CN111925451B

CN111925451B - BCMA (brain cell activating antigen) -targeted Chimeric Antigen Receptor (CAR) and application thereof

Info

Publication number: CN111925451B
Application number: CN202011081141.2A
Authority: CN
Inventors: 郑培朵; 郑玉婷; 刘欢; 杨洋
Original assignee: Youren Cell Engineering Zhejiang Co ltd
Current assignee: Youren cell engineering (Zhejiang) Co.,Ltd.
Priority date: 2020-10-12
Filing date: 2020-10-12
Publication date: 2021-01-29
Anticipated expiration: 2040-10-12
Also published as: CN111925451A

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to a BCMA-targeted Chimeric Antigen Receptor (CAR) and application thereof, wherein the BCMA-targeted chimeric antigen receptor takes a BCMA-targeted single-domain antibody as an antigen binding domain and is prepared by adjusting CD4 in the process of preparing CAR-T cells⁺And CD8⁺The proportion of the T cells is optimized, the obtained novel CAR-T cells can effectively kill tumor cells, and cytokines such as IFN-gamma, IL-10, IL-6 and the like are controlled within an acceptable range, so that the novel CAR-T cells have a good clinical application prospect.

Description

BCMA (brain cell activating antigen) -targeted Chimeric Antigen Receptor (CAR) and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a BCMA-targeted Chimeric Antigen Receptor (CAR) and application thereof.

Background

Statistically, millions of patients are lost each year due to malignancies, one of the most threatening human health, and their associated diseases, while Multiple Myeloma (MM), the second most common hematologic malignancy after non-hodgkin lymphoma, accounts for about 1% of neoplastic diseases, about 13% of hematologic malignancies, with a prevalence varying from 1 to 4 out of every 10 million people, with a higher incidence in men than women. Multiple myeloma has a short survival time, and if no treatment is given, the median survival time of advanced MM patients is only 6 months, while even if chemotherapy or the like is given, the median survival time does not exceed 3 years, and only one-fourth of patients can survive more than 5 years, so MM is still considered to be an incurable disease until now.

Researchers developed a variety of novel therapies for multiple myeloma in order to effectively inhibit the occurrence and development of MM, and also achieved some positive therapeutic effects, wherein the technology of chimeric antigen receptor T cells (CAR-T) became one of the research hotspots in the present year. The technology is a novel cellular immunotherapy starting from the last 90 years, the treatment process mainly comprises the steps of introducing nucleic acid fused with CAR gene into the genome of autologous or allogeneic T lymphocytes to construct CAR-T cells, then infusing the cells back into a patient body, and treating related diseases by activating immune response, and the classical structure of the chimeric antigen receptor comprises an antigen binding domain, a transmembrane region, a costimulatory factor and an intracellular signal domain, wherein the antigen binding domain is used for recognizing target tumor cells, and the rest elements assist CAR-T cells to activate and play an anti-tumor role. Early CAR-T cell research focused on BCMA-targeted second generation CAR-T cells, which generally included a targeting BCMA antigen binding domain, transmembrane region, CD28 or 4-1BB co-stimulatory factor, and CD3 zeta intracellular signaling domain, and, because of their surprising efficacy in leukemia, lymphoma, and myeloma, received high attention from researchers and pharmaceutical companies, developing a number of similar therapeutic cells and therapies, of which kymeriah, gilichata, of norward corporation has been approved for marketing, and several scientific and pharmaceutical companies of our country have also developed similar products.

