CN117736336A - BiTE-secreting CAR-T cells and their applications - Google Patents

Abstract

The invention discloses a BiTE-secreting CAR-T cell and application thereof, in particular discloses a BiTE-secreting chimeric antigen receptor, which comprises a leader peptide, a nano antibody targeting mesothelin, an extracellular hinge region, a transmembrane region, an intracellular signal region, a P2A connecting peptide, an Ig kappa leader peptide, a NKG2D ligand-targeting protein, a connecting peptide and a CD 3-targeting single-chain antibody which are sequentially connected from an amino end to a carboxyl end, and further discloses the BiTE-secreting CAR-T cell as a T cell for expressing the chimeric antigen receptor. The CAR-T cells secreting the BiTE can promote the killing effect of the T cells, reduce the recurrence rate of the CAR-T cells for treating tumors, improve the treatment effect, find a new treatment method and an optimal treatment target for solid tumors, and improve the curative effect of the current CAR-T cell therapy on triple negative breast cancer.

Description

BiTE-secreting CAR-T cells and their applications

Technical field

The invention belongs to the field of biomedicine, and specifically relates to BiTE-secreting CAR-T cells and their applications.

Background technique

Tumors, such as triple-negative breast cancer, are highly aggressive, prone to metastasis and recurrence, and have poor prognosis. Compared with other types of breast cancer, the core difficulty in treatment is the lack of effective treatment targets. At present, surgical treatment has great limitations. It only removes the target tumor mass to achieve a cure effect. However, for tumors that have spread, surgical resection cannot solve this problem well. Treatment should be combined with other methods to achieve the best effect. Currently, more cancer treatments rely on radiotherapy and chemotherapy, and there are no suitable and effective treatment targets and treatment plans. Since there is currently no suitable targeted drug treatment for solid tumor cancer, systemic chemotherapy can only be used. For example, docetaxel (DTX) is a taxane anti-tumor drug that can inhibit the mitosis process of tumor cells by promoting microtubule polymerization in the M phase, thereby achieving anti-tumor purposes. However, tumor cells are generally resistant to chemotherapy drugs, and primary or secondary DTX resistance can lead to chemotherapy failure and affect patient prognosis. The remaining reported anthracyclines, mTOR inhibitors, Src tyrosine kinase inhibitors and vascular endothelial growth factor inhibitors are all reported to have certain effects on triple-negative breast cancer, but there is no conclusion yet on their long-term survival effects on patients. At present, tumors, especially solid tumors, still lack clear treatments and effective drugs.

CAR-T targeted anti-tumor cellular immunity technology includes antibodies that recognize cancer-specific targets, hinge regions, transmembrane regions, intracellular signaling regions (immunoreceptor tyrosine activation motifs, ITAM) and intracellular The chimeric antigen receptor of the signaling domain co-stimulatory molecule CD28 and the CD137 (4-1BB) conduction domain is expressed in a lentiviral vector, and the vector is transfected into autologous T cells, so that the modified CAR-T cells have targeted It can specifically recognize and kill cells expressing specific antigens, and can also proliferate and activate in the body. There are currently a variety of CAR-T studies in the treatment of solid tumors. For example, Li et al. published a report in which their in vitro experiments demonstrated that EphA2-targeting CAR-T cells can lyse EphA2-positive lung cancer cells. Louis et al. used GD2-targeting CAR-T cells to treat 19 cases of neuroblastoma, and 3 patients achieved complete remission without serious adverse reactions. In 2018, Beatty et al also reported that some patients with refractory metastatic pancreatic cancer received mesothelin-targeted CAR-T cell therapy. Recently, the team led by Gianpietro successfully screened B7-H3, a suitable target for CAR-T cells in the treatment of solid tumors. The team successfully constructed B7-H3.CAR-T cells that co-activate 4-1BB. Mouse experiments have proven that this construct can improve the activity of T cells and the efficiency of tumor immunotherapy; in addition, researchers found that the T cells can also reduce the expression of PD-1 in cells, and are positive for PD-(L)1 expression. The tumor treatment effect is better. No side effects were detected in subsequent mouse experiments, and safety is guaranteed. In addition, there are a variety of CAR-Ts in clinical treatment of triple-negative breast cancer, such as CAR-Ts targeting MUC1, c-Met, ROR1, Mesothelin and NKG2D. NKG2D CAR-T has been reported to be used in triple-negative breast cancer research. Data show that NKG2D ligands are widely expressed in triple-negative breast cancer cell lines. NKG2D CAR-T has been shown to be effective in triple-negative breast cancer cell lines in vitro and in mice. In vivo experiments have shown good killing effects. In addition, the Mesothelin target is specifically highly expressed in triple-negative breast cancer, and some researchers have reported that mesothelin-targeted CAR-T cells have a certain effect on triple-negative breast cancer. Although CAR-T cell therapy has achieved great success in the treatment of blood cancers, it is not effective against solid tumors. One of the main reasons is the antigen heterogeneity of solid tumors, which causes single-target CAR-T to easily cause antigen escape, leading to resistance to single-antigen target CAR-T and increasing the recurrence rate of tumors.

