Preparation and application of chimeric antigen receptor targeting bcma

Abstract

The application relates to a preparation and application of a chimeric antigen receptor targeting BCMA. The immunogenicity of scFv is significantly reduced by humanizing the mouse-derived antibody, and the human anti-mouse antibody (HAMA) reaction can be reduced, and the persistence and safety of CAR-T cells in the patient body can be improved. The affinity (KD approximately 1.46E-07 M) of the preferred scFv (such as D12.u21) obtained by the application to BCMA is reduced by about 10,000 times compared with that of a commercial high-affinity scFv (C11D5.3, KD approximately 1.09E-11 M). The "medium-low" affinity characteristic aims to balance the activation strength of CAR-T cells and avoid T cell exhaustion caused by excessive activation. Although the affinity is greatly reduced, the CAR-T cells expressing the preferred scFv (D12.u21) of the application show better killing effect than the high-affinity control (C11D5) CAR-T cells in in-vitro experiments. Under a higher target ratio, the killing efficiency and IFN-gamma cytokine secretion level of the CAR-T cells are both significantly higher, indicating that the CAR-T cells have stronger effector function and activation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the preparation and application of a chimeric antigen receptor targeting BCMA. Background Technology

[0002] Multiple myeloma (MM) is a hematologic malignancy caused by the abnormal proliferation of malignant plasma cells in the bone marrow. B cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor superfamily. Its expression is highly restricted to the surface of plasma cells and mature B lymphocytes, and is almost not expressed in important healthy tissues, making it an ideal target for the treatment of MM.

[0003] Chimeric antigen receptor T-cell (CAR-T) therapy targeting BCMA has shown significant clinical efficacy in the treatment of relapsed / refractory multiple myeloma (R / R MM), with an overall response rate of 73% to 100%. However, existing therapies still face many challenges: (1) the median progression-free survival (PFS) is short (approximately 4.1 to 11.8 months), and about 45% of patients relapse after achieving remission; (2) serious adverse reactions such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) exist; and (3) the in vivo persistence of CAR-T cells is limited.

[0004] In CAR molecule design, the single-chain variable fragment (scFv), as the antigen-binding domain, directly determines the specificity, safety, and function of CAR-T cells. Studies have shown that scFv affinity is a key parameter affecting CAR-T cell function. Excessively high affinity may lead to T cell overactivation, accelerated exhaustion, and reduced durability, while also increasing the risk of toxicity due to off-target attacks on normal cells with low BCMA expression. Therefore, developing novel BCMA scFvs with moderate affinity and low immunogenicity is crucial for constructing safer, more effective, and durable next-generation BCMA-CAR-T therapies. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention discloses a chimeric antigen receptor targeting BCMA, comprising an antigen-binding domain, wherein the antigen-binding domain comprises a single-chain variable region fragment (scFv) having an amino acid sequence as shown in any one of SEQ ID NO: 3 or SEQ ID NO: 4-31, or a single-chain variable region fragment (scFv) comprising an amino acid sequence having at least 90% homology with any one of SEQ ID NO: 3 or SEQ ID NO: 4-31 and retaining BCMA-binding activity.

[0006] Among them, SEQ ID NO:3-31 is the amino acid sequence of the humanized anti-human BCMA antibody, which is obtained by humanization and affinity modification of the amino acid sequence of anti-human BCMA mouse antibody SEQ ID No.2. The heavy chain amino acid sequence of SEQ ID No.2 is: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMNWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSANTAYLRINNLKNEDTATYFCAPAYYGNYVWFPYWGQGTLVSVSA; the light chain amino acid sequence is: DIVLTQSPATLGVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQSPKLLIKNVSNVESGVPARFSGSGSGTDFTLNIHPVQEEDTATYYCQHSWEIPLTFGAGTKLELK.

[0007] As a preferred embodiment of the present invention, the antigen-binding domain comprises a single-stranded variable region fragment (scFv) having an amino acid sequence as shown in SEQ ID NO: 29 or SEQ ID NO: 31.

[0008] As a preferred embodiment of the present invention, the chimeric antigen receptor further comprises a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain, wherein the hinge region is derived from CD8, the transmembrane domain is derived from CD8, the costimulatory domain is derived from 4-1BB, and the intracellular signal transduction domain is derived from CD3ζ.

[0009] As a preferred embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 32, 33, 34, 35, 36 or 37.

[0010] Chimeric antigen receptors targeting BCMA can be used in the preparation of drugs for the treatment of BCMA-positive diseases, namely multiple myeloma.

[0011] A method for preparing a chimeric antigen receptor targeting BCMA includes the following steps: Step 1: Provide the scFv sequence of mouse anti-BCMA antibody D12, the amino acid sequence of which is shown in SEQ ID NO:2; Step 2: The mouse scFv sequence is modified to humanize it to obtain a humanized scFv sequence. The frame region of the humanized scFv is derived from human light chain genes IGKV4-1*01 and IGKJ4*01, and human heavy chain genes IGHV7-4-1*01 and IGHJ4*01. Step 3: Connect the humanized scFv sequence obtained in Step 2 with other domain sequences of CAR to obtain the nucleic acid sequence encoding the chimeric antigen receptor; Step 4: Clone the nucleic acid sequence obtained in Step 3 into the expression vector and transform it into the host cell for expression.

[0012] As a preferred embodiment of the present invention, in step two, the affinity of the scFv is adjusted by reversing mutations on the embedded residues, residues that directly interact with the complementarity-determining region (CDR), or residues that have an important influence on the conformation of the light chain variable region (VL) and the heavy chain variable region (VH), and finally an scFv with an affinity reduced by at least 1000 times compared with the control scFv (C11D5) shown in SEQ ID NO:1 is obtained.