B Cell Maturative Antigen (BCMA), also known as TNF ligand superfamily member 17 (TNFRSF 17), is mainly expressed in plasma cells and mature B lymphocytes and is essentially undetectable in other normal human tissues. In clinical diagnosis and treatment, BCMA is one of the most selectively expressed receptors on MM cell lines, and the expression level of BCMA is gradually increased along with the disease progress of multiple myeloma. Therefore, the BCMA can be used as an important prognosis and monitoring tool for MM patients and is an ideal antigen target, and various BCMA-based immunotherapies have been developed, such as CN111333732A, CN110945026A, WO2017021450a1, WO2016087531a1 and the like which disclose antibodies targeting BCMA, and researchers have proposed chimeric antigen receptors targeting BCMA, for example, CN109153731A discloses a chimeric antigen receptor targeting BCMA, CN109134665A discloses a chimeric antigen receptor based on single domain antibodies, and WO2019195017a1 discloses a CAR-T cell targeting BCMA. In addition, it is appreciated that multiple studies of targeting BCMA chimeric antigen receptors have entered clinical trials, for example, phase i clinical trials targeting BCMA CAR-T cells were first developed by Ali, etc. in 2016, 12 patients participated in this clinical trial, most of patients exhibited varying degrees of remission, and subsequently, Raje, Berdeja, etc. developed second generation CARs targeting BCMA, and autologous T cells were transduced with lentiviral vectors to form BCMA targeting CAR-T cells, and among 33 patients, the overall response rate was up to 85%, and BCMA targeting CAR-T cells developed by tokenian, kojic, etc. in china have also entered clinical trials. However, the above-mentioned CAR-T cells have some problems in their use, including aggravation of Cytokine Release Syndrome (CRS) in patients, and various degrees of side effects such as fever, tachycardia, neutropenia, thrombocytopenia, hypotension and acute kidney injury, which limit the clinical application of CAR-T cells.

In order to solve the technical problems, the invention provides a novel chimeric antigen receptor and application thereof, wherein the novel chimeric antigen receptor comprises a signal peptide, an antigen binding domain targeting BCMA, a transmembrane region, a costimulatory factor and a CD3 zeta intracellular signal domain, particularly a single-domain antigen binding domain targeting BCMA with high specificity is selected, and the proportion of CD4+ and CD8+ T cells in the preparation process is optimized, so that the novel chimeric antigen receptor can stimulate sufficient immune response, reduce the release level of the immune factor, relieve side effects caused by CAR-T cell therapy and provide a new idea for developing novel multiple myeloma therapies.

Disclosure of Invention

The invention mainly aims to provide a novel chimeric antigen receptor targeting BCMA and application thereof, so as to reduce the difficulty of molecular biological operation in the CAR-T cell treatment process, improve the cell transfection efficiency and improve the anti-tumor effect.

The detailed technical scheme of the invention is as follows:

the chimeric antigen receptor targeting BCMA is characterized by comprising a signal peptide, a nano antibody targeting BCMA, a transmembrane region, a costimulatory factor and an intracellular signal domain, wherein the amino acid sequence of the nano antibody targeting BCMA is shown as SEQ ID NO. 1.

Furthermore, the transmembrane region is a CD28 transmembrane region, and the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 2.

Further, the intracellular signal domain is a CD3 zeta intracellular signal domain, and the amino acid sequence of the intracellular signal domain is shown as SEQ ID NO. 3.

Further, the co-stimulatory factor is selected from the group consisting of CD27, CD28, 4-1BB, OX40, ICOS, B7-H3, and/or any combination thereof.

Further, the co-stimulatory factor is selected from 4-1BB, and the amino acid sequence of the co-stimulatory factor is shown as SEQ ID NO. 4.

Furthermore, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 5.

Furthermore, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 6.

Furthermore, the nucleotide sequence of the chimeric antigen receptor is shown as SEQ ID NO. 7.

An expression vector is provided that includes nucleotides encoding amino acids of the chimeric antigen receptors described herein or nucleotides thereof.

Further, the expression vector is a lentiviral vector.

A chimeric antigen receptor T cell is provided that expresses a chimeric antigen receptor described herein.

Furthermore, when the chimeric antigen receptor T cell is prepared, the ratio of CD4+ to CD8+ T cells is 2:1-3: 2.

Provides an application of the chimeric antigen receptor T cell in the preparation of antitumor drugs.

Further, the tumor is a B cell malignancy, preferably multiple myeloma.