Contents of the invention

In order to solve the shortcomings of single-antigen target CAR-T in tumor treatment in the prior art, the present invention aims to provide BiTE-secreting CAR-T cells and their applications.

Mesothelin is a tumor-associated antigen that is overexpressed in most malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and some lung cancers. Although mesothelin has limited expression in normal tissues, it is expressed at low levels on normal peritoneal, pleural, and pericardial mesothelial surfaces. In addition, mesothelin is the target of endogenous immune responses in pleural mesothelioma, ovarian cancer and pancreatic cancer, and it appears that this target has good immunogenicity. Clinical trials using an antibody-based strategy to target mesothelin-overexpressing tumors have shown preliminary safety and potential efficacy, with only serositis being identified as a dose-dependent toxicity of targeting extratumoral antigens. , so the safety of this target is excellent. More importantly, the mesothelin target is specifically highly expressed in triple-negative breast cancer but not in non-triple-negative breast cancer, and this high expression is positively correlated with the progression of triple-negative breast cancer, indicating that it can be used as a triple-negative breast cancer A potential CAR-T target for breast cancer, and this target has good safety in the human body.

NKG2D protein is a C-type lectin-like glycoprotein that is widely expressed on the surface of human CD8 ⁺ T cells, NK cells, γδ T cells and CD4 ⁺ T cell subsets. NKG2D is an activating receptor that plays an important role in the activation of NK cells. However, it cannot directly activate T cells, but only provides costimulatory signals to participate in the activation of T cells. The NKG2D receptor can recognize ligand proteins of various structures, which are mainly divided into six types of MHC-Ⅰ molecule-related molecules A/B, namely MICA/MICB ligand proteins and MHC-Ⅰ class molecule-related molecules, UL16-binding proteins, namely ULBP1 -6 ligand proteins. Since the two major types of NKG2D ligands are generally barely expressed or very lowly expressed on normal cells, but are mainly expressed on the surface of various tumor cells or the expression regulation increases during virus infection, they can be considered as a kind of stress response of the body. Performance. When the NKG2D protein on immune cells in the body combines with cells expressing related NKG2D ligands, it will quickly activate the immune cells and trigger a series of effector reactions, thereby exerting a cytotoxic effect and ultimately killing the cells. NKG2D CAR has good killing power and targeting properties against tumor cells that highly express its ligand, including prostate cancer, ovarian cancer, lymphoma, etc. In addition, studies have shown that NKG2DCAR can not only recognize tumor cells, but also further recognize tumor vascular endothelial cells in the tumor microenvironment.

"BiTE" technology, Bispecific T-cell engager, uses the VH-VL segments of two specific antibodies as two "binding arms". One arm is designed to bind CD3 to bind and activate T cells. cells, and the other arm targets tumor antigens.

