fragment to the BCMA antigen, with specific values ​​as shown in Table 1.

[0025] Table 1. BCMA antigen binding activity gradient detection data .

[0026] Example 2: Comparison of the lethality of heavy-chain recombinant CARs with that of the original ScFv The three high-affinity CARs selected in the previous screening (16, 19, and 20 VH) were recombined. We chose the simplest second-generation CAR structure to prepare CAR T cells. The schematic diagram of the CAR structure is shown below. Figure 2 As shown. Unmodified primitive T cells were used as negative controls, 16VH (171), 19VH (172), 20VH (173), 16VH-19VH (174), and 16VH-20VH (175) were used as experimental groups, and primitive 16-ScFv (170) was used as a positive control. Because 16 showed the best killing efficiency when comparing the killing efficiency of these six humanized BCMA-ScFv cells (numbered 15, 16, 17, 18, 19, and 20) in the previous stage, all VH combinations were based on 16. These groups of CAR T cells were co-incubated with RPMI-8226-LUC tumor cells expressing BCMA antigen at effector-to-target ratios of 1:8, 1:4, 1:2, 1:1, 2:1, and 4:1. After 16 hours, the killing efficiency of CAR T cells on target cells was detected, and the results are shown in the figure. Figure 3As shown in Tables 2 and 3, the specific values ​​indicate that, at an effector-to-target ratio of 1:8 to 1:1, CAR170, CAR171, CAR173, and CAR175 are not significantly different from unmodified primitive T cells. However, CAR172 and CAR174 are significantly better than unmodified primitive T cells within this effector-to-target ratio range. In particular, CAR174 exhibits a much higher killing efficiency than other CARs at a smaller effector-to-target ratio. That is, a small number of CAR174 cells can show a very high killing efficiency. At an effector-to-target ratio of 1:8, CAR174 achieves a killing efficiency of 35%, while the other six CARs are significantly lower.

[0027] Structurally, the effects of 16-VH and 20-VH alone are not significantly different from those of 16-VH and 16-VL in combination. 19-VH alone is more effective than the combination of 16-VH and 16-VL, while the combination of 16-VH and 19-VH is the most effective. The effect of the combination of 16-VH and 20-VH is not significantly different from that of 16-VH and 20-VH alone.

[0028] Table 2. Killing efficiency of VH recombinant CAR T cells .

[0029] Table 3. Killing efficiency of VH recombinant CAR T cells .

[0030] Example 3: Design and Construction of Heavy Chain Recombinant Plasmids Based on the above experiments, we found that the 16VH-19VH combination exhibited the best killing effect. This time, we added a tandem structure of three VHs (16VH, 19VH, and 20VH) to verify whether increasing the number of tandem VHs could further improve CAR T cell efficiency. Furthermore, we added membrane-bound IL15 to the second-generation CAR structure to enhance CAR T cell function.

[0031] 1. Plasmid design (1) A CAR structure of 16VH-19VH-20VH-mbIL15 is connected in series. The three VHs are connected in series with 3×G4S to connect the CD8 transmembrane domain, 41BB co-stimulatory domain, CD3ζ activation domain and membrane-bound IL15 (mbIL15), named 340.

[0032] (2) A 16VH-19VH-mbIL15 CAR structure is formed in series. 16 and 19-VH are connected in series with 3×G4S to connect the CD8 transmembrane domain, 41BB co-stimulatory domain, CD3ζ activation domain and membrane-bound IL15 (mbIL15), named 341.

[0033] (3) A 16VH-20VH-mbIL15 CAR structure is formed in series, with 3×G4S connecting the 16 and 20-VH regions to link the CD8 transmembrane domain, the 41BB co-stimulatory domain, the CD3ζ activation domain, and the membrane-bound IL15 (mbIL15), named 342. A schematic diagram of the specific CAR structure is shown below. Figure 4 As shown.