[0013] The beneficial effects of this invention: By humanizing mouse antibodies, this invention significantly reduces the immunogenicity of scFv, which is expected to reduce the human anti-mouse antibody (HAMA) response and improve the persistence and safety of CAR-T cells in patients. The preferred scFv obtained by this invention (such as D12.u21) has an affinity for BCMA (KD ≈ 1.46E-07 M) that is approximately 10,000 times lower than that of commercially available high-affinity scFv (C11D5.3, KD ≈ 1.09E-11 M). This "medium-low" affinity characteristic is designed to balance the activation intensity of CAR-T cells and avoid T cell exhaustion caused by overactivation. Despite the significant reduction in affinity, CAR-T cells expressing the preferred scFv (D12.u21) of this invention showed superior killing effects compared to high-affinity control (C11D5) CAR-T cells in in vitro experiments. At a higher target-to-cytokine ratio, its killing efficiency and IFN-γ cytokine secretion levels were significantly higher, indicating stronger effector function and activation potential. CAR-T cells derived from the optimized scFv cell line exhibited good expansion capacity and stable CAR expression positivity in vitro, meeting the cell quantity requirements for subsequent functional experiments and potential clinical applications. Attached Figure Description

[0014] Figure 1 This is a flowchart illustrating the construction process of the scFv-Fc expression plasmid in an embodiment of the present invention;

[0015] Figure 2 The image shows the ELISA detection results of the mouse hybridoma positive clone D12 of the present invention binding BCMA to the commercial antibody C11D5.

[0016] Figure 3 The image shows the ELISA detection results of the humanized scFv-Fc protein of this invention binding to BCMA.

[0017] Figure 4 This is a schematic diagram of the CAR-T carrier structure of the present invention;

[0018] Figure 5 The graph shows the fold increase curves of four different BCMA CAR-T cells during in vitro culture according to the present invention.

[0019] Figure 6 This is a graph showing the CAR positivity rate of four different BCMA CAR-T cells detected by BCMA protein and Flag antibody on Day 5 and Day 7 in this embodiment of the invention.

[0020] Figure 7 This is a comparison of the in vitro killing efficiency of Flag-D12.u21 CAR-T and Flag-C11D5 CAR-T against U266 target cells at effector-to-target ratios of 1:1, 3:1, and 10:1 in this embodiment of the invention.

[0021] Figure 8 This is a comparison of the IFN-γ secretion levels in the supernatant of Flag-D12.u21 CAR-T and Flag-C11D5 CAR-T after co-culturing with target cells in an embodiment of the present invention. Detailed Implementation

[0022] Example 1

[0023] This invention relates to the preparation and application of a chimeric antigen receptor targeting BCMA. A monoclonal antibody against human BCMA, named D12, was obtained via hybridoma synthesis. The antigen used for immunization was human BCMA protein. The immunized animals were female 6-8 week old BALB / c mice purchased from Spiefol (Beijing) Biotechnology Co., Ltd. Mice were immunized after emulsifying BCMA protein with Freund's adjuvant. Freund's complete adjuvant was used for the first immunization, while Freund's incomplete adjuvant was used for the second and third immunizations. Mice with high serum titers were selected for booster immunization and then sacrificed, and their spleens were harvested. Hybridoma cells were obtained by fusing spleen cells with Sp2 / 0 cells using the PEG fusion method.

[0024] Hybridoma cells were cultured in 96-well plates for approximately 8-9 days using a limiting dilution method. The supernatant was then used for ELISA screening. Specifically, 100 μL of BCMA-mFc protein (1 μg / mL) was coated onto each well. On the second day, after blocking, 100 μL of hybridoma supernatant or positive antibody sample was added to each well, and the cells were incubated at room temperature for 1 hour. After washing away unbound antibodies, 100 μL of anti-mFc HRP (1:5000) was added to each well, and the cells were incubated at room temperature for 1 hour. After washing away unbound secondary antibodies, TMB chromogenic buffer was added, and the reaction was incubated for approximately 15 minutes. The reaction was then terminated with sulfuric acid, and the OD450 reading was recorded using a microplate reader. Positive hybridoma clone D12 was then selected.

[0025] The hybridoma sequencing method used referenced the PCR amplification of the variable region of mouse antibodies. The determined antibody variable region sequence was constructed into the form of scFv and combined with human Fc to form a fusion protein.

[0026] Specifically, the hybridoma antibody sequence amplification method for mouse anti-human BCMA antibody D12 is as follows: Using the PCR product from hybridoma sequencing as a template, primer pairs D12H-F / D12H-R and D12K-F1 / D12K-R were designed to amplify the heavy and light chains, respectively; and linker sequences (GSTSGSGKPGSGEGSTKG) were added to the 3' end of the light chain and the 5' end of the heavy chain; the two resulting fragments were fused into an scFv sequence via overlap PCR using primer pair D12K-F2 / D12H-R, and a signal peptide sequence was added to the N-terminus. The purified PCR fragment was double-digested with Hind III-BamHI and ligated into the linearized pCDNA3.4_Fc vector, which was then transformed and sequenced for verification (amino acid sequence Seq_ID No. 2), obtaining the expression vector pCDNA3.4-D12scFv-Fc. The construction process is as follows. Figure 1 As shown.

[0027] The scFv sequence (C11D5.3) used in commercial BCMA-CAR-T was obtained through gene synthesis (amino acid sequence Seq_ID No.1), and was also transformed by ligation into a linearized vector after double digestion with Hind III-BamHI.

