CN112062864A - Preparation method and application of targeting BCMA tumor antigen receptor modified T cells - Google Patents

Abstract

The invention relates to a preparation method and application of a target BCMA tumor antigen receptor modified T cell, wherein the target BCMA tumor antigen receptor modified T cell specifically recognizes BCMA molecules on the surface of a tumor cell after being expressed in the T cell through 6 different chimeric antigen receptors for resisting BCMA antigens, can effectively eliminate tumor target cells for expressing the BCMA antigens, has no toxic or side effect on antigen-negative cells, and can be used for the target treatment of tumors, particularly the target treatment of multiple myeloma. Among the targeted BCMA tumor antigen receptor modified T cells, TSN1-CAR-T cells, TSN2-CAR-T cells, TSN5-CAR-T cells, TSN6-CAR-T cells, TSN7-CAR-T cells, TSN8-CAR-T cells and TSN9-CAR-T cells can kill myeloma cells with low BCMA expression.

Description

Preparation method and application of modified T cells targeting BCMA tumor antigen receptor

technical field

The invention belongs to the technical field of cellular immunotherapy, and in particular relates to a preparation method and application of targeting BCMA tumor antigen receptor modified T cells.

Background technique

With the development of tumor immunology theory and clinical technology, Chimeric antigen receptor T-cell immunotherapy (CAR-T) (June CH, Blazar BR, Riley JL, Engineering lymphocyte subsets: tools, trials and tribulations. Nat RevImmunol. 2009; 9:704-16) has become one of the most promising tumor immunotherapies. Generally, a chimeric antigen receptor CAR consists of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, a costimulatory region, and an intracellular signal transduction region. CAR-T cell therapy expresses the fusion protein of single chain fragment variable (scFv) and T cell activation sequence that can specifically recognize tumor-associated antigens on the surface of T cells through exogenous gene transfection technology, so that it can specifically recognize tumor-associated antigens. The scFv that specifically recognizes tumor-associated antigens is coupled to the intracellular activation and proliferation signaling domain of T cells through the transmembrane region. CAR-expressing T cells recognize tumor antigens in an antigen-dependent, but not MHC-restricted, manner, initiating and activating specific tumor-killing responses. So far, CAR-T cell immunotherapy has achieved good results in the treatment of hematological tumors and lymphomas. For B-lineage malignancies, the adoptive T-cell approach with anti-CD19 CAR is most commonly used. Anti-CD19 CAR-T cells have been found to be able to cure mouse leukemia and lymphoma in mouse trials, and some patients have found remission in clinical trials of anti-CD19 CAR-T cell infusions, but CD19 is rarely seen in multiple myeloma. It is expressed in malignant plasma cells, so treating multiple myeloma with CAR-expressing T cells requires finding other better targets.

Multiple myeloma (MM) is a common hematological malignancy caused by the malignant proliferation and accumulation of mature plasma cells in the bone marrow leading to bone destruction and bone marrow failure. Common clinical manifestations include bone pain, anemia, osteolytic lesions, hypercalcemia, renal impairment, cytopenia, and symptomatic plasma cell production. At present, multiple myeloma is the second largest malignant tumor of the hematological system, accounting for 10% of the malignant tumor of the hematological system. It mostly occurs in males, and its incidence increases year by year with age. Siegel, R., et al., Cancerstatistics, 2014. CA Cancer J Clin, 2014. 64(1): p.9-29.]. Multiple myeloma has a high probability of recurrence after treatment and is incurable. Although some monoclonal antibodies have shown promise in the treatment of MM in preclinical studies and early clinical trials, they have not been consistently recognized. Clearly, new immunotherapy studies in MM are clearly very much needed.

Currently, BCMA has become a very popular immunotherapy target for MM and other hematological malignancies. The full name of BCMA is B cell maturation antigen, also known as CD269. It consists of 184 amino acid residues, its intracellular region contains 80 amino acid residues, and the extracellular region sequence is very short. Only one carbohydrate recognition domain is a B cell surface molecule. . BCMA is a type I transmembrane signal protein lacking a signal peptide, expressed only on the surface of mature B cells, and is an important B cell biomarker. BCMA is a member of the tumor necrosis factor receptor family (TNFR), which can bind to two ligands, B cell activating factor BAFF or aproliferation induced ligand (APRIL). Curr Opin Struct Biol, 2004. 14(2): p. 154-60.]. The receptor also binds to various TRAF family members, mediating signals for cell survival and proliferation. Studies have shown that in normal cells, BCMA is mainly expressed by plasma cells and a part of mature B cells, but not expressed in most B cells and other organs. The immune system of BCMA knockout mice is normal, the development of B lymphocytes is normal, but the number of plasma cells is significantly reduced, which proves that BCMA plays an important role in maintaining the survival of plasma cells. Up-regulate anti-apoptotic genes Bcl-2, Mcl-1 and Bclw, etc. to maintain cell growth [BCMA is essential for the survival of long-lived bone marrow plasma cells. J Exp Med, 2004.199(1):p.91-8.] .

Studies have found that BCMA is commonly expressed in multiple myeloma cell lines [Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and surviva. Blood, 2004.103(2): p.689-94.]. In up to 60%-70% of multiple myeloma cases, BCMA is expressed during B cell development and is widely expressed on the surface of MM cells. In view of the expression characteristics of BCMA, we can use BCMA as one of the targets of CAR-T cells for cellular immunotherapy of multiple myeloma.

SUMMARY OF THE INVENTION

In order to solve the above problems existing in the prior art, the present invention provides a preparation method and application of T cells targeting BCMA tumor antigen receptor modified T cells. The present invention provides 6 kinds of chimeric antigen receptors targeting BCMA tumor antigens, and further obtains the modified T cells targeting BCMA tumor antigen receptors, and the modified T cells targeting BCMA tumor antigen receptors specifically recognize tumor cells The surface BCMA molecule mediates the killing of BCMA-positive tumors, and can specifically kill myeloma cells with low BCMA expression, and has no toxic side effects on antigen-negative cells.