The beneficial effects of the invention include:

the invention provides a novel chimeric antigen receptor structure of a targeting BCMA and application thereof, wherein a single domain antibody of the targeting BCMA is taken as an antigen binding domain in the chimeric antigen receptor, so that the chimeric antigen receptor has a relatively simple amino acid sequence structure, is easy to carry out gene operation, and can be effectively combined with a target antigen, and the expression levels of IFN-gamma, IL-10, IL-6 and the like are obviously reduced, which shows that the chimeric antigen receptor structure can effectively inhibit side effects of an immune factor storm and the like; in addition, the invention also optimizes the preparation method of the CAR-T cell, creatively provides that the CAR-T is prepared by selecting the T cell with the proportion of CD4+ to CD8+ of 2:1-3:2 in the preparation process, and the prepared CAR-T cell has better killing effect on tumor cells.

Drawings

FIG. 1 shows the restriction electrophoresis of the chimeric antigen receptor nucleic acid fragment;

FIG. 2 is a graph of the rate of cell killing by U266;

FIG. 3 is a graph showing IFN-. gamma.secretion levels in mice;

FIG. 4 is a graph showing IL-6 secretion levels in mice;

FIG. 5 is a graph showing IL-10 secretion levels in mice;

FIG. 6 is a graph showing the change in tumor volume in tumor-bearing mice.

Detailed Description

Some terms related to the present invention are explained below.

As used herein, "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-or double-stranded form, or a combination of DNA or RNA thereof, and polymers thereof, said nucleic acid molecules being synthetic or recombinant.

In the present invention, the "expression vector" refers to a vector comprising a recombinant nucleotide including an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector includes cis-acting elements sufficient for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, including cosmids, plasmids, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses), among others, that incorporate recombinant polynucleotides.

In the present invention, "lentivirus" refers to a genus of the family Retroviridae, which is unique among retroviruses, is capable of infecting non-dividing cells, can transmit a large amount of genetic information to the DNA of host cells, and thus is one of the most effective methods in gene delivery vectors, such as HIV, SIV, and FIV, among which lentiviruses.

In the present invention, "tumor" refers to a disease caused by uncontrolled growth of abnormal cells, which may spread locally or through the blood stream and lymphatic system to other parts of the body. Various tumors are described herein, including but not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. The terms "tumor," "cancer," and "carcinoma" are used interchangeably herein and include both solid tumors and liquid tumors.

In the present invention, a "chimeric antigen receptor" (CAR) is an artificially engineered receptor that is capable of anchoring a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen onto an immune cell (e.g., a T cell) so that the immune cell recognizes the tumor antigen or a viral antigen and kills the tumor cell or a virally infected cell. Generally, a chimeric antigen receptor comprises, in order, a signal peptide, an antigen binding domain, a transmembrane region, and an intracellular signal region. The chimeric antigen receptor of the present invention can be constructed using signal peptides, transmembrane regions and intracellular signal regions known in the art for constructing CARs. The polypeptide combined with the tumor cell membrane antigen can be combined with tumor cells to widely express the membrane antigen with higher or medium affinity, and the polypeptide is usually inserted with an epitope; between the signal peptide and the polypeptide that binds to the tumor cell membrane antigen, inside the antigen-binding domain, and between the antigen-binding domain and the hinge region; the antigen binding domain is a natural polypeptide or an artificial synthetic polypeptide.

In the present invention, the term "costimulatory molecule" refers to a molecule that is present on the surface of an antigen-presenting cell and that is capable of binding to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal. Proliferation of T lymphocytes requires not only antigen binding but also reception of costimulatory molecular signals. The transmission of the costimulatory signal to the T cell is mainly through the binding of the costimulatory molecules CD80, CD86 expressed on the surface of the antigen presenting cell to the CD28 molecule on the surface of the T cell. The B cells receive a costimulatory signal via a general pathogen component such as LPS, or via a complement component, or via CD40L on the surface of activated antigen-specific Th cells. Co-stimulatory factors known in the art for constructing CARs may be employed, including but not limited to CD27, CD28, 4-1BB, OX40, ICOS, B7-H3, and/or any combination thereof.