[0034] 2. Plasmid construction and identification PCR primers for the recombinant antibody fragment were designed using SnapGene software. The recombinant antibody fragment was amplified using the existing CAR plasmid as a template. The PCR product was then subjected to agarose gel electrophoresis and purified. The MFG retroviral vector was double-digested with XhoI / NotI restriction endonucleases and purified. The recombinant antibody fragment and the linearized vector were seamlessly cloned, and the product was transformed into DH5α *E. coli*. After shaking and overnight incubation, single colonies were picked for shaking and sequencing verification.

[0035] (1) Amplify the target fragment The 16VH-19VH-20VH amino acid sequence was optimized with human codons, and the resulting base sequence was synthesized by Qingke Biotechnology Co., Ltd.

[0036] (2) Insertion of the target fragment into the Z90 vector The 16VH-19VH-20VH amino acid fragment was directly synthesized into our company's proprietary reverse transcription packaging vector plasmid Z90 (XhoⅠ, BamHⅠ) to obtain plasmid 16VH-19VH-20VH CAR-mbIL15 (340). Using plasmid 340 as a template, primers were designed for PCR to obtain the target fragments 16VH-19VH and 16VH-20VH. The target fragments were then ligated to the Z90 vector (XhoⅠ, BamHⅠ) using a seamless cloning reagent. The molar ratio of the target fragment to the vector fragment was 3:1-5:1.

[0037] (3) Plasmid transformation and screening The recombinant plasmid was transformed into DH5α competent cells and cultured overnight at 37°C. The next day, single colonies were picked and colony PCR was performed. Gel electrophoresis was used to verify the size of the target fragment, and the correct bacterial culture was then sent to Qingke Biotechnology for sequencing.

[0038] Table 4. Colony PCR Primers Y239-F1 CATCCTCTAGACTGCCTCGCG(SEQ ID No.9) Y342-R2 GGTGTTGGTGGTCGCGG(SEQ ID No.10) .

[0039] (4) Extraction of endotoxin-free plasmids Take the correctly sequenced bacterial strains, add 60 μL to 15 mL of LB liquid medium (final Amp concentration 100 μg / mL), and incubate at 37 ℃ and 200 rpm for 12-16 h on a shaker. Harvest the bacterial pellet by centrifugation, extract the plasmid using an endotoxin-free plasmid extraction kit, measure the DNA concentration, and store the plasmid at -20 ℃ for later use.

[0040] 3. Experimental Results (1) Colony PCR identification results The target bacterial culture of 16VH-19VH CAR-mbIL15 (341) was identified by PCR, and the fragment size was 0.9 kb (e.g., Figure 5 As shown); the 16VH-20VH CAR-mbIL15 (342) target bacterial culture was identified by PCR, and the fragment size was 0.9kb (as shown). Figure 6 (As shown). The above gel electrophoresis results show that the band sizes are all correct and can be sent for further sequencing verification.

[0041] (2) Successful construction of plasmids 341 and 342: The bacterial culture with correct sequencing results (already sequenced) was used to construct plasmids 341 and 342. Figure 5-6 (After labeling, the plasmids were extracted and sent for sequencing. The sequencing results showed that the sequences of plasmids 341 and 342 were completely correct.)

[0042] Example 4: Preparation of Retroviral Vectors and Detection of Viral Biological Titer The three retroviral vectors mentioned above (340, 341, and 342) were prepared using retroviral vector packaging cell lines. Ampho and BAEV-WT cell lines were selected for retroviral vector packaging. Plasmids were transfected into Ampho cells using Lipomaster 2000 transfection reagent to produce the virus. Supernatants were collected at 48 and 72 hours post-transfection and transduced into BAEV-WT cells using horizontal centrifugation to construct a stable BAEV-WT retroviral vector cell line. The supernatant from the stable BAEV-WT retroviral vector cell line was collected and transduced into 293T cells to detect the viral vector biotiter in the supernatant.