The technical scheme adopted in the present invention is:

The present invention provides a chimeric antigen receptor targeting BCMA. The structure includes a signal peptide, a BCMA antigen recognition region, a hinge region, a transmembrane region, an intracellular signal domain, an extracellular domain III of EGFR and an extracellular domain in series in sequence. Fragments of Domain IV.

Preferably, the BCMA antigen recognition region is an anti-BCMA single-chain antibody;

There are 6 kinds of single-chain antibodies against BCMA, namely TSN1, TSN3, TSN5, TSN7, TSN8 and TSN9; wherein, the amino acid sequence of TSN7 is shown in SEQ ID NO.1; it should be noted that the inventors Pre-screened and experimentally verified that all 6 single-chain antibodies have the ability to bind BCMA protein (the 6 single-chain antibodies are structurally similar homologues), so the 6 single-chain antibodies prepared by using the targeted BCMA tumor antigen Receptor-modified T cells have specific killing effect on BCMA protein-positive target cells.

The signal peptide is a CD8 signal peptide, and the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO. 2; the hinge region and the transmembrane region are the CD8 hinge region and the transmembrane region, and the CD8 hinge region and The amino acid sequence of the transmembrane region is shown in SEQ ID NO.3; the intracellular signal domain is CD3ζ intracellular signal domain, and the amino acid sequence of the CD3ζ intracellular signal domain is shown in SEQ ID NO.5; the EGFR The amino acid sequences of the ectodomain III and ectodomain IV fragments are shown in SEQ ID NO.6.

Preferably, a costimulatory signal domain is also connected between the transmembrane region and the intracellular signal domain; the costimulatory signal domain is a 4-1BB costimulatory signal domain, and the costimulatory signal domain of the 4-1BB costimulatory signal domain is The amino acid sequence is shown in SEQ ID NO. 4; a T2A fragment is also connected between the intracellular signal domain and the fragment of the EGFR, and the amino acid sequence of the T2A fragment is shown in SEQ ID NO. 7.

Preferably, an IL2RB stimulation signal domain is added to the costimulatory signal domain, so that in addition to the 4-1BB costimulatory signal domain, an IL2RB stimulation signal domain is added to the costimulatory signal domain (the amino acid sequence is shown in SEQ ID NO. 8). , the nucleotide sequence is shown in SEQ ID NO. 17), the purpose is to screen out the CAR combination with stable expression and high effectiveness.

The CAR structure targeting the BCMA chimeric antigen receptor is added with EGFR extracellular domain III and extracellular domain IV fragments. The CD8 signal peptide sequence was also added before the fragment of EGFR. The main function of EGFR fragments: they can be used as cell surface markers, and are also suitable for in vivo tracking of T cells. They can be detected by flow and immunohistochemistry, and can also be cleared by cetuximab in vivo. The inventors of the present application found in their research that the introduction of the above EGFR fragments can not only make CAR-T cells well traced in vivo, but also act as a safety switch for CAR-T cells: when you want to stop CAR-T cells When it works, tocilizumab is added to safely and effectively control the infused CAR-T cells targeting the BCMA target to function in vivo. It lays a good foundation for the safety of clinical experiments and clinical treatment.

It should be noted that, in other embodiments, those skilled in the art can change the signal peptide, hinge region, transmembrane region, 4-1BB costimulatory signal domain, IL2RB stimulation signal domain and CD3ζ intracellular domain according to actual conditions or needs. The combination type and sequence of the signal domains, no matter what kind of changes are based, as long as the chimeric antigen receptor has the BCMA antigen recognition region sequence of the present invention, it is within the protection scope of the present invention.

The present invention also provides a nucleic acid molecule encoding the BCMA-targeting chimeric antigen receptor.

Preferably, in the nucleic acid molecule, the nucleotide sequence encoding the TSN7 is shown in SEQ ID NO.9; the nucleotide sequence encoding the CD8 signal peptide is shown in SEQ ID NO.10; The nucleotide sequence of the hinge region and transmembrane region of CD8 is shown in SEQ ID NO. 11; the nucleotide sequence encoding the 4-1BB costimulatory signal domain is shown in SEQ ID NO. 12; the nucleotide sequence encoding the CD3ζ cell The nucleotide sequence of the internal signal domain is shown in SEQ ID NO. 13; the nucleotide sequences encoding the EGFR extracellular domain III and extracellular domain IV fragments are shown in SEQ ID NO. 14; The nucleotide sequence of the T2A fragment is shown in SEQ ID NO.15; the nucleotide sequence encoding the IL2RB stimulation signal domain is shown in SEQ ID NO.17.

The present invention also provides a recombinant vector, which is a recombinant viral vector containing the nucleic acid molecule. Preferably, the recombinant viral vector is one of lentivirus, retrovirus or RNA expression vector.

The present invention also provides a genetically modified T cell, which is characterized in that the T cell contains the recombinant vector or the T cell has the exogenous nucleic acid molecule integrated into the chromosome or expresses the mosaic antigen receptors.

The present invention also provides the preparation method of the modified T cells targeting BCMA tumor antigen receptors, comprising the following steps:

(1) obtaining the nucleic acid sequence fragment targeting the BCMA chimeric antigen receptor by PCR method;

(2) Clone the nucleic acid sequence fragment targeting the BCMA chimeric antigen receptor into an expression vector to obtain an expression plasmid of TSNs-CAR;

(3) transfecting the expression plasmid of the TSNs-CAR described in step (2) into 293T cells, and after packaging and concentrating, a lentivirus concentrate is obtained;

(4) Infecting T cells with the lentivirus concentrate to obtain the T cells modified with the BCMA tumor antigen receptor.

