Humanized bcma antibodies and uses thereof

Abstract

The present application relates to a kind of humanized BCMA antibody and its application.The present application designs whole humanization antibody to BCMA target point, the antibody can be highly specific binding human source BCMA protein, based on this antibody constructs chimeric antigen receptor, prepares CAR-T cell, and experiment proves that, CAR-T cell can be efficiently recognized and kill BCMA positive tumor cell in in vitro and in vivo model, significantly inhibit tumor growth proliferation.In tumor-bearing mouse model, the survival time of treatment group mice is significantly prolonged, indicating that the antibody and the CAR therapy derived from the application have clear targeted therapy effect on the related diseases with BCMA as target point.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a humanized BCMA antibody and its application. Background Technology

[0002] Chimeric antigen receptors (CARs) are receptor proteins artificially synthesized through genetic engineering. They endow immune cells (such as T cells) with the ability to specifically recognize and attack tumor cells. The core function of CARs is to integrate targeted recognition and immune cell activation into a single molecule, combining the high specificity of antibodies with the strong killing ability of immune cells. This allows the modified immune cells to recognize cancer cell surface antigens without relying on the major histocompatibility complex (MHC), thereby achieving a highly efficient and precise anti-tumor effect.

[0003] B cell maturation antigen BCMA (CD269 / TNFRSF17) is a member of the tumor necrosis factor receptor superfamily and is encoded by the TNFRSF17 gene. Unlike CD19, whose expression decreases or disappears after plasma cell differentiation, BCMA is mainly expressed in late-stage B cells and plasma cells and is hardly expressed in normal tissues. It has extremely high tissue selectivity, making it an ideal tumor target.

[0004] Multiple myeloma (MM) is a malignant hematologic malignancy originating from plasma cells in the bone marrow. It is characterized by the abnormal proliferation of monoclonal plasma cells and the secretion of large amounts of monoclonal immunoglobulins (M proteins), leading to a range of clinical manifestations including bone destruction, anemia, kidney damage, and immune dysfunction. In MM patients, BCMA is highly expressed on malignant plasma cells, making it an ideal target for tumor therapy. By genetically engineering the patient's T cells to express chimeric antigen receptors targeting BCMA, precise recognition and elimination of myeloma cells can be achieved.

[0005] Compared to early murine antibodies, humanized or fully human designed antibodies significantly reduce the risk of human anti-mouse antibody response (HAMA). These fragments are usually derived from humanization or fully human screening technologies, retaining the antibody's ability to recognize BCMA while reducing rejection reactions in the human body, thus improving drug safety and efficacy. Humanized BCMA antibodies are a class of antibodies designed against B cell maturation antigen (BCMA), characterized by high specificity, low immunogenicity, and good affinity. They can be used for targeted therapy of BCMA-positive tumors such as multiple myeloma. For example, CN109485733A discloses a fully human anti-BCMA chimeric antigen receptor and its application, which has lower immunogenicity, less rejection reaction, and higher safety, effectively shrinking solid tumor lesions and improving the treatment effect of tumors.

[0006] In conclusion, the development and design of humanized BCMA antibodies that can be effectively applied to CARs is of great significance to the field of cancer therapeutic drug development. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a humanized BCMA antibody and its application, developing a humanized BCMA antibody with high affinity and specificity, and promoting the development of related biomedical fields.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a humanized BCMA antibody, wherein the amino acid sequences of the heavy chains CDR1, CDR2 and CDR3 of the humanized BCMA antibody include the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively, and the amino acid sequences of the light chains CDR1, CDR2 and CDR3 include the sequences shown in SEQ ID NO.4, AAS and SEQ ID NO.5, respectively.

[0009] This invention develops a humanized BCMA antibody with high affinity and specificity, enabling highly efficient targeting of target cells expressing BCMA protein. This holds immense potential for therapeutic drug development. Firstly, based on this highly efficient targeting characteristic, more precise drug delivery systems can be designed. Humanized BCMA antibodies can be conjugated with chemotherapeutic drugs, radionuclides, or other therapeutic molecules to form antibody-drug conjugates (ADCs) or radioimmunotherapy drugs. Secondly, humanized BCMA antibodies can also be used in immunotherapy, activating the body's own immune system to attack tumor cells. For example, they can be used in combination with immune checkpoint inhibitors to enhance the immune system's ability to recognize and kill tumor cells. Furthermore, humanized BCMA antibodies can play an important role in diagnostics. Their high affinity and specificity can be used to develop diagnostic reagents for detecting BCMA expression levels. Detecting BCMA expression in patients can aid in the early diagnosis of multiple myeloma and related diseases, and provide a basis for treatment selection. Simultaneously, in disease monitoring, regular monitoring of BCMA expression changes allows for timely evaluation of treatment effectiveness and adjustment of treatment strategies.

