CN112251412B

CN112251412B - BCMA (brain cell activating antigen) targeted chimeric antigen receptor T cell and application thereof

Info

Publication number: CN112251412B
Application number: CN202011085552.9A
Authority: CN
Inventors: 汤朝阳; 请求不公布姓名; 秦乐; 吴迪; 冯世忠; 冯嘉昆; 王艳艳
Original assignee: Individual
Current assignee: Tang Chaoyang
Priority date: 2020-10-12
Filing date: 2020-10-12
Publication date: 2023-03-28
Anticipated expiration: 2040-10-12
Also published as: CN112251412A

Abstract

The invention provides a BCMA-targeted chimeric antigen receptor T cell and application thereof, wherein the chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain and a signal transduction domain; the antigen binding domain comprises an anti-BCMA single chain antibody; the signaling domains include 4-1BB, CD3 zeta, and TLR2. The chimeric antigen receptor T cell constructed by adopting the 4-1BB, the CD3 zeta and the TLR2 which are connected in series as a signal transduction structural domain of the chimeric antigen receptor and matching with a specific antigen binding structural domain anti-BCMA single-chain antibody has the advantages of remarkably enhanced capability of specifically recognizing and killing BCMA positive tumor cells, high safety and good clinical application prospect in the field of B cell malignant tumor treatment.

Description

BCMA (brain cell activating antigen) targeted chimeric antigen receptor T cell and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a BCMA (B cell-binding antigen receptor) targeted chimeric antigen receptor T cell and application thereof.

Background

A Chimeric Antigen Receptor (CAR) is a recombinant receptor targeting a cell surface antigen, and mainly comprises an extracellular antigen binding region, a transmembrane region and an intracellular signal region. Wherein the extracellular antigen-binding region is responsible for recognizing an antigen, the transmembrane region connects the extracellular antigen-binding region and an intracellular signal region, which affects the expression ability of the introduced CAR gene, and the intracellular signal region is responsible for conducting a signal.

Chimeric antigen receptor T cells (CAR-T) recognize and bind to tumor antigens using CAR molecules, thereby exerting a specific killing effect of T cells on tumor cells, have been developed into mature tumor immunotherapy methods, and have achieved remarkable performance in treating hematological malignancies.

B Cell Maturation Antigen (BCMA), expressed on mature B cells and plasma cells, is a biomarker for the diagnosis and prognosis of Multiple Myeloma (MM). The expression levels of BCMA at different stages of multiple myeloma were found to be similar, indicating that BCMA may be a potent therapeutic target throughout the multiple myeloma process. In the prior art, BCMA antigen is used as an extracellular antigen binding region to construct CAR molecules for treating B cell related diseases such as multiple myeloma, but the prior art has the problems of easy initiation of cytokine storm and the like.

Therefore, there is a need to further optimize the molecular structure of BCMA-targeted CARs for use in the field of multiple myeloma treatment.

Disclosure of Invention

Aiming at the defects and actual needs of the prior art, the invention provides a BCMA-targeted chimeric antigen receptor T cell and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a chimeric antigen receptor T cell targeting BCMA, the chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a transmembrane domain, and a signaling domain;

the antigen binding domain comprises an anti-BCMA single chain antibody;

the signaling domains include 4-1BB, CD3 zeta, and TLR2.

According to the invention, toll-like receptor 2 (TLR 2) is connected to the C-terminal of 4-1BB and CD3 zeta to serve as a signal transduction domain, and the signal transduction domain is matched with an anti-BCMA single-chain antibody to construct a chimeric antigen receptor which efficiently targets BCMA antigen, and T cells expressing the chimeric antigen receptor have specific killing effect on BCMA positive tumors, so that the killing effect is obvious, and the killing efficiency is high.

Preferably, the signal peptide comprises a GM-CSF signal peptide.

Preferably, the transmembrane domain comprises CD28 and/or CD8 α, preferably CD8 α.

Preferably, the chimeric antigen receptor consists of a GM-CSF signal peptide, an anti-BCMA single chain antibody, CD8 α, 4-1BB, CD3 ζ, and TLR2 in tandem.

Preferably, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：

MLLLVTSLLLCELPHPAFLLDIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRQAKRKPRKAPSRNICYDAFVSYSERDAYWVENLMVQELENFNPPFKLCLHKRDFIPGKWIIDNIIDSIEKSHKTVFVLSENFVKSEWCKYELDFSHFRLFDENNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS。

in a second aspect, the present invention provides a nucleic acid molecule comprising a gene encoding a GM-CSF signal peptide, a gene encoding an anti-BCMA single-chain antibody, a gene encoding CD8 α, a gene encoding 4-1BB, a gene encoding CD3 ζ and a gene encoding TLR2.