The chimeric antigen receptor targeting BCMA, the nucleic acid molecule, the recombinant vector, and the modified T cell targeting BCMA tumor antigen receptor are prepared for preventing and/or treating cancer or tumor. application in medicine. Especially in the prevention and/or treatment of BCMA-positive B-cell lymphoma, multiple myeloma or plasma cell leukemia.

The beneficial effects of the present invention are:

(1) The targeted BCMA tumor antigen receptor-modified T cells of the present invention can specifically recognize the BCMA molecules on the surface of tumor cells after 6 different anti-BCMA antigen chimeric antigen receptors are expressed in T cells. It can effectively remove tumor target cells expressing BCMA antigen, and has no toxic and side effects on antigen-negative cells, and can be used for targeted therapy of tumors, especially targeted therapy of multiple myeloma.

(2) Among the BCMA tumor antigen receptor-modified T cells of the present invention: TSN1-CAR-T cells, TSN2-CAR-T cells, TSN5-CAR-T cells, TSN6-CAR-T cells, TSN7-CAR-T cells CAR-T cells, TSN8-CAR-T cells and TSN9-CAR-T cells can specifically kill myeloma cells with low BCMA expression.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

FIG. 1A and FIG. 1B are respectively a schematic structural diagram of the targeting BCMA chimeric antigen receptor according to the present invention;

Fig. 2 is the map of pLVKn-TSNs-CAR expression plasmid of the present invention;

Figure 3 is a comparison chart of the results of killing BCMA tumor cells by different CAR-T cells targeting BCMA;

Figure 4 is a comparison chart of the results of killing BCMA low-expressing tumor cells by different CAR-T cells targeting BCMA;

Figures 5A and 5B are the results of intracellular cytokine staining to detect the expression of effector cytokines after co-culture of CAR-T cells with different BCMA targets and target cells;

Figure 6 is a comparison diagram of the changes in tumor volume in mice after injection of different BCMA-targeting CAR-T cells;

Figure 7 is a graph showing the results of in vivo fluorescence imaging detection of tumor elimination in mice.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

The experimental methods in the following examples, unless otherwise specified, are conventional methods in the art. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

1. Construction of pLVKn-TSNs-CAR expression plasmids targeting BCMA

1. Gene sequence synthesis

The CD8 signal peptide (the nucleotide sequence encoding the CD8 signal peptide is shown in SEQ ID NO. 10, and the amino acid sequence is shown in SEQ ID NO. 2), BCMA single-chain antibodies (TSN1, TSN3, TSN5, TSN7) were synthesized in turn , TSN8, TSN9), CD8 hinge region and transmembrane region (the nucleotide sequence encoding the CD8 hinge region and transmembrane region is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.3), 4-1BB costimulatory signal domain (the nucleotide sequence encoding the 4-1BB costimulatory signal domain is shown in SEQ ID NO.12, and the amino acid sequence is shown in SEQ ID NO.4), IL2RB stimulation signal domain (encoding The nucleotide sequence of the IL2RB stimulation signal domain is shown in SEQ ID NO. 17, and the amino acid sequence is shown in SEQ ID NO. 8), CD3ζ signal transduction domain (the nucleotide encoding the CD3ζ intracellular signal domain) The sequence is shown in SEQ ID NO.13, the amino acid sequence is shown in SEQ ID NO.5), T2A fragment (the nucleotide sequence encoding the T2A fragment is shown in SEQ ID NO.15, and the amino acid sequence is shown in SEQ ID NO. 7), extracellular domain III and extracellular domain IV fragment of EGFR (the nucleotide sequences encoding the extracellular domain III and extracellular domain IV fragment of EGFR are shown in SEQ ID NO.14 shown, the amino acid sequence is shown in SEQID NO.6);

The above sequences were sequentially connected by overlapping PCR method. Eight kinds of chimeric antigen receptor expression cassettes TSNs-CAR were obtained, and the configuration diagrams were shown in Figure 1A and Figure 1B, respectively, named: TSN1-CAR, TSN2-CAR (in the structure of TSN1-CAR, IL2RB stimulation signal was added domain), TSN3-CAR, TSN5-CAR, TSN6-CAR (the IL2RB stimulation signal domain is added to the TSN5-CAR structure), TSN7-CAR (the full-length nucleotide sequence is shown in SEQ ID NO.16, and the amino acid sequence is shown in SEQ ID NO. 18), TSN8-CAR and TSN9-CAR;

2. The above-mentioned TSNs-CAR expression cassette sequence was constructed into pLVKn lentiviral vector to obtain pLVKn-TSNs-CAR expression vector

The two segments of each TSNs-CAR expression cassette sequence contain BamHI and SalI restriction sites respectively. The nucleotide sequence of the CAR molecule and the pLVKn lentiviral vector plasmid were double digested with BamHI and SalI, ligated by T4 ligase, and then the The ligation product was transformed into E. coli competent (stbl3);

Randomly pick 2 single clones from the plate transformed with TSNs-CAR, add them to 5ml LB medium containing ampicillin (100ug/ml) for expansion, and extract plasmids after 12-16 hours, and use BamHI and SalI for expansion. Enzyme digestion identification, and the plasmid with the correct digestion was sent to Shanghai Sangong for further confirmation by sequencing. The correct sequence is the pLVKn-TSNs-CAR expression plasmid, and the expression plasmid map is shown in Figure 2.