[0043] The specific experimental procedure is as follows: 1. Plasmid transfection Using Lipomaster 2000, the constructed plasmids 340, 341, and 342 were transduced into Ampho cells and cultured at 37°C in a 5% CO2 incubator for 16–24 h. When the cell density reached 80%–90%, the medium was changed and the cells were cultured at 32°C in a 5% CO2 incubator. The viral vectors were then collected 24 h, 48 h, and 72 h after the medium change for subsequent cell transduction experiments.

[0044] 2. Construction of stable cell lines The collected viral vectors were transfected into BAEV-WT cells using polybrene to construct three stable cell lines: BAEV-WT-340, BAEV-WT-341, and BAEV-WT-342. These cells were then expanded and cultured. When the cell density reached 80%–90%, the medium was changed, and the cells were cultured at 32°C in a 5% CO2 incubator. Retroviral vectors collected at 24, 48, 72, 96, and 120 hours were collected and named H1, H2, H3, H4, and H5, respectively. The vectors collected after 5 days were mixed, aliquoted, and their titers were determined.

[0045] 3. Titer detection Polybrene was added to the viral vector to promote infection of 239T cells. Infected 293T cells were cultured at 37°C in a 5% CO2 incubator for 48 hours, then digested with trypsin. After APC-G4S antibody staining, the transduction efficiency of 293T cells was detected by flow cytometry. The viral titers produced by the three stable transfected cell lines (BAEV-WT-340, BAEV-WT-341, and BAEV-WT-342) were calculated using the formula: 293T transduction positivity rate × number of 293T cells on the day of transduction / volume of virus used for transduction (ml). Figure 7 shows the retroviral vector biotiter detection, and Table 5 shows the specific biotiter values ​​in TU / ml. A titer above 3E5 TU / ml indicates successful vector preparation and suitability for T cell transduction.

[0046] Table 5. Biotiters of Retroviral Vectors BAEV-WT-340 7.48E+05 BAEV-WT-341 8.14E+05 BAEV-WT-342 3.69E+05 .

[0047] Example 5: Preparation of Heavy Chain Recombinant CAR T Cells Experimental Procedure: CD3 antibody and the cytokine IL2 were added to peripheral blood mononuclear cells (PBMCs) of healthy volunteers to activate T cells. Three days after PBMC activation, a retroviral vector was added to the T cells, and the vector was transduced into the T cells using density gradient centrifugation to prepare CAR T cells. Table 6 shows the specific CAR T positivity rates for 340, 341, and 342 CAR T cells. The transduction positivity rates were all above 30%, allowing for further functional validation.

[0048] Table 6 CAR T Positive Rate 340 CAR T 52.4% 341 CAR T 53.7% 342 CAR T 31.2% .

[0049] Example 6: In vitro study of the anti-tumor function of heavy chain recombinant CAR T cells 1. Luciferase assay for the antitumor activity of CAR T cells against BCMA-positive cells Experimental Procedure: 340 CAR T cells, 341 CAR T cells, and 342 CAR T cells were used as the experimental groups. Untreated T cells served as the sex control, for a total of four T cell groups. Tumor cells NALM6-BCMA (BCMA+) and RPMI (BCMA+) were used as target cells. Different ratios of effector cells to target cells were co-cultured. Untreated T cells were added, and the CAR positivity of the three CAR T cell types was balanced to reduce experimental errors caused by different CAR positivity rates. Finally, the killing efficiency of each group of effector cells against target cells was detected using luciferase assay.

[0050] Experimental Results: Based on the results, when killing RPMI, 341 CAR T cells showed a stronger killing effect than 340 CAR and 342 CAR T cells, and the difference was statistically significant (**). P <0.01), such as Figure 8 In the study of Nalm6-BCMA, 341 CAR T cells showed a stronger killing effect than 340 CAR and 342 CAR T cells, and the results were statistically significant (*). P <0.05), such as Figure 8 The specific experimental data on the killing results of CAR T cells are shown in Tables 7 and 8. Furthermore, 341 CAR T cells exhibited significantly higher killing efficiency than other CAR T cells at a low target-to-cell ratio, indicating that higher killing efficiency can be achieved with fewer CAR T cells.