[0028] Protein preparation: 1) Add 30 μg of expression plasmid to 1.8 mL of Opit-MEM serum-depleted medium and 120 μL of PEI to 1.68 mL of Opit-MEM serum-depleted medium. Gently vortex to mix and incubate at room temperature for 5 min. Add the diluted PEI to the diluted plasmid DNA, gently vortex to mix, and incubate at room temperature for 15-20 min; 2) Slowly add the PEI / DNA complex to 30 mL of 293F cells (1 mL of cells corresponds to 1 μg of DNA transfection). Gently shake the culture flask during the addition process, then place it on a track shaker and culture the cells at 37℃ and 8% CO2 concentration. 18-22 hours after transfection, add Profeed (Opmai) feed to the culture flask to a final concentration of 5% of the total volume; 3) On the fourth day after transfection, detect the viable cell density and cell viability to identify protein expression in the cell supernatant; 4) When the cell viability is around 60% (around day 6), collect the cell supernatant and purify the protein.

[0029] Protein purification: Following the instructions for AmMag ProteinA Magmetic Beads, the beads were mixed with the supernatant and incubated at room temperature for 4 hours. The magnetic beads were then adsorbed and aggregated using a magnetic rack. The supernatant was discarded, and the beads were washed with PBS. 50 mM pH 3.0 citric acid solution was added, and the mixture was eluted for 10 minutes. After removing the magnetic beads with a magnetic rack, 1 M pH 9.0 Tris-HCl was added to the elution buffer to adjust the pH, which is generally ±1 of the protein's pI. The mixture was filtered through a 0.22 μm filter membrane, and the concentration was measured. After aliquoting, the mixture was stored at -80°C.

[0030] Example 2

[0031] Affinity identification of anti-BCMA antibodies

[0032] The binding of anti-BCMA antibody to human BCMA protein was detected by ELISA. The specific method is as follows: The coating protein was BCMA mFc, 0.5 μg / mL, 100 μL / well, incubated overnight at 4℃; the next day, 300 μL / well of 1% BSA was added and incubated at 25℃ for 1 h; the samples were washed three times with 300 μL / well of PBST; for the sample or positive antibody (C11D5) sample, starting from 20 nM, a 3-fold dilution gradient was used, for a total of 11 dilutions, adding 100 μL / well; an equal volume of 1% BSA was added to column H12 as a negative control, incubated at 25℃ for 1 h, and washed three times with 300 μL / well of PBST; anti-hFc was then used. Dilute HRP (1:20000) (Abcam, ab99759) 100 μL / well, incubate at 25℃ for 1 h, wash three times with 300 μL / well PBST; after patting dry, add 100 μL TMB chromogenic solution (Beyotime, PO209-100 ml), incubate for about 15 min, then stop the reaction with ELISA stop solution (Solarbio, C1058-500 ml), and read OD450 on the microplate reader.

[0033] The results are as follows Figure 2 As shown, the mouse anti-human BCMA clone D12 obtained by screening was verified at the ELISA level to have similar binding activity to commercial antibodies, and subsequent modifications were performed on this clone.

[0034] Example 3

[0035] Humanization and affinity modification of anti-BCMA antibodies

[0036] The antibody sequence with clone number D12 was humanized to reduce immunogenicity, and affinity was optimized to obtain a humanized antibody capable of generating highly active CAR-T cells. Based on the obtained murine antibody VH / VL CDR, the variable region sequences of the heavy and light chains were compared with the fully human antibody Germline database to obtain highly homologous human Germline templates. The human Germline light chain framework region was derived from the human K light chain gene, with human germline light chain templates IGKV4-1*01 and IGKJ4*01 being preferred. The human germline heavy chain framework region was derived from the human heavy chain, with human germline heavy chain templates IGHV7-4-1*01 and IGHJ4*01 being preferred. The CDR of the murine antibody was transplanted onto a selected humanized template, replacing the humanized framework region. Then, based on the three-dimensional structure of the murine antibody, reverse mutations were performed on the embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of VL and VH, resulting in a series of humanized light and heavy chain sequences. Since this molecule will ultimately be integrated into CAR-T cells in the form of scFv, to avoid potential affinity changes during the conversion of whole IgG to scFv, humanized molecules in the form of scFv-Fc were directly constructed for screening during the humanization screening stage.

[0037] Specifically, the mouse-derived sequence was cloned from hybridoma cells. Humanized sequence 1 (D12u1scFv) (amino acid sequence Seq_ID No. 3) was obtained through gene synthesis. Other humanized sequences were obtained by introducing relevant mutations based on sequence 1 using QuickChange PCR. The expression and purification process of all scFv-Fc proteins followed the procedures in Example 1. The amino acid sequences of D12u2scFv-Fc to D12u30scFv-Fc were Seq_ID Nos. 4-31. The purified proteins were initially tested for binding to the antigen BCMA using an ELISA method, with the experimental steps following those in Example 2.

[0038] The results are as follows Figure 3 As shown, by adjusting the sequences of the FR and CDR regions, a series of mutant clones with different affinities were obtained. Among them, such as... Figure 3As shown in Figure A, D12u1scFv-Fc, D12u2scFv-Fc, and D12u10scFv-Fc lost their activity after mutation; among other mutations, D12u4scFv-Fc, D12u6scFv-Fc, D12u8scFv-Fc, D12u23scFv-Fc, and D12u25scFv-Fc showed the most significant decrease in activity; followed by D12u5scFv-Fc, D12u7scFv-Fc, and D12u20scFv-Fc; and clones such as D12u17scFv-Fc, D12u21scFv-Fc, D12u29scFv-Fc, and D12u30scFv-Fc showed affinity close to that of the parent D12. Figure 3 (B, C, D).

[0039] Affinity of humanized anti-BCMA antibodies was detected by SPR.