2. Packaging, concentration and titer determination of lentivirus

1. Packaging of lentivirus

(1) Cell treatment: 24h before transfection, collect the 3rd-10th passage 293T cells in the logarithmic growth phase, inoculate the 293T cells in a 10cm cell culture dish, and the inoculation volume is 1×10 ⁷ . Grow in DMEM medium of FBS, place in a 37°C 5% CO ₂ cell incubator for 18 hours, and transfect when the cell density reaches 60-80%; (2) Co-transfect 293T cells with a lentiviral expression vector plasmid TSNs-CAR and its packaging plasmids (pSPAX2, pMD2-G); (3) 293T culture supernatant was collected 72 hours after transfection, centrifuged at 3000 rpm for 15 minutes, the cells and supernatant were collected separately, and the cells were lysed by repeated freezing and thawing three times. The supernatant was collected by centrifugation, and the cell pellet was discarded; (4) The virus supernatant was filtered with a 0.45 μm filter to obtain TSN1-CAR, TSN2-CAR, TSN3-CAR, TSN5-CAR, TSN6-CAR, TSN7-CAR, TSN8-CAR, respectively. CAR and TSN9-CAR virus solutions;

2. Lentivirus concentration and virus titer determination

(1) Use PEG6000 to concentrate the virus in the virus solution obtained above, add 1/4 volume of PEG6000/NaCl solution (25% PEG6000+4.4% NaCl) to the virus solution, mix well, and let stand at 4°C for 1 hour; (2) ) at 4°C, 5000rpm for 30 minutes; (3) Discard the supernatant, add 2ml of virus lysis solution (10mM Tris-HCl (pH8.0), 2mM MgCl ₂ , 5% sucrose) to dissolve the lentivirus pellet, and at -80 Store at ℃ to obtain a lentivirus concentrate; (4) measure the virus titer, inoculate BHK21 cells to a 24-well plate in advance, 10 ⁵ /well, and place in a 37 ℃ 5% CO ₂ cell incubator for 18 hours; (5) dissolve Virus, prepare a 10-fold dilution of virus samples from 10 ^-2 to 10 ^-7 ; (6) remove the cell culture medium, add cell culture medium containing different amounts of virus, and add 6ug/ml polybrene to the culture medium (polybrene), placed in a 37°C 5% CO ₂ cell incubator for 2 hours; (7) Removed the cell culture medium, added 1% FBS DMEM medium, and placed it in a 37° C 5% CO ₂ cell incubator for 48 hours (8) Remove the cell culture medium, add DMEM medium containing 2ug/ml puromycin and 1% FBS, and place it in a 37°C 5% CO ₂ cell incubator for 72 hours; (9) Remove the cell culture medium and add Crystal violet staining solution, count the number of stained clones, and calculate the virus titer; (10) Dilute the virus to 10 ⁷ TU/ml and store at -80°C.

3. Isolation and culture of T cells and lentiviral transduction of T cells

1. Use Ficoll density gradient centrifugation to obtain human PBMC cells; 2. Use T cell separation reagent of MiltenyiBiotec to separate T lymphocytes from human PBMC cells; 3. Add CD3/CD28 magnetic beads to the cells, The cells were cultured in RPMI-1640 medium for 24 hours; 4. The cells were transferred to RetroNectin pre-coated MOI=10 lentivirus (with lentivirus concentrate) culture flask, placed at 37°C, 5% CO _2. Culture in a cell incubator, change the medium after 24 hours, and continuously add IL-2 200IU/ml. Different viral fluids were used to obtain different chimeric antigen receptor-modified T cells that could express targeting human BCMA antigens; they were named TSN1-CAR-T, TSN2-CAR-T, TSN3-CAR-T, TSN5- CAR-T, TSN6-CAR-T, TSN7-CAR-T, TSN8-CAR-T and TSN9-CAR-T cells; 5. Flow cytometry to detect the expression of CAR on the surface of T cells.

Example 1

This embodiment provides a preparation method for targeting BCMA tumor antigen receptor-modified T cells (TSN7-CAR-T), including the following steps:

(1) Obtaining the nucleic acid sequence fragment TSN7-CAR targeting the BCMA chimeric antigen receptor by PCR, specifically: synthesizing CD8 signal peptide, anti-BCMA single-chain antibody TSN7 (the nucleotide encoding the TSN7) in turn The sequence is shown in SEQ ID NO.9, the amino acid sequence of the TSN7 is shown in SEQ ID NO.1), CD8 hinge region and transmembrane region, 4-1BB costimulatory signal domain, CD3ζ intracellular signal domain, T2A and EGFR The nucleotide sequences of the extracellular domain III and extracellular domain IV fragments of the It is TSN7-CAR, the full-length nucleotide sequence is shown in SEQ ID NO.16, and the amino acid sequence is shown in SEQ ID NO.18;

(2) Clone the TSN7-CAR expression cassette sequence into the pLVKn lentiviral vector plasmid to obtain the expression plasmid pLVKn-TSN7-CAR; the map is shown in Figure 2; the operation method is detailed in "Clone the above TSNs-CAR expression cassette sequence" Constructed into the pLVKn lentiviral vector to obtain the pLVKn-TSNs-CAR expression vector" as described in this part;

(3) The expression plasmid pLVKn-TSN7-CAR described in step (2) was transfected into 293T cells, and after packaging and concentration, a lentivirus concentrate was obtained; for the operation method, please refer to "Packaging, concentration and titer determination of lentivirus" ” records in that part;

(4) Infect T cells with the lentivirus concentrate to obtain the BCMA tumor antigen receptor-modified T cells (TSN7-CAR-T). For details of the operation method, please refer to "Isolation and Culture of T Cells and Lentivirus" Transducing T Cells" section.