Preferably, the nucleic acid molecule comprises the nucleic acid sequence shown as SEQ ID NO. 2;

SEQ ID NO:2：

atgcttctcctggtgacaagccttctgctctgtgagttaccacacccagcattcctcctggacatcgtcctgacccagtcacctgccagcctggccgtcagcctgggcgagagagccaccattaattgccgcgctagcgaatcagtgtcagtcatcggcgctcacctgatccactggtatcagcaaaagcctgggcagccaccaaagctgctcatctatctcgcctccaacctggagacaggcgtgcctgctcgctttagtggttctggtagcggcaccgatttcaccctgactatttcaagcctgcaggcagaggacgcagccatatactattgcctgcagtcccggattttccctcgcacattcggccagggcacaaaactggaaatcaaaggatctacctccggctccgggaagcccggaagcggcgaaggctcaaccaaaggccaggttcagctcgtgcagtctggttctgaactgaagaaacccggtgcatctgtgaaagtctcctgcaaggcttccgggtatactttcacagactacagtattaattgggttcgccaagctcctggacagggactggagtggatgggatggataaacacagaaactagggagcctgcatacgcctatgatttcagaggtcgcttcgtgttcagtctggatacaagtgtttcaacagcctatctgcaaattagctccctgaaagccgaggacaccgcagtttactactgtgcacgggattattcttacgctatggactattgggggcagggcacactcgtgacagtgtctagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgccaggccaaaaggaagcccaggaaagctcccagcaggaacatctgctatgatgcatttgtttcttacagtgagcgggatgcctactgggtggagaaccttatggtccaggagctggagaacttcaatccccccttcaagttgtgtcttcataagcgggacttcattcctggcaagtggatcattgacaatatcattgactccattgaaaagagccacaaaactgtctttgtgctttctgaaaactttgtgaagagtgagtggtgcaagtatgaactggacttctcccatttccgtctttttgatgagaacaatgatgctgccattctcattcttctggagcccattgagaaaaaagccattccccagcgcttctgcaagctgcggaagataatgaacaccaagacctacctggagtggcccatggacgaggctcagcgggaaggattttgggtaaatctgagagctgcgataaagtcc。

in a third aspect, the present invention provides an expression vector which is a viral vector comprising the nucleic acid molecule of the second aspect.

Preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a lentiviral vector.

In a fourth aspect, the invention provides a recombinant lentivirus prepared by co-transfecting a mammalian cell with the expression vector of the third aspect and a packaging helper plasmid.

In a fifth aspect, the present invention provides a method for producing a chimeric antigen receptor T cell according to the first aspect, the method comprising the step of introducing the recombinant lentivirus according to the fourth aspect into a T cell.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor T cell of the first aspect.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a seventh aspect, the present invention provides a chimeric antigen receptor T cell of the first aspect, a nucleic acid molecule of the second aspect, an expression vector of the third aspect, a recombinant lentivirus of the fourth aspect, or a pharmaceutical composition of the sixth aspect, for use in the preparation of a medicament for the treatment of a B-cell malignancy.

Compared with the prior art, the invention has the following beneficial effects:

(1) The chimeric antigen receptor T cell constructed by adopting the 4-1BB, the CD3 zeta and the TLR2 which are connected in series as a signal transduction structural domain of the chimeric antigen receptor and matching with a specific antigen binding structural domain anti-BCMA single-chain antibody has the remarkably enhanced capacity of specifically recognizing a tumor antigen, and the anti-tumor capacity of the T cell is more remarkably improved;

(2) The chimeric antigen receptor T cell has low immunogenicity, high safety and low occurrence probability of side reaction, and has good clinical application prospect in the field of B cell malignant tumor treatment.

Drawings

FIG. 1 is a schematic diagram of a chimeric antigen receptor structure;

FIG. 2 shows the killing efficiency of different CAR-T cells against tumor cell K562-BCMA-GL at different E: T ratios;

FIG. 3 shows the secretion of IFN-. Gamma.by different CAR-T cells after co-culture with test cells.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and that no limitation of the invention is intended.