In order to reduce the recurrence rate of CAR-T cells in treating tumors and improve the therapeutic effect, the present invention uses modern genetic engineering technology to express Mesothelin VHH on human killer T cells and secrete "BiTE" to prepare Mesothelin CART·BiTE cells, and combine Mesothelin When CART·BTE cells are reinfused into the patient's body, they can kill cancer cells efficiently and specifically, and can be used to improve the current therapeutic effect of immune cell therapy on tumors.

The specific technical solutions of the present invention are as follows:

The first aspect of the present invention provides a chimeric antigen receptor that secretes BiTE. The chimeric antigen receptor includes a leader peptide connected sequentially from the amino terminus to the carboxyl terminus, a nanobody targeting mesothelin, and an extracellular hinge. region, transmembrane region, intracellular signal region, P2A connecting peptide, Igκ leader peptide, protein targeting NKG2D ligand, connecting peptide, single chain antibody targeting CD3.

Further, the mesothelin-targeting Nanobody comprises the amino acid sequence shown in SEQ ID NO.1;

The protein targeting NKG2D ligand includes the amino acid sequence shown in SEQ ID NO.3;

The CD3-targeting single-chain antibody includes the amino acid sequence shown in SEQ ID NO. 5.

Further, the leader peptide is selected from one or more combinations of CD8α signal peptide, GM-CSF signal peptide, CD28 signal peptide, CD4 signal peptide, CD5 signal peptide, CD134 signal peptide and CD137 signal peptide;

The extracellular hinge region is selected from one or more combinations of CD8α hinge region, CD28 hinge region, CD4 hinge region, CD5 hinge region, CD134 hinge region, CD137 hinge region and ICOS hinge region;

The transmembrane region is selected from one or a combination of more of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region and CD28 transmembrane region;

The intracellular signal region is selected from one of the 4-1BB signal region, CD3ζ signal region, ICOS signal region, CD27 signal region, OX40 signal region, CD28 signal region, IL1R1 signal region, CD70 signal region and TNFRSF19L signal region, or Various combinations;

Preferably, the leader peptide is selected from CD8α signal peptide;

Preferably, the extracellular hinge region is selected from the CD8α hinge region;

Preferably, the transmembrane region is selected from the CD8 transmembrane region;

Preferably, the intracellular signal region is selected from 4-1BB and CD3ζ, and the 4-1BB and CD3ζ are connected in sequence.

A second aspect of the invention provides nucleotide sequences encoding said chimeric antigen receptors.

A third aspect of the invention provides an expression vector containing the nucleotide sequence.

Further, the expression vector is selected from a lentiviral expression vector, a retroviral expression vector or an adenoviral expression vector.

A fourth aspect of the present invention provides a BiTE-secreting CAR-T cell, where the cell is a T cell expressing the chimeric antigen receptor.

A fifth aspect of the present invention provides a pharmaceutical composition comprising the expression vector or the BiTE-secreting CAR-T cell.

A sixth aspect of the present invention provides the use of the BiTE-secreting CAR-T cells in the preparation of tumor therapeutic drugs.

Further, the tumor is a tumor with high mesothelin expression;

Preferably, the tumor is a solid tumor;

Preferably, the tumor includes pleural mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, and breast cancer.

Preferably, the tumor is triple negative breast cancer.

The beneficial effects of the present invention are:

The BiTE-secreting chimeric antigen receptor Mesothelin CAR·BiTE provided by the present invention is a CAR designed to target multiple antigens of Mesothelin and NKG2DL by combining BiTE technology, and further utilizes modern genetic engineering technology to express Mesothelin on human killer T cells. CAR·BiTE, prepare Mesothelin CART·BiTE cells, and infuse MesothelinCART·BiTE cells back into the patient's body. Not only can it avoid problems such as antigen escape of tumor cells, but also when T cells secrete "BiTE", it interacts with NKG2D on tumor cells. When combined, the intracellular signaling area is activated, thereby promoting the expansion of T cells in the patient's body and exerting a killing effect, reducing the recurrence rate of CAR-T cell therapy tumors, improving the therapeutic effect, and finding new treatments and treatments for solid tumors. The best therapeutic target to improve the efficacy of current CAR-T cell therapy in triple-negative breast cancer.