Experimental example

1. To verify the ability of tumor antigen receptor-modified T cells (CAR-T) targeting BCMA to kill BCMA target cells in vitro

T cells (TSN1-CAR-T, TSN2-CAR-T, TSN3-CAR-T, TSN5-CAR-T, TSN6-CAR-T, TSN7-CAR-T, TSN7-CAR-T, TSN7-CAR-T, TSN7-CAR-T, TSN7-CAR-T, TSN7-CAR-T, TSN7-CAR-T, TSN7-CAR-T , TSN8-CAR-T and TSN9-CAR-T) and LAMA84 (BCMA expression negative) and KMS11 (BCMA expression positive) cell count, adjust the cell density to 1 × 10 ⁶ /ml, according to the effect-target ratio of 10:1, 20 :1, 30:1 co-culture. After 4 h of co-culture, cells were stained with Annexin V and PI kits, and a control group was set at the same time. Two control groups were set up. The first control group added target cells but also added uninfected CAR T cells (UNT), and the second control group added target cells but also added cells infected with EGFR ectodomain III and cells. Viral T cells (CON) of the ectodomain IV fragment. Cell killing was detected using flow cytometry. The results are shown in Figure 3, where TSN1 is TSN1-CAR-T cells, TSN2 is TSN2-CAR-T cells; TSN3 is TSN3-CAR-T cells, TSN5 is TSN5-CAR-T cells, and TSN6 is TSN6-CAR-T cells -T cells, TSN7 is TSN7-CAR-T cells, TSN8 is TSN8-CAR-T cells, TSN9 is TSN9-CAR-T cells; UNT is untransfected CAR-T cells as control; CON is only transfected with EGFR ectodomain III and ectodomain IV fragments of virus T cells; LAMA84 is a BCMA expression-negative cell; KMS11 is a BCMA expression-positive cell. As can be seen from Figure 3, although UNT and CON transfected cells have non-specific killing, but TSNs-CAR-T (where s represents 1, 2, 3, 5, 6, 7, 8, 9) cells have no effect on KMS11 cells. It shows a stronger killing effect, and the killing effect becomes larger with the increase of the effect-target ratio.

The TNS1-CAR-T, TNS2-CAR-T, TNS5-CAR-T, TNS6-CAR-T, TNS7-CAR-T, TNS8-CAR-T, TNS9-CAR-T cells prepared by the present invention can also kill specifically Myeloma cells with low BCMA expression. Take T cells 9 days after infection (TNS1-CAR-T, TNS2-CAR-T, TNS5-CAR-T, TNS6-CAR-T, TNS7-CAR-T, TNS8-CAR-T, TNS9-CAR-T ) and AMO-1 cells (very low expression of BCMA), ARD (low expression of BCMA) and RPMI-8226 (low expression of BCMA), and the cell density was adjusted to 1×10 ⁶ /ml, according to the effective target Co-culture ratios 5:1, 10:1, 15:1, 20:1, 25:1. After 4 h of co-culture, cells were stained with Annexin V and PI kits, and cell killing was detected by flow cytometry. The results are shown in Figure 4, the TNS1-CAR-T, TNS2-CAR-T, TNS5-CAR-T, TNS6-CAR-T, TNS7-CAR-T, TNS8-CAR-T, TNS9-CAR prepared by the present invention -T cells can specifically kill BCMA low-expressing myeloma cells.

2. Detection of cytokine secretion ability of tumor antigen receptor-modified T cells (CAR-T) targeting BCMA to kill tumor cells

The specific operations are as follows:

(1) Take the prepared BCMA-targeting CAR-T cells, resuspend them in Lonza medium, and adjust the cell concentration to 1×10 ⁶ /mL; (2) Each well of the experimental group contains target cells (K562-CD269) 2 × 10 ⁵ cells, 2 × 10 ⁵ cells of CAR-T cells, and 200 μl of Lonza medium without IL-2, mixed well and added to a 96-well plate. At the same time, add the intracellular membrane kit BD Golgiplug (containing BFA, add 1 μl of BD Golgiplug to each 1 ml of cell culture medium), and after thorough mixing, incubate at 37°C for 5-6 hours; collect the cells as the experimental group; (3) Each Wash the cells once with 1 mL of PBS, centrifuge at 300G for 5 min, carefully aspirate or discard the supernatant; (4) After washing the cells with PBS, add 250 μl/EP tube fixation/permeabilization solution, and incubate at 4°C for 20 min; membrane rupture. Wash the cells twice with 1×BD permeabilization/prewash solution, 1mL/time; (5) For intracellular factor staining, take an appropriate amount of cytokine fluorescent antibody, and dilute to 50μl with BD permeabilization/prewash solution; use this antibody The diluent was used to fully resuspend the fixed permeabilized cells, incubate at 4°C for 30 min in the dark, wash the cells twice with 1 mL/time of 1×BD permeation membrane/prewash, and then resuspend with PBS. (6) Detection by flow cytometry. The results are shown in Figures 5A and 5B, TSNs-CAR-T cells co-cultured with BCMA-positive cells produced effector cytokines, and the relative contents of secreted cytokines IL-2, IFN-γ, and TNF-α were relatively high.

3. To verify the ability of BCMA-targeting CAR-T cells to inhibit the growth of BCMA-positive tumors in a mouse subcutaneous tumor model

Using 6-week-old NSG immunodeficient mice, ³ ×10 ⁶ KMS11 cell lines were injected subcutaneously, and the tumors were allowed to grow for about 15 days. The size of the tumor was measured with a caliper. where V represents the tumor volume; L represents the longest diameter of the tumor mass; W represents the longest transverse diameter perpendicular to the longest diameter. When the tumor size reached 20-50 mm, 5 x 10 ⁶ control T cells (CON group and PBS group) or 5 x 10 ⁶ CAR-positive BCMA-targeting CAR-T cells were injected via tail vein: TSN1 ^- CAR -T, TSN2-CAR-T, TSN3-CAR-T and TSN7-CAR-T, observe and record the changes in tumor volume. The tumor growth curve is shown in Figure 6. In the KMS11 subcutaneous tumor model, infusion of TSN7-CAR-T cells produced a strong antitumor effect and completely eliminated the subcutaneous tumor. Infusion of TSN1-CAR, TSN2-CAR, and TSN3-CAR cells had no significant difference in tumor inhibitory effect compared with infusion of CON cells, and compared with the PBS group, there was a slight anti-tumor effect.