[0040] The affinity of different humanized BCMA antibodies for BCMA molecules was determined using the surface plasmon resonance (SPR) method. The specific experimental procedure is as follows:

[0041] The instrument used was a Biacore 8K with the Protein A S-series sensor chip. Antibodies were injected into the Protein A chip and captured by the chip. Binding phase: The antigen (BCMA-His protein) was serially diluted two-fold from 200 nM to 3.125 nM using running buffer (1×HBS-EP+ buffer, pH 7.4) and injected into the flow cell on the chip surface. The binding time was 120 seconds. Dissociation phase: Immediately after binding, running buffer was injected, and the dissociation time was set to 600 seconds. Analysis was performed using Biacore Insight Evaluation on the Biacore 8K. The results are shown in Table 1. The affinity of the control antibody C11D5scFv-Fc was approximately 1.09E-11, and the affinity of the D12 mouse antibody D12scFv-Fc was approximately 3.62E-09. After modification, the affinity became 1.46E-07. Compared with the ELISA results, the affinity of the D12 mouse antibody was about 300 times lower than that of the control antibody. After modification, the affinity levels of D12u21scFv-Fc and D12u30scFv-Fc were at the micromolar level, and the kinetic curves showed that their binding mode was "fast up and fast down". Table 1: SPR results of BCMA antibody Ligand Analyte ka (1 / Ms) kd (1 / s) KD (M) C11D5scFv-Fc BCMA-His 4.85E+06 5.29E-05 1.09E-11 D12scFv-Fc BCMA-His 8.58E+05 3.11E-03 3.62E-09 D12u21scFv-Fc BCMA-His 9.05E+05 1.32E-01 1.46E-07 D12u27scFv-Fc BCMA-His 1.78E+06 1.92E-2 1.07E-7 D12u29scFv-Fc BCMA-His 1.68E+06 2.62E-03 1.56E-09 D12u30scFv-Fc BCMA-His 1.76E+06 2.76E-01 1.56E-07

[0042] Example 4

[0043] Constructing CAR-T vectors

[0044] After obtaining the scFv sequences of anti-human BCMA proteins with different affinities, the scFv sequences were subsequently integrated into the CAR molecule as antigen-binding domains. The overall construction is as follows: Figure 4 As shown, a second-generation CAR molecule construction method was used. The CD8, 4-1BB, and CD3ζ domain sequences were all derived from previously constructed CAR molecules. In this invention, only the scFv region was replaced. To ensure consistency in subsequent CAR-T detection, a Flag tag was uniformly added to the N-terminus of the scFv region. Specifically, C11D5 scFv, D12-scFv, and humanized D12 scFv were amplified by PCR using primer pairs C11D5-F / C11D5-R, D12-F / D12-R, and D12U-F / D12U-R (Seq_ID No. 78-83), respectively. Then, multiple rounds of PCR were performed using different 5' primers: MS1-F, MS2-F, and FT-F (Seq_ID No. 84-86). A Flag tag sequence (G4S-Flag-G4S) and a signal peptide were fused to the 5' end. The resulting PCR products were purified, double-digested with Xba I and Nhe I, ligated into a linearized pCDH-CMV-EF1 vector, transformed, and sequenced for verification. The complete CAR molecules were identified as Flag-C11D5 CAR, Flag-D12 CAR, Flag-D12.u21 CAR, and Flag-D12.u27. CAR, Flag-D12.u29 CAR, and Flag-D12.u30 CAR correspond to the amino acid sequences Seq_ID No. 32-37.

[0045] CAR-T preparation

[0046] Recombinant virus preparation: Passage and plate healthy 293T cells. When the cells have been cultured for 16-18 hours and the cell density reaches 50-70%, transfect the cells. Mix the target plasmid, helper plasmids REV, RRE, and VSVG in a ratio of 2.5:1:1.25:1 and add CaCl2 solution. Add an equal volume of 2×HBS (Hankes balanced salt solution) to the prepared solution by spiral pouring. Quickly pipette the solution until it becomes a mist. Then add the mixture dropwise to the culture dish and shake well. Incubate for 8-10 hours and replace with fresh medium. 48 hours after transfection, collect the supernatant and concentrate and purify it. Finally, aliquot the virus and store it at -80℃.

[0047] Viral titer detection: Specific procedure: Jurkat cells in good growth condition were taken and resuspended to a concentration of 5 × 10⁵ / mL. 1 mL was plated per well, and 0.1 μL, 0.3 μL, 1 μL, 3 μL, 10 μL, 30 μL, and 100 μL of viral load were added, respectively. The cells were centrifuged at 2000g, 32°C for 90 minutes, and the cell clumps were reconstituted. 1 mL of additional fluid was added. Cells were collected after 48 hours, and the infection efficiency was detected by flow cytometry. The positive rate was identified using self-labeled fluorescent BCMA protein and Flag antibody. Because the affinity of different humanized BCMA antibodies obtained in this invention for BCMA molecules varies, the positive rate results of BCMA protein identification may not be able to simultaneously assess the recombinant viral titer of each antibody group. The final recombinant viral titer result is based on the Flag antibody detection result.

[0048] The flow cytometry results for viral titer detection are shown in Table 2. The viral titer of Flag-C11D5 is 8.4×10⁶ TU / mL, the viral titer of Flag-D12 is 4.51×10⁵ TU / mL, the viral titer of Flag-D12.u21 is 1.41×10⁷ TU / mL, and the viral titer of Flag-D12.u30 is 1.13×10⁶ TU / mL.