[0010] Optionally, the amino acid sequence of the heavy chain variable region of the humanized BCMA antibody includes the sequence shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.7.

[0011] Secondly, the present invention provides the use of the humanized BCMA antibody described in the first aspect in the preparation of formulations targeting BCMA antigen.

[0012] Thirdly, the present invention provides a chimeric antigen receptor, the chimeric antigen receptor comprising a signal peptide, a BCMA antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain; the BCMA antigen-binding domain comprising the humanized BCMA antibody described in the first aspect.

[0013] Optionally, the signal peptide includes any one of CD8α signal peptide, IgGκ light chain signal peptide or insulin signal peptide, preferably CD8α signal peptide.

[0014] Optionally, the hinge region includes any one of CD8α, CD28, human IgG1, IgG2, IgG4 or IgA, preferably the CD8α hinge region.

[0015] Optionally, the transmembrane domain includes a CD8α transmembrane region and / or a CD28 transmembrane region, preferably a CD8α transmembrane region.

[0016] Optionally, the signal conduction structure domain includes the CD3ζ signal conduction domain.

[0017] Optionally, the signal transduction domain further includes an intracellular co-stimulatory domain.

[0018] Preferably, the intracellular co-stimulatory domain includes any one or at least two combinations of 4-1BB, CD28 intracellular region, DAP10, or OX40.

[0019] Preferably, the chimeric antigen receptor comprises a CD8α signal peptide, the humanized BCMA antibody described in the first aspect, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ signal transduction domain.

[0020] Optionally, the amino acid sequence of the CD8α signal peptide includes the sequence shown in SEQ ID NO.15.

[0021] Optionally, the amino acid sequence of the CD8α hinge region includes the sequence shown in SEQ ID NO.17.

[0022] Optionally, the amino acid sequence of the CD8α transmembrane region includes the sequence shown in SEQ ID NO.18.

[0023] Optionally, the amino acid sequence of the 4-1BB intracellular co-stimulatory domain includes the sequence shown in SEQ ID NO.19.

[0024] Optionally, the amino acid sequence of the CD3ζ signal transduction domain includes the sequence shown in SEQ ID NO.20.

[0025] Optionally, the amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.21.

[0026] Fourthly, the present invention provides a nucleic acid molecule that encodes the humanized BCMA antibody described in the first aspect or the chimeric antigen receptor described in the third aspect.

[0027] Fifthly, the present invention provides a recombinant vector containing the nucleic acid molecule described in the fourth aspect.

[0028] In a sixth aspect, the present invention provides a chimeric antigen receptor cell, said chimeric antigen receptor cell comprising immune cells expressing the chimeric antigen receptor described in the third aspect.

[0029] This invention further designs a chimeric antigen receptor with a specific structure based on a humanized BCMA antibody, and introduces it into immune cells to construct chimeric antigen receptor immune cells. These modified immune cells can specifically recognize and bind to BCMA-expressing tumor cells, using their powerful killing ability to precisely target tumor cells. In in vitro experiments, their killing activity against BCMA-expressing tumor cells and cytokine secretion were observed to evaluate their efficacy and safety. Further validation of the therapeutic effect in mouse models revealed that they can inhibit tumor cell growth and significantly prolong the survival time of tumor-bearing mice.

[0030] Optionally, the immune cells include either T cells or NK cells.

[0031] In a seventh aspect, the present invention provides the use of the chimeric antigen receptor described in the third aspect or the chimeric antigen receptor immune cell described in the sixth aspect in the preparation of a medicament for treating tumors, wherein the tumors include tumors expressing BCMA positively.

[0032] Optionally, the tumor includes any one of multiple myeloma, plasma cell leukemia, or B-cell lymphoma / leukemia.