[0051] Table 7 CAR-T cell killing efficiency .

[0052] Table 8 CAR-T cell killing efficiency .

[0053] 2. Comparison of killing effects between 341 CAR T cells and CT103A (ADI-34860) Experimental Procedure: The ScFv sequence of CT103A is derived from patent US 11807663 B2. The structure of CT103A CAR is the same as that of the aforementioned heavy chain recombinant CAR. CT103A CAR T cells were prepared using the same method described above, and the CAR T cell positivity rate was detected by flow cytometry. Three groups of T cells—CT103A CAR T cells, 341 CAR T cells, and untreated T cells—were co-cultured with NALM6-BCMA (BCMA+). Different effector cell to target cell ratios were set for co-culture. Untreated T cells were added to balance the CAR positivity rates of the two CAR T cell types, reducing experimental errors caused by different CAR positivity rates. Finally, the killing efficiency of each group of effector cells against target cells was detected by flow cytometry.

[0054] Experimental results: Figure 9 The CAR T transduction positivity rate is shown in Table 9. The specific values ​​of the CAR T positivity rate are all above 50%. Figure 10 Table 10 shows the specific values ​​of CAR-T cell killing efficiency. Based on the results, the heavy chain recombinant CAR T (341) we independently developed has a higher killing efficiency than CT103A CAR T, and exhibits higher killing efficiency at a low target-to-efficiency ratio. It can effectively kill and clear a large number of target cells with a small number of effector cells. The specific experimental data of the killing results are shown in Table 9.

[0055] Table 9 CAR T Positive Rate CT103A CAR T 50.31% 341 CAR T 63.26% .

[0056] Table 10 CAR-T cell killing efficiency .

[0057] 3. Conclusion Among the three types of heavy chain recombinant CAR T cells we independently developed, there were structures with two VH tandem and structures with three VH tandem. The results showed that 341 had the best killing efficiency. 341 is a 16VH-19VH combination, which has a better killing effect than the CAR structure with three VH tandem (16VH-19VH-20VH). This indicates that the number of tandem VHs is not directly proportional to the killing efficiency. Furthermore, 341 has better anti-tumor efficacy than the commercially available CT103A CAR T cells and exhibits higher killing efficiency at low target-to-cell ratios, showing broad application prospects.

[0058] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A receptor targeting BCMA chimeric antigen, characterized in that, The receptor comprises two tandem ScFv heavy chains VH targeting BCMA, excluding the ScFv light chain VL; the two tandem ScFv heavy chains VH targeting BCMA have the structures SEQ ID No. 1 and SEQ ID No. 2, which are linked by one or more G4S sequences.

2. The BCMA-targeting chimeric antigen receptor according to claim 1, characterized in that, The receptor includes an upstream signal peptide and a myc tag for detection, tandemly connected; SEQ ID No. 1 and SEQ ID No. 2, which are linked by one or more G4S sequences; a CD8 hinge-transmembrane domain; a 4-1BB coactivation domain and a CD3ζ intracellular signal transduction domain.

3. The BCMA-targeting chimeric antigen receptor according to claim 2, characterized in that, The CD3ζ intracellular signal transduction domain is followed by a membrane-bound IL15.

4. A chimeric antigen receptor T cell targeting BCMA, characterized in that, It expresses the BCMA chimeric antigen receptor as described in any one of claims 1-3.

5. A drug for treating tumors, characterized in that, It contains chimeric antigen receptor T cells targeting BCMA as described in claim 4, wherein the tumor is multiple myeloma.

6. The application of the BCMA-targeted chimeric antigen receptor according to any one of claims 1-3 in the preparation of chimeric antigen receptor T cells.

7. The use of the targeted BCMA chimeric antigen receptor according to any one of claims 1-3 in the preparation of a drug for treating tumors, wherein the tumor is multiple myeloma.

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