[0049] The results showed that Flag-C11D5 and Flag-D12.u21 viruses had high titers and met the experimental requirements, while Flag-D12 and Flag-D12.u30 viruses had low titers. Therefore, Flag-D12 and Flag-D12.u30 viruses were used for secondary infection in the subsequent virus infection process. Table 2 Virus titer results name Viral titer (TU / mL) Flag-C11D5 <![CDATA[8.4×10 6 ]]> Flag-D12 <![CDATA[4.51×10 5 ]]> Flag-D12.u21 <![CDATA[1.41×10 7 ]]> Flag-D12.u30 <![CDATA[1.13×10 6 ]]>

[0050] Peripheral blood mononuclear cells (PBMCs) resuscitation and activation: PBMCs were removed from the liquid nitrogen tank, resuscitated in a 37°C water bath, and then added to centrifuge tubes containing X-vivo15 (serum-free culture medium for immune cells). The cells were centrifuged at 1800 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in X-vivo15 to a concentration of 1×10⁷ / mL. The cells were seeded into 24-well plates (2 mL per well) and incubated overnight. Cells were collected and counted the following day. Two × 10⁵ cells were used for flow cytometry antibody staining to identify the proportion of CD3 T cells, and the amount of magnetic beads used was calculated (beads:T cells = 3:1). Centrifuge cells at 1800 rpm for 10 minutes. Simultaneously, add 120 μL of beads to a cryovial containing 1 mL of X-vivo15 medium. Wash away the buffer from the beads. Place the cryovial on a magnetic rack to attract the beads. Discard the supernatant completely. Resuspend the cell pellet in 1.6 mL of X-vivo15 medium and add it to the cryovial containing only beads (co-incubate cells with beads at a density of 1 × 10⁷ cells / mL). Mix the cells and beads thoroughly. Seal the cryovial with sealing film and incubate at room temperature on a shaker for 45 minutes to allow the beads to fully bind to the CD3 T cells. Finally, place the cryovial on a magnetic rack for 2 minutes, discard the supernatant, and the CD3 T cells bound to the beads will be attracted by the magnetic beads. Resuspend the cells in 8 mL of X-vivo15 medium at a density of 2 × 10⁶ cells / mL. Cells were cultured at a density of cells / mL, with 2 mL placed in each well of a 24-well plate and activated for 24 hours.

[0051] Viral infection: Cells were collected into centrifuge tubes, mixed, and counted. Two × 10⁵ cells were taken for flow cytometry to identify T cell activation levels. PBMCs were then centrifuged, resuspended, and counted. X-vivo15 medium (containing 200 IU of interleukin-2) was added to resuspend the cells to 1 × 10⁶ cells / mL. 0.5 mL of cell suspension was added to each well of a 24-well plate. The viral load was calculated based on the viral titer and MOI. A control well (mock) without virus was included. The required Flag-C11D5, Flag-D12, Flag-D12.U21, and Flag-D12.U30 viruses were added to each well. Additionally, 2 μL of polybrene (a sensitizing agent) was added to each well. Mix the solutions sequentially (ug / mL); finally, wrap the wells with plastic wrap and centrifuge at 2000g, with a 3-minute acceleration to the target speed and a 1-minute deceleration to a stop, at 32°C for 90 minutes. After centrifugation, add liquid to a final volume of 2mL and incubate statically. The next day, perform a real-time secondary infection of cells with Flag-D12 and Flag-D12.U30 viruses, as described above. Finally, discard the virus 48 hours after infection and completely replace the culture medium with X-vivo15.

[0052] Cell passage and proliferation statistics: The culture density of BCMA CAR-T cells was maintained at 5×105 cells / mL. CAR-T cells were collected and counted on day 4, day 5 and day 7 respectively. After counting, fresh culture medium was added to adjust the cell density to 5×105 cells / mL and culture continued.

[0053] Plotting CAR-T cell doubling curves as follows Figure 5 As shown, by day 7 of culture, Flag-C11D5 BCMA CAR-T cells had expanded approximately 41-fold, Flag-D12 BCMA CAR-T cells had expanded approximately 34-fold, Flag-D12.U21 BCMA CAR-T cells had expanded approximately 34-fold, and Flag-D12.U30 BCMA CAR-T cells had expanded approximately 26-fold.

[0054] The results showed that all four types of BCMA CAR-T cells could proliferate normally, but the Flag-D12.U30 BCMA CAR-T cells had a relatively slower overall proliferation rate compared to the other three types of CAR-T cells.

[0055] BCMA CAR-T positivity rate identification: BCMA CAR-T cells from Day 5 and Day 7 were counted under a microscope. Two × 10⁵ cells from each cell were added to flow cytometry antibodies for detecting CAR (chimeric antigen receptor) positivity rate. The cells were incubated at 4°C in the dark for 15 min, washed with 1 mL of PBS (phosphate buffered saline), centrifuged at 4500 rpm for 3 min, and the supernatant was discarded. The cells were then resuspended in 0.2 mL of PBS and analyzed. The flow cytometry antibodies used were APC anti-DYKDDDDK tag antibody (Biolegend-637307) and PE BCMA-mFc protein (laboratory-produced).

[0056] Positive rate results for Day 5 and Day 7 are as follows: Figure 6 As shown, the positivity rates of the four BCMA CAR-T cells were detected on Day 5 and Day 7, respectively. On Day 5, the positivity rates of Flag-C11D5, Flag-D12, Flag-D12.U21, and Flag-D12.U30 detected using BCMA protein were 30.6%, 8.28%, 38.6%, and 30.4%, respectively, while the positivity rates of Flag-C11D5, Flag-D12, Flag-D12.U21, and Flag-D12.U30 detected using Flag antibody were 50.7%, 31.6%, 62.1%, and 65.9%, respectively. On Day 7, the positive rates of Flag-C11D5, Flag-D12, Flag-D12.U21, and Flag-D12.U30 detected by BCMA protein were 22.5%, 0.77%, 36.4%, and 3.68%, respectively. The positive rates of Flag antibody detection for Flag-C11D5, Flag-D12, Flag-D12.U21, and Flag-D12.U30 were 38.4%, 7.18%, 58.5%, and 23.3%, respectively. Because the affinity between the different humanized BCMA antibodies obtained in this invention and the BCMA molecule varies, the positive rate results of BCMA protein identification may not accurately assess the positive rate of each CAR-T group. Therefore, the final CAR-T positive rate results are based on the Flag antibody detection results.