[0033] Compared with the prior art, the present invention has at least the following beneficial effects: This invention presents a fully humanized antibody targeting BCMA. This antibody binds to human BCMA protein with high specificity and exhibits minimal cross-reactivity with other non-target proteins, demonstrating excellent selectivity and binding specificity. Based on this antibody, a chimeric antigen receptor was further constructed, and CAR-T cells were successfully prepared. Experiments confirmed that these CAR-T cells efficiently recognized and killed BCMA-positive tumor cells in both in vivo and in vitro models, significantly inhibiting tumor growth and proliferation. In a tumor-bearing mouse model, the survival time of mice treated with CAR-T cells was significantly prolonged, indicating that this antibody and its derived CAR-T therapy have a clear targeted therapeutic effect on BCMA-targeted diseases, demonstrating good clinical translational potential and broad application prospects. Attached Figure Description

[0034] Figure 1 This is a flow cytometry result of BCMA antibody binding to natural antigen.

[0035] Figure 2 This is a schematic diagram of the structure of a chimeric antigen receptor.

[0036] Figure 3 Map of chimeric antigen receptor lentiviral plasmid expression vectors.

[0037] Figure 4 This is a diagram showing the expression results of a chimeric antigen receptor lentiviral plasmid.

[0038] Figure 5 This is a graph showing the positive rate of chimeric antigen receptor T cells.

[0039] Figure 6 The graph shows the IFN-γ factor detection results 6 and 8 days after chimeric antigen receptor T cells kill BCMA-positive cells.

[0040] Figure 7 The figures show the inhibition of tumor growth and mouse survival by chimeric antigen receptor T cells in mice. Figure A shows the in vivo imaging results in mice, Figure B shows the bioluminescence intensity results in mice, and Figure C shows the mouse survival rate results. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0042] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0043] Example 1 This embodiment designs a humanized BCMA antibody.

[0044] As shown in Table 1, the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 of the humanized BCMA antibody are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the amino acid sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO.4, AAS, and SEQ ID NO.5, respectively. The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.6, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.7.

[0045] Table 1 Heavy chain variable region amino acid sequence (SEQ ID NO.6): EVQLVESGGGLVQPGGSLRLSCAASGFSLNVYYMSWVRQAPGKGLEWIGFINTGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAPGYITNLWGPGTLVTVSS.

[0046] Light chain variable region amino acid sequence (SEQ ID NO.7): DIQMTQSPSSSLSASVGDRVTITCQASQSISNELSWYQQKPGKVPKLLIYAASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGGYLGGYVSTIYVGAFGQGTKLTVL.

[0047] Heavy chain variable region nucleotide sequence (SEQ ID NO.13): GAAGTGCAGCTGGTGGAGTCCGGCGGTGGACTGGTGCAACCGGGAGGCTCACTCAGATTGTCATGCGCCGCCTCTGGCTTTAGTCTCAATGTCTACTACATGAGCTGGGTGAGGCAGGCACCCGGCAAGGGCCTGGAATGGATCGGCTTCATTAATACTGGTGGTAGCGCATACTACGCGAGCTGGGCGAAGGGCAGGTTTACCATCAGCCGCGACAACAGCAAGAATACCCTGTACCTGCAGATGAATAGCCTGAGGGCCGAAGACACGGCGGTCTATTATTGTGCACGGGCTCCTGGTTATATTACGAACCTGTGGGGCCCTGGGACCCTCGTAACTGTGAGCAGC。

[0048] Nucleotide sequence of the light chain variable region (SEQ ID NO.14): GACATCCAGATGACCCAGTCCCCAAGTTCCCTGAGCGCCTCCGTGGGCGACCGCGTGACCATAACCTGTCAGGCCAGTCAGAGCATTAGCAATGAATTATCCTGGTATCAACAGAAGCCCGGTAAGGTGCCCAAACTGCTCATTTACGCTGCATCCACTCTGGCATCCGGGGTGCCAAGCCGCTTCTCCGGGAGTGGGTCTGGCACCGACTTCACCCTGACCATATCTTCCCTGCAGCCCGAGGACGTGGCAACGTACTATTGCCTAGGCGGTTATCTAGGCGGTTATGTTAGTACTATTTATGTTGGTGCTTTCGGGCAGGGCACGAAGTTGACCGTGCTG。

[0049] Antibody affinity was detected using the Biacore 8K (Cytiva) chip: First, an Fc-tagged anti-BCMA scFv antibody fragment was captured using a Protein A chip (Cytiva). Then, the antibody to be tested was diluted to an appropriate concentration (2 μg / mL) and immobilized on the chip. The antigen (human BCMA extracellular fragment) was then serially diluted (6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM) as the analyte. Next, a single-cycle kinetics mode was used, allowing different concentrations of analyte to flow sequentially across the chip surface, monitoring the binding and dissociation processes in real time, and the system automatically generated a sensor map. Finally, the data were fitted and analyzed to calculate the affinity constant (KD). The results are shown in Table 2. The equilibrium dissociation constant KD value for the humanized BCMA scFv antibody fragment binding to its specific antigen protein BCMA was 9.73E-08 M, indicating that the humanized antibody has high antigen affinity.