The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 preparation of CAR vectors

The structural schematic diagram of the chimeric antigen receptor targeting BCMA constructed in the embodiment is shown in figure 1, the amino acid sequence is shown in SEQ ID NO. 1, and the coding gene is shown in SEQ ID NO. 2;

respectively adding restriction enzyme Pme1 enzyme cutting site and protective base thereof, restriction enzyme Spe1 enzyme cutting site and protective base thereof at the C end and the N end of the coding gene;

the coding gene is subjected to double enzyme digestion by utilizing restriction enzymes Pme1 and Spe1, an enzyme digestion product containing a sticky end is obtained by agar gel electrophoresis recovery, and is connected into a linearized pWPXld-eGFP plasmid (containing the sticky end) which is also subjected to double enzyme digestion by Pme1 and Spe1, and the connection reaction is carried out in the presence of T4 DNA polymerase (Invitrogen company), so that a lentivirus vector containing the coding gene of the alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2 is obtained.

In this example, a lentiviral vector containing the gene encoding α BCMA scFv-4-1BB-CD3 ζ was constructed at the same time.

Example 2 recombinant lentivirus packaging

In this example, 293T cells were used to prepare recombinant lentiviruses, and when 293T cells were plated on 100mm petri dishes to the bottom of 80-90%, lentivirus packaging was performed:

2h before virus packaging, the culture medium is changed into DMEM containing 1% fetal calf serum, and the addition amount is 6mL/100mm culture dish;

preparing a plasmid mixed solution as shown in table 1, wherein pWPXld-expression plasmid is a lentiviral vector for expressing CAR molecules, and pWPXld-eGFP plasmid is an empty vector without CAR molecule encoding genes;

TABLE 1

Adding 36 μ g PEI into another 500 μ L opti-MEM medium, mixing, and standing at room temperature for 5min;

mixing the plasmid mixed solution shown in the table 1 with PEI, blowing, beating and uniformly mixing, and standing at room temperature for 25-30 min;

dropwise adding the mixed solution to 293T cells cultured in a 100mm culture dish;

after culturing for 6h, changing the culture medium into DMEM containing 1% fetal calf serum, and adding the DMEM into a culture dish with the volume of 7mL/100 mm;

collecting virus supernatant 24h, 48h and 72h after packaging, and simultaneously supplementing a culture medium to 293T cells, wherein the addition amount is 7mL/100mm culture dish;

1000g was centrifuged for 10min, filtered through a 0.45 μm filter to obtain recombinant lentiviruses expressing CAR, and stored at 4 ℃ for further use.

Example 3T cell activation and lentivirus transfection

Separating Peripheral Blood Mononuclear Cells (PBMC) from whole blood by using a Ficoll density gradient centrifugation kit (GE company), removing red blood cells, and then separating T cells by using MACS Pan-T magnetic beads;

the sorted T cells were diluted with medium (AIM-V medium +5% FBS + penicillin 100U/mL + streptomycin 0.1 mg/mL) to a cell concentration of 2.5X 10 ⁶ Per mL for standby;

t cells are activated by adopting a CD2/CD3/CD 28T cell activation and amplification kit (America whirlwind company), namely, coated magnetic beads are mixed with the T cells according to the proportion of 1 ⁶ Per mL/cm ² After mixing, the mixture was left at 37 ℃ and 5% CO ₂ Culturing and stimulating for 48h in an incubator;

after 48h of T cell activation, beads were removed, centrifuged at 300g for 5min, supernatant removed, T cells resuspended in fresh medium and CAR-expressing recombinant lentivirus or blank added separatelyControl lentivirus (MOI = 10), and adding 8. Mu.g/mL polybrene and 300IU/mL IL-2, at 37 ℃, 5% CO ₂ Culturing in an incubator;

after 24h, centrifuging for 5min at 300g, removing supernatant, and resuspending T cells with fresh culture medium containing 300IU/mL IL-2 to obtain CAR-T cells;

maintenance of CAR-T cell density at 1X 10 ⁶ About one/mL, half the liquid change is carried out every 2-3 days, and after two weeks, the number of CAR-T cells is expanded by 100 times.

CAR-T cells constructed in this example included α BCMA scFv-4-1BB-CD3 zeta-TLR 2-T and α BCMA scFv-4-1BB-CD3 zeta-T, while the WT control group (transfection blank lentivirus) was set.