Example 1 determination of single domain antibodies targeting BCMA

The method for screening BCMA-targeted single domain antibodies by utilizing humanized BCMA protein to immunize alpaca and through a phage display library comprises the following steps:

antigen immunization. Selecting healthy adult monomodal camel, uniformly mixing human BCMA protein according to the ratio of 15 mu g/Kg and Freund's adjuvant according to the ratio of 1:1, and immunizing for 6-8 times by adopting a back subcutaneous multipoint injection mode at intervals of 2 weeks. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for each of the remaining immunizations. 100mL of unimodal camel neck peripheral blood is collected every week after immunization is finished, the titer of the BCMA antibody in the alpaca serum is detected by using ELISA, and when the concentration of the BCMA antibody reaches above 128K, the requirements for constructing an antibody phage display library are met, and the polyclonal antibody of the BCMA is obtained primarily.

And (II) constructing a phage display library.

Collecting 100mL of immunized alpaca Peripheral blood, separating by using lymphocyte separation liquid to obtain Peripheral Blood Mononuclear Cells (PBMC), extracting PBMC total RNA, performing reverse transcription reaction by using an RT-PCR kit to obtain cDNA, and then obtaining a specific VHH fragment through two rounds of nesting. And (3) carrying out enzyme digestion and purification on the obtained VHH fragment, connecting the VHH fragment to a phage vector, and then constructing a phage library through click transformation. Randomly selecting 50 clones for identification, and calculating the insertion rate to be more than 98.5%.

(III) Single Domain antibody screening and obtaining

Screening positive clones by an enzyme-linked immunosorbent assay method by taking soluble human BCMA as an antigen. Extracting positive clone plasmid and transforming to escherichia coli competent cell, inducing nano antibody protein expression by 100mM IPTG, and separating and purifying by using resin to collect target protein. The affinity of the target protein and human BCMA is measured by a BIACORE3000 biomacromolecule interaction instrument (purchased from GE company), the result shows that the affinity of the screened antibody is between 3.52E-07 and 7.89E-09, and the single-domain antibody with the highest affinity is selected for subsequent experiments.

(four) Single Domain antibody sequence analysis

The amino acid sequence structure of the BCMA single domain antibody is determined, as shown in SEQ ID NO:1, the sequence structure is analyzed by IMGT, and the sequence of the CDR1 region is GGSVRTGE, the sequence of the CDR2 region is ARELVGW, and the sequence of the CDR3 region is TGVYYCTANVEGNRIY.

EXAMPLE 2 determination of the sequence of the chimeric antigen receptor Gene

The amino acid and nucleotide sequences of a signal peptide, a CD28 transmembrane region, a 4-1BB costimulatory factor, a CD3 zeta intracellular signal region, a human CD28 hinge region are searched and obtained by a bioinformatics method, wherein the amino acid sequence of the CD28 transmembrane region is shown as SEQ ID NO. 2, the amino acid sequence of the 4-1BB amino acid is shown as SEQ ID NO. 4, the amino acid sequence of the signal peptide is shown as SEQ ID NO. 5, and the amino acid sequence of the CD3 zeta intracellular signal region is shown as SEQ ID NO. 3. In order to facilitate the analysis and the comparison, a targeting BCMA ssFv fragment with a common antibody structure kept by the laboratory is selected as a positive control to carry out the subsequent experiment, and a chimeric antigen receptor gene sequence is constructed in a gene total synthesis mode, wherein the CAR-VHH structure with a single-domain antibody antigen binding domain functional element is signal peptide-anti BCMA VHH-CD 28 TM-4-1 BB-CD3 zeta, the CAR-AN structure with a common antibody antigen binding domain functional element is signal peptide-anti BCMA ssFC-CD 28 TM-4-1 BB-CD3 zeta, the CAR-VHH amino acid sequence is shown as SEQ ID NO:6, and the nucleotide sequence is shown as SEQ ID NO: 7.

EXAMPLE 3 preparation of plasmid vector carrying chimeric antigen receptor Gene

3.1 digestion of the target product and linearization of the plasmid vector

CAR-VHH and CAR-AN gene sequences are amplified and obtained by a PCR method, and Xba I and BamH I enzyme cutting sites are added at two ends of the sequences. The target gene fragment and a lentiviral vector plasmid pCDH-CMV-MCS-EF1-GFP-T2A-Puro (purchased from Addgene company) are subjected to double enzyme digestion reaction of Xba I and BamH I, wherein the enzyme digestion reaction conditions are as follows: the enzyme was cleaved at 37 ℃ for 40 min.