[0057] The results showed that although Flag-D12 BCMA CAR-T cells underwent two viral infections, the CAR positivity rate remained low and could not meet the requirements for functional testing. Furthermore, the positivity rate of Flag-D12.U30 BCMA CAR-T cells on Day 7 was significantly lower than that on Day 5, indicating that CAR-positive cells in Flag-D12.U30 BCMA CAR-T cells proliferated slowly, their cellular composition was not stable enough, and they could not meet the requirements for long-term functional testing. The positivity rate of Flag-D12.U21 was more stable and consistent across the two testing results. Therefore, Flag-C11D5 and Flag-D12.U21 BCMA CAR-T cells were ultimately used for subsequent functional identification.

[0058] Example 5

[0059] BCMA CAR-T killing efficiency and cytokine release assay

[0060] CAM (fluorescein acetylated methoxylate) labeling of target cells for cytotoxicity identification: CAR-T cells and target cells were counted separately, and target cells were labeled with CAM. In this invention, U266 cells expressing BCMA were selected as target cells. This experimental system contained only CAR-T cells and target cells, with effector-to-target ratios set at 1:1, 3:1, and 10:1. The positive control was CAM-labeled target cells added to the sample wells with 1% Triton, while the negative control wells (background wells) were also CAM-labeled target cells. After mixing the cells according to the effector-to-target ratio, they were added to 96-well plates and cultured for 6 hours. The supernatant was collected and centrifuged, and the supernatant was transferred to a blank 96-well plate. The supernatant was then analyzed using an EnSpire microplate reader, and the cytotoxicity was calculated based on the detection values.

[0061] The results are as follows Figure 7 As shown, at an effector-to-target ratio of 1:1, both Flag-D12.u21 BCMA CAR-T cells and Flag-C11D5 BCMA CAR-T cells maintained relatively consistent killing effects, with a killing efficiency of around 10%. When the effector-to-target ratio increased to 3:1, the killing efficiency of Flag-C11D5 BCMA CAR-T cells increased to around 20%, while the killing efficiency of Flag-D12.u21 BCMA CAR-T cells increased to around 30%. When the effector-to-target ratio increased to 10:1, the killing efficiency of Flag-C11D5 BCMA CAR-T cells increased to around 30%, while the killing efficiency of Flag-D12.u21 BCMA CAR-T cells increased to around 40%.

[0062] The results showed that, at a low effector-to-target ratio of 1:1, both Flag-D12.u21 BCMA CAR-T cells and Flag-C11D5 BCMA CAR-T cells had low killing efficiency, with no difference between them. As the effector-to-target ratio increased, the killing efficiency of both CAR-T cells significantly improved. At an effector-to-target ratio of 3:1, the killing efficiency of Flag-D12.u21 BCMA CAR-T cells was significantly higher than that of Flag-C11D5 BCMA CAR-T cells.

[0063] IFN-γ (interferon-γ) secretion assay: mock, C11D5, D12.u21 and target cells in good growth condition were centrifuged separately, and then resuspended in X-vivo15 for counting. CAR-T (CAR+) and target cells were mixed at a ratio of 1:2 and added to 96-well plates for 24 h of culture. The supernatant was collected to detect the release of effector cytokine IFN-γ. The cytokine assay was performed using the Human IFN Gamma Uncoated ELISA Kit (invitrogen, cat: 88-7316-88, lot: 423448-004).

[0064] The results are as follows Figure 8 As shown, the level of IFN-γ secreted by Flag-C11D5 is around 20 ng / mL, while the level of IFN-γ secreted by Flag-D12.U21BCMA CAR-T is around 30 ng / mL.

[0065] The results showed that Flag-D12.U21 BCMA CAR-T cells secreted significantly higher levels of the effector cytokine IFN-γ than Flag-C11D5 BCMA CAR-T cells. Therefore, humanized and affinity-modified Flag-D12.u21 BCMA CAR-T cells exhibited enhanced cytotoxicity and effector cytokine secretion compared to Flag-C11D5 BCMA CAR-T cells.

[0066] The above amino acid sequence is as follows:

[0067] Amino acid sequence of C11D5-scFv (Seq_ID No.1): DIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKP GSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 38.

[0068] Amino acid sequence of D12-scFv (Seq_ID No.2): DIVLTQSPATLGVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQSPKLLIKNVSNVESGVPARFSGSGSGTDFTLNIHPVQEEDTATYYCQHSWEIPLTFGAGTKLELKGSTSGSGKPG SGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMNWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSANTAYLRINNLKNEDTATYFCAPAYYGNYVWFPYWGQGTLVSVSA Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 39.

[0069] Amino acid sequence of D12.u1-scFv (Seq_ID No.3): DIVMTQSPASLAVSLGERATISCRASQSVSTSGNNYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWSIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCARAYYGDYVYFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 40.

[0070] Amino acid sequence of D12.u2-scFv (Seq_ID No.4): DIVMTQSPASLAVSLGERATISCRASQSVSTSGNNYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCARAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 41.