Example 4 in vitro testing of the killing function of CAR-T cells on tumor cells K562-BCMA-GL

WT, alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2-T and alpha BCMA scFv-4-1BB-CD3 zeta-T prepared in example 3 were compared with 1X 10 ⁴ The individual tumor cells K562-BCMA-GL were mixed in the ratio E: T of 4, 1, 2, 1, 4, 1 ₂ Co-culturing for 18h in an incubator;

after 18h, adding 100 μ L/well Luciferase substrate (1 ×), resuspending and mixing the cells, immediately measuring RLU (relative light unit) by a multifunctional microplate reader for 1 second, using a Luciferase (Luciferase) quantitative killing efficiency evaluation method, comparing the killing effect of WT, alpha BCMA scFv-4-1BB-CD3 ζ -TLR2-T and alpha BCMA scFv-4-1BB-CD3 ζ -T on K562-BCMA-GL in vitro, wherein the killing ratio calculation formula is as follows:

100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)

The result is shown in figure 2, the in vitro killing efficiency of the alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2-T and the alpha BCMA scFv-4-1BB-CD3 zeta-T on K562-BCMA-GL is obviously higher than that of WT, the killing efficiency of the alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2-T is stronger, and the alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2-T can also show stronger tumor killing activity under the condition that the E: T is very small, namely the tumor target cells are far larger than effector T cells, which indicates that the TLR2 plays a role in expanding the extracellular region transmission signal and causing the cascade amplification effect of the lower-level cell killing effect.

Example 5

Respectively mixing K562 and K562-BCMA-GL according to 5 × 10 ⁵ Inoculating 24-well plates at the density of cells/well, adding WT, alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2-T and alpha BCMA scFv-4-1BB-CD3 zeta-T, and co-culturing in an incubator for 12h; and detecting the co-culture supernatant by adopting an IFN-gamma ELISA detection kit.

As shown in FIG. 3, the IFN-gamma cytokine level in the supernatant co-cultured with BCMA-CAR-T and K562-BCMA was significantly increased compared to that in the supernatant co-cultured with K562, wherein the IFN-gamma cytokine level in the supernatant co-cultured with alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2-T and K562-BCMA was higher than that in alpha BCMA scFv-4-1BB-CD3 zeta-T, indicating that the killing ability of alpha BCMA scFv-4-1BB-CD3 zeta-TLR 2-T was stronger.

In conclusion, the invention adopts the 4-1BB, the CD3 zeta and the TLR2 which are connected in series as the signal transduction structural domain of the chimeric antigen receptor, and the signal transduction structural domain is matched with the specific antigen binding structural domain to resist the BCMA single-chain antibody, so that the constructed chimeric antigen receptor T cell has the capability of remarkably enhancing the specificity recognition of the tumor antigen and the killing of the tumor cell, and has wide application prospect in the field of treatment of B cell malignant tumor.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangdong Shoutai in vivo biomedical science and technology Co., ltd

<120> BCMA-targeted chimeric antigen receptor T cell and application thereof

<130> 202009

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 648

<212> PRT

<213> Artificial sequence

<400> 1

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

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

20 25 30

Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser

35 40 45

Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr

65 70 75 80

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

85 90 95

Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr Tyr Cys

100 105 110

Leu Gln Ser Arg Ile Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu

115 120 125

Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu

130 135 140

Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu

145 150 155 160

Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Ser Tyr Ala Met Asp Tyr Trp

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Leu His Met Gln Ala Leu Pro Pro Arg Gln Ala Lys Arg Lys Pro Arg