3.2 recovery of the cleavage product

The DNA fragments containing CAR-VHH, CAR-AN fragment and pCDH-CMV-MCS-EF1-GFP-T2A-Puro were electrophoresed on 1.5% agarose gel, and after the electrophoresis, the agarose gel bands containing the above nucleic acid fragments were cut off and placed in two clean EP tubes. The DNA in the agarose gel was purified and concentrated using DNA Extraction kit Ver 4.0 (purchased from Taraka).

3.3 product ligation and transformation

Connecting the DNA fragments at 16 ℃ overnight under the action of T4 ligase, transferring the obtained connecting product into DH5 alpha competent cells by a click method, adding the competent cells into 900 mu L of liquid LB culture medium without antibiotic, culturing for 2h by a shaker at 37 ℃ at 200 rpm, centrifuging for 2 min at 4000 rpm, discarding the supernatant, carrying out basic suspension precipitation by using 100 mu L of liquid LB culture medium, smearing the bacterial liquid on Amp-resistant solid LB plates, placing the plates in a bacterial incubator at 37 ℃, culturing overnight, and picking out positive clones.

3.4 identification of Positive clones

Extracting plasmids in the positive clones, carrying out double enzyme digestion by Xba I and BamH I, identifying target fragments by agarose gel electrophoresis of enzyme digestion products, wherein the size of the CAR-VHH target fragment is about 950bp, the size of the CAR-AN target fragment is about 1600bp, and the sequencing identification of the plasmids shows that the target sequences are correct.

Example 4 packaging, concentration and titer determination of lentiviruses

4.1 packaging of Lentiviral vectors

The original strain solution with correct sequencing was inoculated into 50 mL of Amp-resistant liquid LB medium, shaken at 37 ℃ at 200 rpm, cultured overnight, centrifuged at 4000 rpm to collect the cells, and Plasmid was extracted using Takara MiniBEST Plasmid Purification Kit Ver 4.0 (purchased from Takara).

Mixing with 5% CO at 37 deg.C in six-hole plate₂293T cells were cultured under the conditions, and the fresh medium was replaced when the cell confluence reached 80%. Plasmid was packaged as dR8.9: VSVG envelope protein plasmids were mixed at a ratio of 4:3:1, followed by addition of Opti-MEM medium to make up the volume of 5 mL. The mixed solution of the plasmid and the transfection reagent is dropwise added into 293T cell culture solution at 37 ℃ and 5% CO₂Culturing under the condition for 48 h.

4.2 concentration and titer determination of lentiviruses

Centrifuging at 2500 rpm for 10 min, collecting supernatant, sequentially filtering with 0.45 μm filter membrane and 0.22 μm filter membrane to remove cell debris and other impurities, dialyzing and concentrating the virus-containing supernatant, and detecting titer of the virus by TCID50 method, wherein the results show that titer of lentivirus carrying CAR-VHH and CAR-AN genes reaches 10⁷TCID50/mL or more.

Example 5 preparation of targeting BCMA CAR-T cells

Healthy volunteers were recruited, 20mL of peripheral blood was collected, separated by flow cytometry and CD4 in the peripheral blood was obtained⁺、CD8⁺T cells. CD4 is currently the most commonly used cell in CAR-T cell production⁺：CD8⁺T cells with a ratio of 1:1, however, in initial experiments, the inventors found that it is difficult to adapt the ratio of T cells to the chimeric antigen receptor structure with a single domain antibody antigen binding domain of the present invention, and the killing efficiency of the chimeric antigen receptor structure against tumor cells is low, so that CD4 is adjusted during CAR-T cell preparation⁺：CD8⁺The proportion of T cells is 1:1, 2:1 and 3:2 respectively.

Culturing the obtained T cells in RPMI1640 medium (containing 10% FBS) for 2-3 passages, collecting cells and distributing in 6-well plate (1 × 10 per well) after the cells are in logarithmic growth phase⁷The cells, although each containing a lentivirus carrying the CAR-VHH and CAR-AN genes, were cultured at 37 ℃ for 7 daysThe cells are collected and preserved.