[0050] Table 2 Specific binding of the antibody to BCMA antigen on MM.1s cells (human multiple myeloma cells, BCMA-positive cells naturally expressing BCMA): 1 × 10^6 MM.1s cells were incubated with the antibody (final concentration 1 μg / mL) in PBS + 5% BSA at room temperature in the dark for 30 min. After centrifugation and washing, the cells were incubated with secondary antibody (Alexa Fluor 647 labeled, 1:1000 dilution, Invitrogen #A21445) at room temperature in the dark for another 30 min. Cells were then washed and analyzed by flow cytometry. Results are as follows: Figure 1 As shown, using the commercial BCMA antibody (Biolegend #357506) as a positive control, its positive binding rate to BCMA antigen on MM.1s cells was 99.56%, while the positive binding rate of the humanized BCMA antibody to BCMA antigen on MM.1s cells was 73.32%, indicating that the humanized BCMA antibody of the present invention can also effectively bind to BCMA antigen on cells.

[0051] Example 2 This embodiment designs a humanized anti-BCMA chimeric antigen receptor (Anti-BCMA CAR).

[0052] The schematic diagram of the chimeric antigen receptor structure is shown below. Figure 2As shown, the chimeric antigen receptor includes a signal peptide (Leader), a BCMA antigen-binding domain, a hinge region, a transmembrane domain (TM), and a signal transduction domain. The signal peptide is a CD8α signal peptide (SEQ ID NO. 15). The antigen-binding domain includes the humanized BCMA antibody (anti-BCMA scFv) designed in Example 1. The light chain variable region and the heavy chain variable region are connected by a linker, the amino acid sequence of which is shown in SEQ ID NO. 16. The hinge region is a CD8α hinge region (SEQ ID NO. 17). The transmembrane domain includes a CD8α transmembrane region (SEQ ID NO. 18). The signal transduction domain includes a 4-1BB intracellular co-stimulatory domain (SEQ ID NO. 19) and a CD3ζ signal transduction domain (SEQ ID NO. 20). The overall amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 21.

[0053] The amino acid sequence of the CD8α signal peptide (SEQ ID NO.15): MALPVTALLLPLALLLHAARP.

[0054] The amino acid sequence of the linker (SEQ ID NO.16): GGGGSGGGGSGGGGS.

[0055] The amino acid sequence of the CD8α hinge region (SEQ ID NO.17): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0056] The amino acid sequence of the CD8α transmembrane region (SEQ ID NO.18): IYIWAPLAGTCGVLLLSLVITLYC.

[0057] The amino acid sequence of the intracellular co-stimulatory domain of 4-1BB (SEQ ID NO.19): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0058] Amino acid sequence of the CD3ζ signal transduction domain (SEQ ID NO.20): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0059] Anti-BCMA CAR amino acid sequence (SEQ ID NO.21): MALPPVTALLLPLALLLHAARPDIQMTQSPSSSLSASVGDRVTITCQASQSISNELSWYQQKPGKVPKLLIYAASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGGYLGGYVSTIY VGAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLNVYYMSWVRQAPGKGLEWIGFINTGGSAYYASWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARAPGYITNLWGPGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0060] The specific nucleotide sequences of each element are as follows: The nucleotide sequence of the CD8α signal peptide (SEQ ID NO.22): ATGGCCCTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTGCTCCTGCACGCCGCTCGCCCC.

[0061] Nucleotide sequence of the linker (SEQ ID NO.23): GGCGGCGGCGGCTCTGGAGGAGGAGGCAGCGGCGGAGGAGGCTCC.

[0062] The nucleotide sequence of the CD8α hinge region (SEQ ID NO.24): ACCACCACCCCTGCCCCGCGGCCGCCTACCCCTGCTCCTACCATCGCCTCTCAGCCTCTGAGCCTGCGGCCCGAGGCCTGCAGACCCGCCGCTGGCGGCGCCGTGCACACCAGAGGCCTGGACTTCGCCTGTGAT.