[0071] Amino acid sequence of D12.u3-scFv (Seq_ID No.5): DIVMTQSPASLAVSLGERATISCRASQSVSTSGNNYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 42.

[0072] Amino acid sequence of D12.u4-scFv (Seq_ID No.6): DIVMTQSPASLAVSLGERATISCRASQSVSTSGNNYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYADDFKGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 43.

[0073] Amino acid sequence of D12.u5-scFv (Seq_ID No.7): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYADDFKGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 44.

[0074] Amino acid sequence of D12.u6-scFv (Seq_ID No.8): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIYNVSNVESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYADDFKGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 45.

[0075] Amino acid sequence of D12.u7-scFv (Seq_ID No.9): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 46.

[0076] Amino acid sequence of D12.u8-scFv (Seq_ID No.10): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIYNVSNVESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 47.

[0077] Amino acid sequence of D12.u9-scFv (Seq_ID No.11): DIVMTQSPASLAVSLGERATISCRASQSVSTSGNNYLHWYQQKPGQPPKLLIYNVSNVESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 48.

[0078] Amino acid sequence of D12.u10-scFv (Seq_ID No.12): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCARAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 49.

[0079] Amino acid sequence of D12.u11-scFv (Seq_ID No.13): DIVMTQSPASLAVSLGERATISCRASQSVSTSGNNYLHWYQQKPGQPPKLLIYNVSNVESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCARAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 50.

[0080] Amino acid sequence of D12.u12-scFv (Seq_ID No.14): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 51.

[0081] Amino acid sequence of D12.u13-scFv (Seq_ID No.15): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 52.

[0082] Amino acid sequence of D12.u14-scFv (Seq_ID No.16): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 53.

[0083] Amino acid sequence of D12.u15-scFv (Seq_ID No.17): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVKQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 54.

[0084] Amino acid sequence of D12.u16-scFv (Seq_ID No.18): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQSPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 55.

[0085] Amino acid sequence of D12.u17-scFv (Seq_ID No.19): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYLHWYQQKPGQSPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 56.

[0086] Amino acid sequence of D12.u18-scFv (Seq_ID No.20): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYLHWYQQKPGQSPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVKQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 57.

[0087] Amino acid sequence of D12.u19-scFv (Seq_ID No.21): DIVMTQSPASLAVSLGERATISCKSSQSVSTSGNNYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYADDFKGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 58.

[0088] Amino acid sequence of D12.u20-scFv (Seq_ID No.22): DIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYMHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 59.

[0089] Amino acid sequence of D12.u21-scFv (Seq_ID No.23): DIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 60.

[0090] Amino acid sequence of D12.u22-scFv (Seq_ID No.24): DIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYLHWYQQKPGQSPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 61

[0091] Amino acid sequence of D12.u23-scFv (Seq_ID No.25): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYMHWYQQKPGQPPKLLINNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 62.

[0092] Amino acid sequence of D12.u24-scFv (Seq_ID No.26): DIVMTQSPASLAVSLGERATISCRASQSVSTSRYSYLHWYQQKPGQPPKLLINNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 63.

[0093] Amino acid sequence of D12.u25-scFv (Seq_ID No.27): DIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYMHWYQQKPGQPPKLLINNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFDYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 64.

[0094] Amino acid sequence of D12.u26-scFv (Seq_ID No.28): DIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYLHWYQQKPGQPPKLLINNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 65.

[0095] Amino acid sequence of D12.u27-scFv (Seq_ID No.29): DIVLTQSPATLGVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQSPKLLIKNVSNVESGVPARFSGSGSGTDFTLNIHPVQEEDTATYYCQHSWEIPLTFGAGTKLELKGSTSGSGKPG SGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMNWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSANTAYLRINNLKNEDTATYFCAPAYYGNYVWFGYWGQGTLVSVSA Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 66.

[0096] Amino acid sequence of D12.u29-scFv (Seq_ID No.30): DIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYLHWYQQKPGQPPKLLINNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFGYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 67.

[0097] Amino acid sequence of D12.u30-scFv (Seq_ID No.31): EIVLTQSPATLSLSPGERATLSCRASQSVSTSRYSYMHWYQQKPGQAPRLLINNVSNVESGIPARFSGSGSGTDFTLTISSLEPEDTAVYYCQHSWEIPLTFGGGTKVEIKGSTGSGKPG SGEGSTKGQVQLVQSGSELKKPGESVKVSCKASGYTFTDYSMNWVRQAPGKGLKWMGWINTETGEPTYADDFKGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCAPAYYGNYVWFGYWGQGTLVTVSS Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 68.

[0098] Amino acid sequence of Flag-C11D5 CAR (Seq_ID No.32): MALPVTALLLPLALLLHAARPGGGGSDYKDDDDKGGGGSDIVLTQSPPSLAMSLGKRATISCRASESVTILGSHLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDD VAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYAYDFRGRFAFSLETSASTAYLQINNLKYED TATYFCALDYSYAMDYWGQGTSVTVSSASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 69.