Example 6 targeting BCMA CAR-T cells in vitro anti-tumor Effect

In order to detect the tumor killing effect of the CAR-T cells provided by the invention, myeloma cell line U266 cells capable of expressing BCMA antigens are selected as in-vitro experimental objects to verify the in-vitro tumor killing effect. U266 cells were cultured in RPMI1640 medium, 5% CO₂Then, the cells were cultured in an incubator at 37 ℃ for 48 hours, and then digested with trypsin to collect the cells.

CAR-T VHH, CAR-T AN cells and PBS (negative control) were mixed with U266 cells at a 1:1 ratio, added to 96-well culture plates and placed in 5% CO₂After culturing in an incubator at 37 ℃ for 24 hours, adding 20 mu L of CCK-8 into each well, continuously incubating for 2 hours, detecting the wavelength of 450nm by using an enzyme-linked immunosorbent assay, and measuring the OD value, wherein the killing rate is [1- (experiment group OD value-effector cell control group OD value)/target cell control group OD value ═ 1- (experiment group OD value-effector cell control group OD value)/target cell control group OD value]X 100%. As shown in FIG. 2, the CAR-T cells can effectively kill U266 cells, and the killing rate is over 60%. The results also indicate that the antigen binding domain using the single domain antibody appears to have a stronger tumor killing effect than the antigen binding domain using the normal antibody, especially in increasing CD4⁺：CD8⁺This trend is more pronounced after T cell ratios. This illustrates CD4⁺：CD8⁺The T cell ratio has a significant effect on the killing effect of CAR-T cells, CD4 compared to the currently common 1:1 ratio⁺The increased specific gravity of T cells appears to be more favorable for BCMA-targeted CAR-T cells to exert anti-tumor effects in CAR-T VHH from CD4 in a 3:2 ratio⁺：CD8⁺The therapeutic cells obtained by T cell preparation have the highest tumor cell killing efficiency, up to about 86%, while the highest killing efficiency in CAR-T AN is represented by CD4 in the ratio of 2:1⁺：CD8⁺T cell preparation the therapeutic cells obtained were approximately 72%. In order to obtain convincing experimental data, CAR-T cells with high killing efficiency are selected for subsequent in-vivo animal experiments, namely CD4 with the ratio of 2:1⁺：CD8⁺T cell preparation of the therapeutic cells.

Example 7 targeting GPC3 in vivo anti-tumor Effect

In order to further verify the anti-tumor effect of the CAR-T cells, the relevant pharmaceutical experiments using Balb/c mouse transplantation tumor model (loaded with hepatoma cell line U266 cells) as biological material in this example were performed by the following specific experimental procedures and results:

7.1 secretion of immune factors

Taking U266 cells in logarithmic growth phase, injecting BALB/C mice subcutaneously, each 1X 10⁶And selecting mice forming obvious tumor masses as experimental objects after 7-10 days. Selecting 30 tumor-bearing mice, randomly dividing into 3 groups, namely CAR-T VHH, CAR-T AN and physiological saline group, and injecting 2 x 10 mice respectively in tail vein injection mode⁶Injecting the cells and physiological saline with the same volume once every 7 days for 2 times, taking blood from tail vein 3 weeks after the second injection, separating to obtain serum, and determining IFN-gamma, IL-6 and IL-10 levels in the serum of the mouse by adopting an Elisa kit instruction. In FIG. 3, it is shown that IFN- γ levels in the plasma of mice are significantly increased after CAR-T treatment, while the levels in the CAR-T VHH, CAR-T AN groups are similar and not significantly different; in FIG. 4 it is shown that IL-6 levels in the plasma of mice are significantly increased after CAR-T treatment and the CAR-T VHH group has lower IL-6 expression levels with significant differences; FIG. 5 shows that the level of IL-10 in the plasma of mice was significantly increased after CAR-T treatment, and the CAR-T VHH group had a lower level of IL-10 expression, with significant differences and a more significant reduction. The above results demonstrate that in vivo experiments, both CAR-T VHH and CAR-T AN were able to effectively activate immune responses in vivo to suppress tumor development and development, and although no significant difference in IFN- γ expression levels was seen, CAR-T VHH appeared to have a relatively mild activating effect on interleukins, which were expressed at a lower level than the CAR-T AN group, IL-6 and IL-10, and which were among the major immune factors in the immune factor storm during CAR-T therapy, suggesting that CAR-T VHH has a lower risk of toxic side effects during therapy.