Thirty-one days after the injection of the BCMA-targeting CAR-T cells TSN7-CAR-T, the mice were confirmed by in vivo fluorescence imaging that 3 of the 5 mice had complete tumor eradication, and 2 mice had small residual tumors. Among them, the No. 2 mouse (the ventral image in Figure 7, the second mouse from the top) lost weight significantly and had the possibility of GVHD. After being sacrificed and dissected, it was found that no visible tumor mass was found at the original tumor inoculation site. The results are shown in the figure. 7 is shown.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited to this. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention. should be included within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

SEQUENCE LISTING

<110> Pu Shimai Biotechnology (Beijing) Co., Ltd.

<120> Preparation method and application of modified T cells targeting BCMA tumor antigen receptor

<130> 2010

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<170> PatentIn version 3.3

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Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly

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Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala

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Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser

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Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr

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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 4

<211> 42

<212> PRT

<213> Artificial sequences

<400> 4

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 5

<211> 113

<212> PRT

<213> Artificial sequences

<400> 5

Pro Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 6

<211> 335

<212> PRT

<213> Artificial sequences

<400> 6

Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu

1 5 10 15

Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile

20 25 30

Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe

35 40 45

Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr

50 55 60

Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn

65 70 75 80

Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg

85 90 95

Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile

100 105 110

Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val

115 120 125

Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp

130 135 140

Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn

145 150 155 160

Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu

165 170 175

Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser

180 185 190

Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu

195 200 205

Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln

210 215 220

Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly

225 230 235 240

Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro

245 250 255

His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr

260 265 270

Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His

275 280 285

Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro

290 295 300

Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala

305 310 315 320

Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met

325 330 335

<210> 7

<211> 25

<212> PRT

<213> Artificial sequences

<400> 7

Leu Glu Ser Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

1 5 10 15

Asp Val Glu Glu Asn Pro Gly Pro Arg

20 25

<210> 8

<211> 94

<212> PRT

<213> Artificial sequences

<400> 8

Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn

1 5 10 15

Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly

20 25 30

Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro Ser Ser Ser Phe

35 40 45

Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu

50 55 60

Arg Asp Lys Val Thr Gln Leu Leu Pro Leu Asn Thr Asp Ala Tyr Leu

65 70 75 80

Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr His Leu Val

85 90

<210> 9

<211> 729

<212> DNA

<213> Artificial sequences

<400> 9

caaattcagc tggtccagag cggacctgag ctgaaaaaac ccggcgagac tgttaagatc 60

agttgtaaag catctggcta taccttcacc gactacagca taaattgggt gaaacgggcc 120

cctggaaagg gcctcaaatg gatgggttgg atcaataccg aaactaggga gcctgcttat 180

gcatatgact tccgcgggag attcgccttt tcactcgaga catctgcctc tactgcttac 240

ctccaaataa acaacctcaa gtatgaagat acagccactt acttttgcgc cctcgactat 300

agttacgcca tggactactg gggacaggga acctccgtta ccgtcagttc cggtggcggt 360

ggctcgggcg gtggtgggtc gggtggcggc ggatctgaca ttgtgctcac tcagtcacct 420

cccagcctgg ccatgagcct gggaaaaagg gccaccatct cctgtagagc cagtgagtcc 480

gtcacaatct tggggagcca tcttattcac tggtatcagc agaagcccgg gcagcctcca 540

acccttctta ttcagctcgc gtcaaacgtc cagacgggtg tacctgccag attttctggt 600

agcgggtccc gcactgattt tacactgacc atagatccag tggaagaaga cgatgtggcc 660

gtgtattatt gtctgcagag cagaacgatt cctcgcacat ttggtggggg tactaagctg 720

gagattaag 729

<210> 10

<211> 63

<212> DNA

<213> Artificial sequences

<400> 10

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 11

<211> 207

<212> DNA

<213> Artificial sequences

<400> 11

accactaccc cagcaccgag gccacccacc ccggctccta caattgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatcta catttgggcc cctctggctg gtacttgcgg ggtcctgctg 180

ctttcactcg tgatcactct ttactgt 207

<210> 12

<211> 126

<212> DNA

<213> Artificial sequences

<400> 12

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 60

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 120

gaactg 126

<210> 13

<211> 336

<212> DNA

<213> Artificial sequences

<400> 13

cgcgtgaaat tcagccgcag cgcagatgct ccagcctacc agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