[0063] The nucleotide sequence of the CD8α transmembrane region (SEQ ID NO.25): ATCTACATCTGGGCCCCACTGGCCGGAACATGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTATTGC.

[0064] Nucleotide sequence of the 4-1BB intracellular co-stimulatory domain (SEQ ID NO.26): AAGAGAGGCAGAAAAAAGCTGCTGTACATCTTCAAGCAGCCTTTCATGAGACCCGTCCAGACCACACAGGAGGAGGACGGATGTAGCTGCAGATTCCCTGAGGAAGAAGAAGGCGGATGTGAACTG.

[0065] Nucleotide sequence of the CD3ζ signaling domain (SEQ ID NO.27).

[0066] AGAGTGAAGTTCTCTAGAAGCGCTGATGCCCCTGCCTACCAGCAGGGCCAGAACCAGCTGTATAATGAGCTGAACCTGGGCAGGCGCGAGGAATACGACGTGCTGGACAAGCGGCGGGGCCGGGACCCGAGATGGGCGGGAAACCTAGACGGAAGAACCCCCAGGAG GGCCTGTACAACGAGCTGCAGAAAGATAAGATGGCTGAAGCCTACAGCGAGATCGGCATGAAAGGCGAGCGGAAGAGGCAAGGGCCACGACGGCCTGTATCAGGGCCTCTCCACCGCCACAAAGGACACCTACGATGCCCTTCATATGCAGGCCCTGCCTCCTCGG.

[0067] Anti-BCMA CAR nucleotide sequence (SEQ ID NO.28):

[0068] Example 3 This embodiment constructs a chimeric antigen receptor expression vector.

[0069] (1) The theoretical protein sequence of the CAR gene was designed according to Example 2, the CAR gene was optimized to enable it to be expressed efficiently in human cells, the CAR gene was prepared by codon optimization and whole gene synthesis method, and the whole gene was synthesized at Guangzhou Aiji Biotechnology Co., Ltd. (2) The CAR gene synthesized from the whole gene was digested with EcoRI and BamHI and the empty vector pCDH-EF1-MCS was digested in a water bath at 37℃ for 30 min. Then, DNA was electrophoresed on a 1.5% agarose gel and purified and recovered using Tiangen's agarose gel kit. (3) Connect the pCDH-EF1-MCS vector to the CAR gene fragment. The connection system is shown in Table 3.

[0070] Table 3 Ligation was performed at 22℃ for 1 h. The ligation product was directly transformed into Stbl3 competent E. coli cells. 200 μL of the transformation product was plated onto kanamycin-resistant LB agar plates and incubated upside down overnight at 37℃. The next morning, three single clones were randomly selected for colony PCR identification, and positive clones were sent for sequencing.

[0071] (4) The constructed Anti-BCMA CAR lentiviral expression vector map is as follows: Figure 3 As shown.

[0072] Example 4 In this embodiment, flow cytometry was used to detect the expression of Anti-BCMA CAR in 293T cells after transient transfection with CAR plasmid.

[0073] First, 293T cells were prepared and cultured to an appropriate density. Then, the CAR plasmid DNA was mixed with the transfection reagent according to the manufacturer's instructions. The mixture was added to the culture medium containing 293T cells, and the culture dish was gently shaken to ensure even distribution. Next, the cells were placed in an incubator and cultured under appropriate conditions to promote transfection efficiency. 24 h post-transfection, cells were collected and washed with PBS. Cells were then labeled with Protein L-FITC and incubated. Flow cytometry was then used to analyze the binding of the labeled cells to the Protein L-FITC-detected protein in the CAR plasmid-transfected 293T cells. Finally, flow cytometry data were collected and analyzed to assess transfection efficiency and CAR protein expression levels. The results are shown below. Figure 4As shown, after 293T cells were transfected with the CAR plasmid, the Anti-BCMA CAR molecule could bind to the Protein L-FITC detection protein (positive rate 41.35%), indicating that the Anti-BCMA CAR molecule could be correctly expressed on the cell membrane.

[0074] Example 5 This embodiment involves lentivirus packaging.