[0099] Amino acid sequence of Flag-D12 CAR (Seq_ID No.33): MALPVTALLLPLALLLHAARPGGGGSDYKDDDDKGGGGSDIVLTQSPATLGVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQSPKLLIKNVSNVESGVPARFSGSGSGTDFTLNIHPVQEEDTATYYCQHSWEIPLTFGAGTKLELKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMNWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSANTAYLRINNLKNEDTATYFCAPAYYGNYVWFPYWGQGTLVSVSAASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR The corresponding nucleotide sequence is nucleotide sequence Seq_ID No.70

[0100] Amino acid sequence of Flag-D12.u21 CAR (Seq_ID No.34): MALPVTALLLPLALLLHAARPGGGGSDYKDDDDKGGGGSDIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYLHWYQQKPGQPPKLLIYNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFPYWGQGTLVTVSSASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No.71

[0101] Amino acid sequence of Flag-D12.u27 CAR (Seq_ID No.35): MALPVTALLLPLALLLHAARPGGGGSDYKDDDDKGGGGSDIVLTQSPATLGVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQSPKLLIKNVSNVESGVPARFSGSGSGTDFTLNIHPVQEEDTATYYCQHSWEIPLTFGAGTKLELKGSTSGSGKPGSGEGSTKGQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMNWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSANTAYLRINNLKNEDTATYFCAPAYYGNYVWFGYWGQGTLVSVSAASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No.72

[0102] Amino acid sequence of Flag-D12.u29 CAR (Seq_ID No.36): MALPVTALLLPLALLLHAARPGGGGSDYKDDDDKGGGGSDIVMTQSPASLAVSLGERATISCKSSQSVSTSRYSYLHWYQQKPGQPPKLLINNVSNRESGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCQHSWEIPLTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKISCKASGYTFTDYSMNWVRQAPGKGLEWMGWINTETGEPTYAQDFTGRFVFSLDTSANTAYLQINNLKAEDTAVYFCAPAYYGNYVWFGYWGQGTLVTVSSASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No.73

[0103] Amino acid sequence of Flag-D12.u30 CAR (Seq_ID No.37): MALPVTALLLPLALLLHAARPGGGGSDYKDDDDKGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSTSRYSYMHWYQQKPGQAPRLLINNVSNVESGIPARFSGSGSGTDFTLTISSLEPEDT AVYYCQHSWEIPLTFGGGTKVEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGESVKVSCKASGYTFTDYSMNWVRQAPGKGLKWMGWINTETGEPTYADDFKGRFVFSLDTSVNTAYLQISSLKAEDTA VYYCAPAYYGNYVWFGYWGQGTLVTVSSASFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCELRVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Its corresponding nucleotide sequence is nucleotide sequence Seq_ID No. 74.

[0104] Components not described in detail in this article are existing technologies.

[0105] While the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and modifications or variations without creative effort are still within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor targeting BCMA, characterized in that, The antibody includes an antigen-binding domain comprising a single-chain variable region fragment (scFv) having an amino acid sequence as shown in any one of SEQ ID NO: 3 or SEQ ID NO: 4-31, or a single-chain variable region fragment (scFv) comprising an amino acid sequence having at least 90% homology to any one of SEQ ID NO: 3 or SEQ ID NO: 4-31 and retaining BCMA-binding activity; SEQ ID NO: 3-31 is a humanized amino acid sequence of an anti-human BCMA mouse antibody, obtained by humanization and affinity modification of the anti-human BCMA mouse antibody amino acid sequence SEQ ID No.

2. The heavy chain amino acid sequence of No. 2 is: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMNWVKQAPGKGLKWMGWINTETGEPTYADDFKGRFAFSLETSANTAYLRINNLKNEDTATYFCAPAYYGNYVWFPYWGQGTLVSVSA; the light chain amino acid sequence is: DIVLTQSPATLGVSLGQRATISCRASQSVSTSRYSYMHWYQQKPGQSPKLLIKNVSNVESGVPARFSGSGSGTDFTLNIHPVQEEDTATYYCQHSWEIPLTFGAGTKLELK.

2. The chimeric antigen receptor targeting BCMA according to claim 1, characterized in that: The antigen-binding domain comprises a single-stranded variable region fragment (scFv) having an amino acid sequence as shown in SEQ ID NO: 29 or SEQ ID NO:

31.

3. A chimeric antigen receptor targeting BCMA according to claim 2, characterized in that: It also includes a hinge region, a transmembrane domain, a costimulatory domain, and an intracellular signal transduction domain. The hinge region is derived from CD8, the transmembrane domain is derived from CD8, the costimulatory domain is derived from 4-1BB, and the intracellular signal transduction domain is derived from CD3ζ.

4. A chimeric antigen receptor targeting BCMA according to any one of claims 3, characterized in that: The amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 32, 33, 34, 35, 36 or 37.

5. The use of a chimeric antigen receptor targeting BCMA as described in any one of claims 1-4 in the preparation of a medicament for treating BCMA-positive diseases.

6. The application according to claim 5, characterized in that: The BCMA-positive disease is multiple myeloma.

7. A method for preparing a chimeric antigen receptor targeting BCMA according to any one of claims 1-4, characterized in that: Includes the following steps: Step 1: Provide the scFv sequence of mouse anti-BCMA antibody D12, the amino acid sequence of which is shown in SEQ ID NO:2; Step 2: Humanize the mouse scFv sequence to obtain a humanized scFv sequence. The frame region of the humanized scFv is derived from human light chain genes IGKV4-1*01 and IGKJ4*01, and human heavy chain genes IGHV7-4-1*01 and IGHJ4*01. Step 3: Connect the humanized scFv sequence obtained in Step 2 with other domain sequences of CAR to obtain the nucleic acid sequence encoding the chimeric antigen receptor; Step 4: Clone the nucleic acid sequence obtained in Step 3 into the expression vector and transform it into the host cell for expression.

8. The method for preparing a chimeric antigen receptor targeting BCMA according to claim 7, characterized in that: In step two, the affinity of the scFv is adjusted by reverting mutations to the embedded residues, residues that directly interact with the complementarity-determining region (CDR), or residues that have a significant impact on the conformation of the light chain variable region (VL) and the heavy chain variable region (VH), and finally, an scFv with an affinity reduced by at least 1000 times compared to the control scFv (C11D5) shown in SEQ ID NO:1 is obtained.

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