7.2 tumor volume determination in tumor-bearing mice

The U266 cell tumor-bearing mice were treated with CAR-T VHH, CAR-T AN and physiological saline and then assayed every 2 daysTumor volume was measured once for 20 days. Tumor volume calculation formula volume = (a)²xb)/2, a is the length of the widest part of the tumor and b is the length of the longest part of the tumor.

As shown in figure 6, CAR-T cell therapy was able to effectively inhibit tumor growth in tumor-bearing mice experiments, the tumor volume in the treated group was significantly less than in the saline group, and CAR-T VHH appeared to be more effective in inhibiting tumor growth processes, and the tumor volume in the CAR-T VHH group was significantly less than in the CAR-T AN group two weeks after treatment, demonstrating that constructing CAR-T cells with the single domain antibodies provided in the present invention as antigen binding domains is more advantageous in tumor therapy.

Sequence listing

<110> Beijing Guangdong Biotechnology Ltd

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Ala Arg Glu Leu Val Gly Trp Tyr Arg Gln Val Ser Gly Met Gln His

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agctatgtgg aactggaagt ggtggcgacc cagatgaaca gcaccggcgt gtattattgc 300

accgcgaacg tggaaggcaa ccgcatttat gcgctgccgg cgccgggcag ccgctatccg 360

acctggggcc agggcaccca gggcaccccg gatgatgtga ccgtgagcag catttatatt 420

tgggcgccgc tggcgggcac ctgcggcgtg ctgctgctga gcctggtgat taccctgtat 480

tgcaaacgcg gccgcaaaaa actgctgtat atttttaaac agccgtttat gcgcccggtg 540

cagaccaccc aggaagaaga tggctgcagc tgccgctttc cggaagaaga agaaggcggc 600

tgcgaactgg cggtgctgcc gagcgcgagc gcggcggcgc cggcgaccgg cggcggcggc 660

gcgggcctga ccgcgggcct ggcgctgggc gcggcgggcg gcaccgcggt gctggcgctg 720

gcggcgggcg cggcgccggg catgggcggc ctgccggcgg cgctggcgcc gggcggcggc 780

ctgaccgcgg gcctgggcct ggcgctgatg gcgggcgcga ccagcggcat tggcatgctg 840

ggcggcgcgg cggcgggcct gggccatgcg ggcctgaccg gcggcctgag caccgcgacc 900

ctggcgacca ccgcggcgct gcatatgggc gcgctgccgc cggcg 945

Claims

1. The BCMA-targeted chimeric antigen receptor is characterized by comprising a signal peptide, a BCMA-targeted nano antibody, a transmembrane region, a costimulatory factor and an intracellular signal domain, wherein the amino acid sequence of the BCMA-targeted nano antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 5.

2. The chimeric antigen receptor according to claim 1, wherein the transmembrane region is a CD8 transmembrane region, and the amino acid sequence thereof is shown in SEQ ID NO 2; the intracellular signal domain is CD3 zeta intracellular signal domain, the amino acid sequence of the intracellular signal domain is shown as SEQ ID NO. 3, the costimulatory factor is 4-1BB, and the amino acid sequence of the costimulatory factor is shown as SEQ ID NO. 4.

3. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO 6.

4. The chimeric antigen receptor according to claim 1, wherein the nucleotide sequence is represented by SEQ ID NO 7.

5. An expression vector comprising a nucleotide sequence encoding the chimeric antigen receptor of claim 1, said nucleotide sequence being set forth in SEQ ID No. 7.

6. A chimeric antigen receptor T cell expressing the chimeric antigen receptor of any one of claims 1 to 4, wherein the chimeric antigen receptor T cell is prepared using CD4+ and CD8+ T cells in a ratio of 2:1 to 3: 2.

7. Use of the chimeric antigen receptor T cell of claim 6 in the preparation of a medicament for resisting multiple myeloma.