[0075] The lentiviral expression vectors from Example 3 were packaged into lentiviruses using a four-plasmid system, and the specific steps are as follows: (1) The four plasmid systems express gag / pol, Rev, VSV-G and the CAR expression vector constructed in this invention respectively for packaging lentiviral vectors: the four plasmids are transiently transfected into 293T cells with a DNA content of 2 μg / mL. (2) Mix the above plasmid with PEI transfection reagent, add it to a certain volume of serum-free DMEM, mix well and let stand for 15 min, add the above mixture to T75 culture flasks containing 293T cells, mix gently, and culture at 37℃ and 5% CO2 cell culture incubator for 6 h. (3) Replace with fresh culture medium after 6 h and continue culturing. Add 10 mM sodium butyrate solution. Collect the culture supernatant of lentivirus after 72 h for purification and detection.

[0076] Example 6 This embodiment involves lentivirus infection of T cells.

[0077] After isolating PBMCs, they were activated with X-VIVO containing 50 ng / mL OKT3 and 300 IU / mL IL-2. Two days later, the culture medium was replaced with X-VIVO containing 300 IU / mL IL-2 for expansion culture. RetroNectin was used to enhance the infection efficiency of lentivirus on T cells. 30 μg of RetroNectin was coated into 6-well plates and incubated at 37°C for 2 h. RetroNectin was then used to block the coated 6-well plates with Hank's solution containing 2.5% BSA, and the plates were incubated at 37°C for 0.5 h. The blocking solution was then removed, and the 6-well plates were washed with Hank's solution containing 2% Hepes. X-VIVO medium was added, along with an appropriate amount of lentivirus solution. The plates were centrifuged at 2000×g for 2 h. The supernatant was discarded, and 1×10⁻⁶ ppm of the lentivirus solution was added. 6 Activated PBMC cells were centrifuged at 1000×g for 10 min and cultured in a cell culture incubator at 37℃, 5% CO2, and controlled humidity. Cell counts were performed every two days, and X-VIVO at 300 IU / mL was replaced, maintaining a cell concentration of 0.5×10⁻⁶ cells / mL. 6-1×10 6 The cells were cultured at / mL for 8 consecutive days. CAR-T cell expansion was assessed using a Countstar IC1000 automated cell counter (USA). Results showed that all CAR-T groups expanded well with no significant differences.

[0078] Example 7 This embodiment evaluates the expression and function of CAR. (1) Flow cytometry was used to detect the expression of CAR molecules on the surface of CAR-T cells and their binding ability to the corresponding antigen protein 6 days after lentiviral infection. T cell populations were labeled with APC-anti-CD3 antibody, and the CAR expression positivity rate was then detected. Results are as follows: Figure 5 As shown, the CAR positivity rates of CAR-T cells measured by the targeting linker antibody (Anti-GS4linker) and BCMA antigen (ACRO Biosystems) were 65.51% and 64.33%, respectively. The results indicate that Anti-BCMA CAR can be expressed efficiently, while the CAR positivity rates of the control group Mock-T cells were only 1.77% and 0.61%.

[0079] (2) Identification of the cytotoxic function of CAR-T cells at 6 and 8 days after lentiviral infection: MM.1s-luc was selected as the target cell with high BCMA expression, and Raji-luc was selected as the target cell with low BCMA expression. The in vitro cytotoxic effects of CAR-T and Mock T cells on the above target cells at effector-target ratios of 1:1, 2:1, and 1:2 were observed; Mock T cells were used as non-transduced control cells. The results are as follows: Figure 6 It can be seen that CAR-T cells can significantly kill Raji-luc cells and MM.1s-luc cells, and secrete a large amount of IFN-γ factor, especially in the 2:1 effector-to-target ratio group, which is the most obvious. This indicates that CAR-T cells prepared with humanized BCMA antibody have significant specific killing ability against BCMA positive cells.

[0080] Example 8 This study aimed to detect the anti-tumor effect of CAR-T cells in vivo.

[0081] To explore the in vivo antitumor effect of CAR-T therapy in an immunodeficient tumor-bearing mouse model, B-NDG mice (Biocytok Jiangsu Gene Biotechnology Co., Ltd.) were randomly divided into two groups: a Mock T group (n=4) and a CAR-T treatment group (n=4). Each mouse was injected with 1×10⁻⁶ CAR-T cells via the tail vein. 6 MM.1s-Luc (human MM.1s cells overexpressing luciferase) were injected via tail vein into the Mock-T group the day after inoculation with 3×10⁻⁶ cells. 6 / 200 μL of untransduced CAR plasmid T cells, CAR-T treatment group received 3×10 6 / 200μL CAR-T cells. Day 0 was the day of the first administration. All animals were administered the drug via a single tail vein injection. Monitoring indicators: routine clinical observation once daily. Chemiluminescence signals were captured using a small animal imaging system on days 0, 7, 14, 21, 27, and 33 after administration to monitor changes in tumor size in mice.