<210> 14

<211> 1008

<212> DNA

<213> Artificial sequences

<400> 14

cgcaaagtgt gtaacggaat aggtattggt gaatttaaag actcactctc cataaatgct 60

acgaatatta aacacttcaa aaactgcacc tccatcagtg gcgatctcca catcctgccg 120

gtggcattta ggggtgactc cttcacacat actcctcctc tggatccaca ggaactggat 180

attctgaaaa ccgtaaagga aatcacaggg tttttgctga ttcaggcttg gcctgaaaac 240

aggacggacc tccatgcctt tgagaaccta gaaatcatac gcggcaggac caagcaacat 300

ggtcagtttt ctcttgcagt cgtcagcctg aacataacat ccttgggatt acgctccctc 360

aaggagataa gtgatggaga tgtgataatt tcaggaaaca aaaatttgtg ctatgcaaat 420

acaataaact ggaaaaaact gtttgggacc tccggtcaga aaaccaaaat tataagcaac 480

agaggtgaaa acagctgcaa ggccacaggc caggtctgcc atgccttgtg ctcccccgag 540

ggctgctggg gcccggagcc cagggactgc gtctcttgcc ggaatgtcag ccgaggcagg 600

gaatgcgtgg acaagtgcaa ccttctggag ggtgagccaa gggagtttgt ggagaactct 660

gagtgcatac agtgccaccc agagtgcctg cctcaggcca tgaacatcac ctgcacagga 720

cggggaccag acaactgtat ccagtgtgcc cactacattg acggccccca ctgcgtcaag 780

acctgcccgg caggagtcat gggagaaaac aacaccctgg tctggaagta cgcagacgcc 840

ggccatgtgt gccacctgtg ccatccaaac tgcacctacg gatgcactgg gccaggtctt 900

gaaggctgtc caacgaatgg gcctaagatc ccgtccatcg ccactgggat ggtgggggcc 960

ctcctcttgc tgctggtggt ggccctgggg atcggcctct tcatgtga 1008

<210> 15

<211> 75

<212> DNA

<213> Artificial sequences

<400> 15

ctcgagagtg gaagcggcga gggcagagga agtcttctaa catgcggtga cgtggaggag 60

aatcccggcc ctagg 75

<210> 16

<211> 2619

<212> DNA

<213> Artificial sequences

<400> 16

ggatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60

gccaggccgc aaattcagct ggtccagagc ggacctgagc tgaaaaaacc cggcgagact 120

gttaagatca gttgtaaagc atctggctat accttcaccg actacagcat aaattgggtg 180

aaacggggccc ctggaaaggg cctcaaatgg atgggttgga tcaataccga aactagggag 240

cctgcttatg catatgactt ccgcgggaga ttcgcctttt cactcgagac atctgcctct 300

actgcttacc tccaaataaa caacctcaag tatgaagata cagccactta cttttgcgcc 360

ctcgactata gttacgccat ggactactgg ggacagggaa cctccgttac cgtcagttcc 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgacat tgtgctcact 480

cagtcacctc ccagcctggc catgagcctg ggaaaaaggg ccaccatctc ctgtagagcc 540

agtgagtccg tcacaatctt ggggagccat cttattcact ggtatcagca gaagcccggg 600

cagcctccaa cccttcttat tcagctcgcg tcaaacgtcc agacgggtgt acctgccaga 660

ttttctggta gcgggtcccg cactgatttt acactgacca tagatccagt ggaagaagac 720

gatgtggccg tgtattattg tctgcagagc agaacgattc ctcgcacatt tggtgggggt 780

actaagctgg agattaagac cactacccca gcaccgaggc cacccacccc ggctcctaca 840

attgcctccc agcctctgtc cctgcgtccg gaggcatgta gacccgcagc tggtggggcc 900

gtgcataccc ggggtcttga cttcgcctgc gatatctaca tttgggcccc tctggctggt 960

acttgcgggg tcctgctgct ttcactcgtg atcactcttt actgtaagcg cggtcggaag 1020

aagctgctgt acatctttaa gcaacccttc atgaggcctg tgcagactac tcaagaggag 1080

gacggctgtt catgccggtt cccagaggag gaggaaggcg gctgcgaact gcgcgtgaaa 1140

ttcagccgca gcgcagatgc tccagcctac cagcaggggc agaaccagct ctacaacgaa 1200

ctcaatcttg gtcggagaga ggagtacgac gtgctggaca agcggagagg acgggaccca 1260

gaaatgggcg ggaagccgcg cagaaagaat ccccaagagg gcctgtacaa cgagctccaa 1320

aaggataaga tggcagaagc ctatagcgag attggtatga aaggggaacg cagaagaggc 1380

aaaggccacg acggactgta ccagggactc agcaccgcca ccaaggacac ctatgacgct 1440

cttcacatgc aggccctgcc gcctcggctc gagagtggaa gcggcgaggg cagaggaagt 1500

cttctaacat gcggtgacgt ggaggagaat cccggcccta ggatggcctt accagtgacc 1560

gccttgctcc tgccgctggc cttgctgctc cacgccgcca ggccgcgcaa agtgtgtaac 1620

ggaataggta ttggtgaatt taaagactca ctctccataa atgctacgaa tattaaacac 1680

ttcaaaaact gcacctccat cagtggcgat ctccacatcc tgccggtggc atttaggggt 1740

gactccttca cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta 1800

aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac ggacctccat 1860

gcctttgaga acctagaaat catacgcggc aggaccaagc aacatggtca gttttctctt 1920

gcagtcgtca gcctgaacat aacatccttg ggattacgct ccctcaagga gataagtgat 1980

ggagatgtga taatttcagg aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 2040