485 490 495

Lys Ala Pro Ser Arg Asn Ile Cys Tyr Asp Ala Phe Val Ser Tyr Ser

500 505 510

Glu Arg Asp Ala Tyr Trp Val Glu Asn Leu Met Val Gln Glu Leu Glu

515 520 525

Asn Phe Asn Pro Pro Phe Lys Leu Cys Leu His Lys Arg Asp Phe Ile

530 535 540

Pro Gly Lys Trp Ile Ile Asp Asn Ile Ile Asp Ser Ile Glu Lys Ser

545 550 555 560

His Lys Thr Val Phe Val Leu Ser Glu Asn Phe Val Lys Ser Glu Trp

565 570 575

Cys Lys Tyr Glu Leu Asp Phe Ser His Phe Arg Leu Phe Asp Glu Asn

580 585 590

Asn Asp Ala Ala Ile Leu Ile Leu Leu Glu Pro Ile Glu Lys Lys Ala

595 600 605

Ile Pro Gln Arg Phe Cys Lys Leu Arg Lys Ile Met Asn Thr Lys Thr

610 615 620

Tyr Leu Glu Trp Pro Met Asp Glu Ala Gln Arg Glu Gly Phe Trp Val

625 630 635 640

Asn Leu Arg Ala Ala Ile Lys Ser

645

<210> 2

<211> 1944

<212> DNA

<213> Artificial sequence

<400> 2

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

gacatcgtcc tgacccagtc acctgccagc ctggccgtca gcctgggcga gagagccacc 120

attaattgcc gcgctagcga atcagtgtca gtcatcggcg ctcacctgat ccactggtat 180

cagcaaaagc ctgggcagcc accaaagctg ctcatctatc tcgcctccaa cctggagaca 240

ggcgtgcctg ctcgctttag tggttctggt agcggcaccg atttcaccct gactatttca 300

agcctgcagg cagaggacgc agccatatac tattgcctgc agtcccggat tttccctcgc 360

acattcggcc agggcacaaa actggaaatc aaaggatcta cctccggctc cgggaagccc 420

ggaagcggcg aaggctcaac caaaggccag gttcagctcg tgcagtctgg ttctgaactg 480

aagaaacccg gtgcatctgt gaaagtctcc tgcaaggctt ccgggtatac tttcacagac 540

tacagtatta attgggttcg ccaagctcct ggacagggac tggagtggat gggatggata 600

aacacagaaa ctagggagcc tgcatacgcc tatgatttca gaggtcgctt cgtgttcagt 660

ctggatacaa gtgtttcaac agcctatctg caaattagct ccctgaaagc cgaggacacc 720

gcagtttact actgtgcacg ggattattct tacgctatgg actattgggg gcagggcaca 780

ctcgtgacag tgtctagcac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgccag gccaaaagga agcccaggaa agctcccagc 1500

aggaacatct gctatgatgc atttgtttct tacagtgagc gggatgccta ctgggtggag 1560

aaccttatgg tccaggagct ggagaacttc aatcccccct tcaagttgtg tcttcataag 1620

cgggacttca ttcctggcaa gtggatcatt gacaatatca ttgactccat tgaaaagagc 1680

cacaaaactg tctttgtgct ttctgaaaac tttgtgaaga gtgagtggtg caagtatgaa 1740

ctggacttct cccatttccg tctttttgat gagaacaatg atgctgccat tctcattctt 1800

ctggagccca ttgagaaaaa agccattccc cagcgcttct gcaagctgcg gaagataatg 1860

aacaccaaga cctacctgga gtggcccatg gacgaggctc agcgggaagg attttgggta 1920

aatctgagag ctgcgataaa gtcc 1944

Claims

1. A chimeric antigen receptor T cell targeting BCMA, wherein said chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a transmembrane domain, and a signaling domain;

the antigen binding domain comprises an anti-BCMA single chain antibody;

the signaling domain comprises 4-1BB, CD3 ζ, and TLR2;

the signal peptide comprises a GM-CSF signal peptide;

the transmembrane domain is CD8 α;

the chimeric antigen receptor is formed by connecting GM-CSF signal peptide, anti-BCMA single-chain antibody, CD8 alpha, 4-1BB, CD3 zeta and TLR2 in series;

the chimeric antigen receptor is an amino acid sequence shown in SEQ ID NO. 1.

2. A nucleic acid molecule comprising a GM-CSF signal peptide-encoding gene, an anti-BCMA single chain antibody-encoding gene, a CD8 α -encoding gene, a 4-1 BB-encoding gene, a CD3 ζ -encoding gene, and a TLR 2-encoding gene;

the nucleic acid molecule is a nucleic acid sequence shown as SEQ ID NO. 2.

3. An expression vector which is a viral vector comprising the nucleic acid molecule of claim 2.

4. The expression vector of claim 3, wherein the viral vector comprises any one of a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.

5. The expression vector of claim 4, wherein the viral vector is a lentiviral vector.

6. A recombinant lentivirus prepared by co-transfecting a mammalian cell with the expression vector of any one of claims 3 to 5 and a packaging helper plasmid.

7. A method for producing the chimeric antigen receptor T cell according to claim 1, which comprises the step of introducing the recombinant lentivirus according to claim 6 into a T cell.

8. A pharmaceutical composition comprising the chimeric antigen receptor T cell of claim 1.

9. The pharmaceutical composition of claim 8, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

10. Use of the chimeric antigen receptor T cell of claim 1, the nucleic acid molecule of claim 2, the expression vector of any one of claims 3-5, the recombinant lentivirus of claim 6, or the pharmaceutical composition of claim 8 or 9 for the preparation of a medicament for the treatment of a B-cell malignancy.