[0082] In vivo imaging results in mice as follows Figure 7 As shown, the bioluminescence intensity of mice in the Mock-T group gradually increased, and the tumors continued to grow until all mice died at 42 days; while the CAR-T treatment group could significantly inhibit tumor growth and the mice continued to survive.

[0083] In summary, this invention designs a fully humanized BCMA antibody that specifically binds to human BCMA protein and has virtually no binding ability to other proteins, exhibiting high specificity. Further development of CAR and CAR-T cells demonstrates that these antibodies significantly kill BCMA-positive tumors both in vivo and in vitro, inhibit tumor cell growth, and significantly prolong the survival time of tumor-bearing mice, indicating a specific therapeutic effect on diseases targeting BCMA protein and possessing broad application prospects.

[0084] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A humanized BCMA antibody, characterized in that, The amino acid sequences of the heavy chains CDR1, CDR2, and CDR3 of the humanized BCMA antibody include the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively, and the amino acid sequences of the light chains CDR1, CDR2, and CDR3 include the sequences shown in SEQ ID NO.4, AAS, and SEQ ID NO.5, respectively.

2. The humanized BCMA antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the humanized BCMA antibody includes the sequence shown in SEQ ID NO. 6, and the amino acid sequence of the light chain variable region includes the sequence shown in SEQ ID NO.

7.

3. The use of the humanized BCMA antibody according to claim 1 or 2 in the preparation of formulations targeting BCMA antigen.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor includes a signal peptide, a BCMA antigen-binding domain, a hinge region, a transmembrane domain, and a signal transduction domain. The BCMA antigen-binding domain includes the humanized BCMA antibody as described in claim 1 or 2.

5. The chimeric antigen receptor according to claim 4, characterized in that, The signal peptide includes any one of CD8α signal peptide, IgGκ light chain signal peptide or insulin signal peptide, preferably CD8α signal peptide. Optionally, the hinge region includes any one of CD8α, CD28, human IgG1, IgG2, IgG4 or IgA, preferably the CD8α hinge region; Optionally, the transmembrane structural domain includes a CD8α transmembrane region and / or a CD28 transmembrane region, preferably a CD8α transmembrane region; Optionally, the signal conduction structure domain includes the CD3ζ signal conduction domain; Optionally, the signal transduction domain further includes an intracellular co-stimulatory domain; Preferably, the intracellular co-stimulatory domain includes any one or at least two combinations of 4-1BB, CD28 intracellular region, DAP10, or OX40.

6. The chimeric antigen receptor according to claim 4 or 5, characterized in that, The chimeric antigen receptor includes a CD8α signal peptide, the humanized BCMA antibody as described in claim 1 or 2, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ signal transduction domain. Optionally, the amino acid sequence of the CD8α signal peptide includes the sequence shown in SEQ ID NO.15; Optionally, the amino acid sequence of the CD8α hinge region includes the sequence shown in SEQ ID NO.17; Optionally, the amino acid sequence of the CD8α transmembrane region includes the sequence shown in SEQ ID NO.18; Optionally, the amino acid sequence of the 4-1BB intracellular co-stimulatory domain includes the sequence shown in SEQ ID NO.19; Optionally, the amino acid sequence of the CD3ζ signal transduction domain includes the sequence shown in SEQ ID NO.20; Optionally, the amino acid sequence of the chimeric antigen receptor includes the sequence shown in SEQ ID NO.

21.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the humanized BCMA antibody of claim 1 or 2 or the chimeric antigen receptor of any one of claims 4-6.

8. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 7.

9. A chimeric antigen receptor cell, characterized in that, The chimeric antigen receptor cells include immune cells expressing the chimeric antigen receptor as described in any one of claims 4-6.

10. The use of the chimeric antigen receptor according to any one of claims 4-6 or the chimeric antigen receptor cell according to claim 9 in the preparation of a medicament for treating tumors, characterized in that, The tumors include those that express BCMA positively.

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