aaactgtttg ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc 2100

tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg ctggggcccg 2160

gagcccaggg actgcgtctc ttgccggaat gtcagccgag gcagggaatg cgtggacaag 2220

tgcaaccttc tggagggtga gccaagggag tttgtggaga actctgagtg catacagtgc 2280

cacccagagt gcctgcctca ggccatgaac atcacctgca caggacgggg accagacaac 2340

tgtatccagt gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga 2400

gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca tgtgtgccac 2460

ctgtgccatc caaactgcac ctacggatgc actgggccag gtcttgaagg ctgtccaacg 2520

aatgggccta agatcccgtc catcgccact gggatggtgg gggccctcct cttgctgctg 2580

gtggtggccc tggggatcgg cctcttcatg tgagtcgac 2619

<210> 17

<211> 282

<212> DNA

<213> Artificial sequences

<400> 17

aactgcagga acaccgggcc atggctgaag aaggtcctga agtgtaacac cccagacccc 60

tcgaagttct tttcccagct gagctcagag catggaggag acgtccagaa gtggctctct 120

tcgcccttcc cctcatcgtc cttcagccct ggcggcctgg cacctgagat ctcgccacta 180

gaagtgctgg agagggacaa ggtgacgcag ctgctccccc tgaacactga tgcctacttg 240

tccctccaag aactccaggg tcaggaccca actcacttgg tg 282

<210> 18

<211> 868

<212> PRT

<213> Artificial sequences

<400> 18

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu

20 25 30

Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr

35 40 45

Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys

50 55 60

Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala

65 70 75 80

Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser

85 90 95

Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr

100 105 110

Ala Thr Tyr Phe Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser

145 150 155 160

Pro Pro Ser Leu Ala Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys

165 170 175

Arg Ala Ser Glu Ser Val Thr Ile Leu Gly Ser His Leu Ile His Trp

180 185 190

Tyr Gln Gln Lys Pro Gly Gln Pro Pro Thr Leu Leu Ile Gln Leu Ala

195 200 205

Ser Asn Val Gln Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser

210 215 220

Arg Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val

225 230 235 240

Ala Val Tyr Tyr Cys Leu Gln Ser Arg Thr Ile Pro Arg Thr Phe Gly

245 250 255

Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro

260 265 270

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

275 280 285

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

305 310 315 320

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

325 330 335

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

340 345 350

Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu

355 360 365

Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp

370 375 380

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

385 390 395 400

Leu Gly Arg Arg Glu Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

405 410 415

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

420 425 430

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

435 440 445

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

450 455 460

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

465 470 475 480

Met Gln Ala Leu Pro Pro Arg Leu Glu Ser Gly Ser Gly Glu Gly Arg

485 490 495

Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Arg

500 505 510

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

515 520 525

His Ala Ala Arg Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu

530 535 540

Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys

545 550 555 560

Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe

565 570 575

Arg Gly Asp Ser Phe Thr His Thr Pro Leu Asp Pro Gln Glu Leu

580 585 590

Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln

595 600 605

Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu

610 615 620

Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val

625 630 635 640

Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile

645 650 655

Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala

660 665 670

Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr

675 680 685

Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln

690 695 700

Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro

705 710 715 720

Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val

725 730 735

Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn

740 745 750

Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn

755 760 765

Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His

770 775 780

Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met

785 790 795 800

Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val

805 810 815

Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly

820 825 830

Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr

835 840 845

Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile

850 855 860

Gly Leu Phe Met

865

Claims (10)

1. a chimeric antigen receptor targeting BCMA is characterized in that, the structure comprises the signal peptide, BCMA antigen recognition region, hinge region, transmembrane region, intracellular signal domain and the extracellular domain III of EGFR successively connected in series and fragments of extracellular domain IV. 2. the chimeric antigen receptor of targeting BCMA according to claim 1, is characterized in that, described BCMA antigen recognition region is the single chain antibody of anti-BCMA; There are 6 kinds of single-chain antibodies against BCMA, namely TSN1, TSN3, TSN5, TSN7, TSN8 and TSN9; Wherein, the amino acid sequence of the TSN7 is shown in SEQ ID NO.1; The signal peptide is a CD8 signal peptide, and the amino acid sequence of the CD8 signal peptide is shown in SEQ ID NO. 2; the hinge region and the transmembrane region are the CD8 hinge region and the transmembrane region, and the CD8 hinge region and The amino acid sequence of the transmembrane region is shown in SEQ ID NO.3; the intracellular signal domain is CD3ζ intracellular signal domain, and the amino acid sequence of the CD3ζ intracellular signal domain is shown in SEQ ID NO.5; the EGFR The amino acid sequences of the ectodomain III and ectodomain IV fragments are shown in SEQ ID NO.6. 3. The chimeric antigen receptor targeting BCMA according to claim 1, wherein a costimulatory signal domain is also connected between the transmembrane region and the intracellular signal domain; the costimulatory signal The domain is a 4-1BB costimulatory signal domain, and the amino acid sequence of the 4-1BB costimulatory signal domain is shown in SEQ ID NO. 4; a T2A fragment is also connected between the intracellular signal domain and the fragment of the EGFR, The amino acid sequence of the T2A fragment is shown in SEQ ID NO.7. 4. the chimeric antigen receptor of targeting BCMA according to claim 3, is characterized in that, described costimulatory signal domain also adds IL2RB stimulation signal domain, and the aminoacid sequence of described IL2RB stimulation signal domain is such as SEQ ID NO. 8 shown. 5. A nucleic acid molecule, wherein the nucleic acid molecule encodes the BCMA-targeting chimeric antigen receptor according to any one of claims 1-5. 6. The nucleic acid molecule according to claim 5, wherein the nucleotide sequence encoding the TSN7 is shown in SEQ ID NO.9; The nucleotide sequence encoding the CD8 signal peptide is shown in SEQ ID NO.10; the nucleotide sequence encoding the CD8 hinge region and transmembrane region is shown in SEQ ID NO.11; The nucleotide sequence encoding the 4-1BB costimulatory signal domain is shown in SEQ ID NO.12; the nucleotide sequence encoding the CD3ζ intracellular signal domain is shown in SEQ ID NO.13; The nucleotide sequences encoding the EGFR extracellular domain III and extracellular domain IV fragments are shown in SEQ ID NO.14; The nucleotide sequence encoding the T2A fragment is shown in SEQ ID NO.15; The nucleotide sequence encoding the IL2RB stimulatory signal domain is shown in SEQ ID NO.17. 7. A recombinant vector, characterized in that the recombinant vector contains the nucleic acid molecule described in claim 6 or 7. 8. A genetically modified T cell, characterized in that the T cell contains the recombinant vector of claim 7 or the chromosome of the T cell is integrated with the nucleic acid molecule of claim 5 or an exogenous nucleic acid molecule or The chimeric antigen receptor of claim 1 is expressed. 9. a preparation method of the described targeting BCMA tumor antigen receptor modified T cell of claim 8, is characterized in that, comprises the steps: (1) obtaining the nucleic acid sequence fragment targeting the BCMA chimeric antigen receptor by PCR method; (2) clone the nucleic acid sequence fragment of described targeting BCMA chimeric antigen receptor into the carrier plasmid, obtain the expression plasmid of TSNs-CAR; (3) transfecting the expression plasmid of the TSNs-CAR described in step (2) into 293T cells, and after packaging and concentrating, a lentivirus concentrate is obtained; (4) Infecting T cells with the lentivirus concentrate to obtain the T cells modified with the BCMA tumor antigen receptor. 10. The chimeric antigen receptor targeting BCMA according to any one of claims 1-4, the nucleic acid molecule according to claim 5 or 6, the recombinant vector according to claim 7, and the target according to claim 8 Use of T cells modified with BCMA tumor antigen receptors in the preparation of drugs for preventing and/or treating cancer or tumors.

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