Description of drawings

Figure 1: Schematic representation of lentiviral plasmid construction.

Figure 2: Mesothelin CART·BTE cell preparation process.

Figure 3: Positive rate of Mesothelin CART·BTE cells.

Figure 4: Schematic diagram of targeted tumor killing by Mesothelin CART·BTE.

Figure 5: The in vitro tumor killing effect of Mesothelin CART·BTE cells on MDA231 cells. (A) After about 24 hours of killing, E:T=10:1; (B) After about 24 hours of killing, E:T=5:1.

Detailed ways

In order to understand the present invention more clearly, the present invention will now be further described with reference to the following examples and accompanying drawings. The examples are for explanation only and do not limit the invention in any way. In the examples, each original reagent material is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

Example 1

1. Structure of BiTE-secreting chimeric antigen receptor

This embodiment provides a chimeric antigen receptor secreting BiTE, which targets mesothelin (Mesothelin) and NKG2D ligand (NKG2DL), and is designated as Mesothelin CAR·BiTE. Mesothelin CAR·BiTE includes a leader peptide connected sequentially from the amino terminus to the carboxyl terminus, a nanobody targeting mesothelin, an extracellular hinge region, a transmembrane region, an intracellular signal region, a P2A connecting peptide, an Igκ leader peptide, and a target Proteins, linker peptides, and CD3-targeting single-chain antibodies to NKG2D ligands.

In a specific embodiment, the leader peptide is selected from CD8α signal peptide, the extracellular hinge region is selected from CD8α hinge region, the transmembrane region is selected from CD8 transmembrane region, and the intracellular signal region is selected from 4-1BB and CD3ζ, said 4-1BB and CD3ζ are connected in sequence.

Nanobody targeting mesothelin (MSLN VHH) (amino acid sequence) SEQ ID NO.1:

QVQLVQSGGGLVHPGGSLRLSCAASGIDLSLYRMRWYRQAPGKERDLVALITDDGTSYYEDSVKGRFTITRDNPSNKVFLQMNSLKPEDTAVYYCNAETPLSPVNYWGQGTQVTVS

Nanobody targeting mesothelin (MSLN VHH) (nucleotide sequence) SEQ ID NO.2:

CAAGTACAACTCGTGCAAAGTGGAGGCGGATTGGTGCATCCAGGAGGGAGCCTCAGACTGTCATGCGCTGCCAGCGGCATAGATCTTTCTTGTACCGGATGAGATGGTACAGGCAGGCGCCAGGAAAGGAGAGATCTCGTCGCACTGATCACCGACGATGGGACCAGCTACTACGAAGACAGTGTCAAGGGCCGGTTCACAATCACCAGAGACAACCCCAGCAACAAGGTGTTTCTCCAAATGAACAGCCTTAAACCAGAGGA CACCGCCGTGATTATTGCAACGCAGAGACACCTCTGTCTCCTGTAAACTACTGGGGGCAGGGAACTCAGGTGACCGTGAGC Protein targeting NKG2D ligand (NKG2D) (amino acid sequence) SEQ ID NO.3:

VTRQMCIYTNPTSCNEIYGKFSSAYLACDGKQMEIITLLNPSLISGDEWQWSGNTPIHVLGMWHYSKVLKLLDQDEKSYVKLLSANQSMCSAQSEYWNKSEDFFQYCNNKYCIWNKPCPGCYSETLPIQVEQNFLSNLFVASWI

Protein targeting NKG2D ligand (NKG2D) (nucleotide sequence) SEQ ID NO.4:

GTGACCAGGCAGATGTGTATTTATACAAACCCTACTTCATGCAATGAGATTTACGGAAAATTCAGTAGCGCCTATCTCGCCTGCGATGGCAAGCAAATGGAGATCATTACCCTTCTGAACCCAAGTCTGATTTCCGGGGATGAATGGCAATGGTCAGGAAATACCCCCATTCACGTGCTGGGCATGTGGCATTACTCCAAGGTGCTTAAGCTTCTGGATCAGGACGAGAAAAGCTACGTAAAACTCTTGTCTGCTAATCAATCTA TGTGTAGCGCTCAGAGCGAGTACTGGAATAAATCTGAAGATTTCTCCAGTATTGTAACAATAAGTACTGTATTTGGAACAAACCTTGTCCCGGCTGTTATTCTGAGACCTTGCCTATTCAGGTGGAGCAGAACTTCTTGTCCAACCTGTTCGTGGCCTCCTGGATT

Single chain antibody targeting CD3 (amino acid sequence) SEQ ID NO.5:

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGS GSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK

Single chain antibody targeting CD3 (nucleotide sequence) SEQ ID NO.6:

GATATTAAACTGCAACAGAGCGGAGCCGAACTGGCCAGACCTGGTGCATCCGTGAAAATGTCATGCAAGACAAGCGGCTATAACCTTCACCAGGTACACCATGCACTGGGTCAAACAGAGACCTGGACAGGGGCTGGAATGGATTGGTTACATTAATCCCTCCCGAGGGTATACAAATTATAATCAGAAATTCAAAGACAAGGCTACTCTGACCACCGATAAATCCTCATCCACCGCCTACATGCAGTTGTCCTCCCTGACCTCCGAGGATT CAGCCGTCTATTACTGCGCCAGGTACTATGACGACCACTACTGTCTGGACTACTGGGGACAGGGTACCACGCTGACTGTTTCATCAGTGGAGGGAGGATCCGGCGGTTCAGGAGGTAGCGGAGGTTCAGGAGGTGGACGACATTCAGTTGACACAGTCTCCAGCCATTATGTCTGCAAGCCCAGGGGAAAAAGTCACGATGACCTGCAGGGCGTCCTCAAGTGTCTCTTATATGAACTGGTATCAGCAGAAATCAGGTACCAG CCCCAAGAGGTGGATATACGACACATCCAAGGTGGCGAGCGGCGTGCCTTACAGGTTTAGTGGGTCCGGCTCCGGTACGTCTTATAGCCTTACTATCAGTAGTATGGAAGCTGAAGATGCCGCTACCTATTACTGCCAGCAGTGGAGCTCCAACCCCCTGACTTTTGGGGCCGGCACTAAGCTCGAATTGAAA

Leader peptide (Leader) (nucleotide sequence) SEQ ID NO.7:

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC

Extracellular hinge region (hinge) (nucleotide sequence) SEQ ID NO.8:

ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATC

Transmembrane region (TM) (nucleotide sequence) SEQ ID NO.9:

TACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGT

Intracellular costimulatory region (4-1BB) (nucleotide sequence) SEQ ID NO.10:

CGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG

Intracellular signal transduction region (CD3ζ) (nucleotide sequence) SEQ ID NO.11:

CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG

P2A connecting peptide (nucleotide sequence) SEQ ID NO.12:

GCAACAAACTTCTCTCTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCG

Igκ leader peptide (nucleotide sequence) SEQ ID NO.13:

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC

Connecting peptide (nucleotide sequence) SEQ ID NO.14:

GGAGGAGGAAGT

2.Construction of Mesothelin CAR·BTE lentiviral plasmid

This embodiment further provides a method for constructing a Mesothelin CAR·BiTE lentiviral plasmid. The structure of the lentiviral plasmid is shown in Figure 1. The vector of the Mesothelin CAR·BiTE lentiviral plasmid is derived from the NL4-3 clone of HIV. The internal structure of HIV has been destroyed to the greatest extent to remove its pathogenicity, so the vector only retains a part of the conserved regions of HIV. The Mesothelin CAR·BiTE recognition region structure was incorporated into the SIN vector, plus elongation factor 1α (EF1α) as a promoter. In addition, the virus is packaged using the vesicular stomatitis virus glycoprotein (VSV-G) capsid. To ensure safety, VSV-GDNA and vector DNA use different plasmids and are only co-transfected into HEK293T cells when producing viral vectors. The VSV-G capsid will assist the adhesion of the lentiviral vector to the cell membrane and maintain the infectivity of the lentivirus. Specific steps are as follows:

(1) Construction of pCDH recombinant plasmid containing CAR encoding gene

The above-mentioned CAR encoding gene was inserted into the pCDH vector and located behind the elongation factor 1α (EF1α) of the vector. When the CAR encoding gene is inserted into the pCDH vector, a start codon (such as ATG) can be added to the 5' end of the CAR-encoding gene, and a stop codon (such as TAA) can be added to the 3' end. Then, it was transferred into E. coli competent cells DH5α, and positive clones were identified by PCR and sequencing. After gel electrophoresis detection and sequencing of the PCR product, the size and sequence of the target fragment were confirmed, and the pCDH recombinant plasmid, that is, the recombinant expression plasmid, was successfully constructed.

(2) Construction of recombinant lentivirus

The pCDH-CAR recombinant plasmid obtained above, the envelope plasmid pMD2G, and the packaging plasmid (pMDLg/pRRE and pRSV-REV) were co-transfected into the cultured HEK293T cells using the lipofectamine transfection reagent Lipofectamine 3000, and centrifuged after 48 hours. Harvest recombinant lentivirus.

3. Preparation method of Mesothelin CART·BTE cells

To prepare CAR-T cells that secrete BiTE, lentiviral vectors, retroviral vectors, adenoviral vectors, etc. that express the chimeric antigen receptor Mesothelin CAR·BiTE can be used to transfect T cells. When preparing lentiviral vectors, retroviral vectors, and adenoviral vectors that express chimeric antigen receptors, transfection methods such as liposomes and calcium phosphate can be used. The preparation of Mesothelin CART·BTE cells using the recombinant lentivirus constructed above will be described below. The preparation process is shown in Figure 2, which specifically includes the steps:

a) Isolation of PBMC (Peripheral Blood Mononuclear Cells)

PBMC are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, etc. It is best to come from fresh peripheral blood or bone marrow collected from cancer patients one month after surgery and one month after radiotherapy and chemotherapy.

The patient's blood is extracted and the sample is sent to the blood separation chamber; peripheral blood mononuclear cells are collected, centrifuged by Ficoll and the middle layer cells are taken; after washing with PBS, PBMC are obtained.

b) Isolation of antigen-specific T lymphocytes using immunomagnetic beads

Take the above PBMC, add serum-free basal medium to prepare a cell suspension; add CD3/CD28 immunomagnetic beads according to the ratio of magnetic beads to cells: 1:1, and incubate at room temperature for 1-2 hours; use a magnet to incubate well Cells using magnetic beads were screened; after washing with PBS and removing the immunomagnetic beads, CD3-positive T lymphocytes were obtained.

c) Preparation of antigen-specific T lymphocytes by viral transfection

Use the recombinant lentivirus in step (2) above to co-culture the CD3-positive T lymphocytes obtained by immunomagnetic bead separation in step b). On the third day of culture, perform cell counting and medium replacement, and adjust the cell concentration to 0.5 ×10 ⁶ cells/mL, inoculate, and culture; on the 5th day of culture, observe the cell status. If the cell density increases, dilute the cell concentration to 0.5 × 10 ⁶ cells/mL, detect cell viability, and continue culturing. After expansion and culture to 9-11 days, the cells were collected and the expression of chimeric antigen receptor was detected by flow cytometry. The results are shown in Figure 3.

Example 2

The schematic diagram of targeted tumor killing by Mesothelin CART·BTE cells is shown in Figure 4. After the in vitro expansion of Mesothelin CART·BTE cells is completed, a preliminary efficacy experiment is conducted on them. The specific method of the RTCA killing experiment is as follows:

Cell killing experiments were performed using a real-time cell analyzer (xCElligence RTCA SP). First, add the complete culture medium of 5,000 Mesothelin-overexpressing MDA-231 cells (both positive for Mesothelin and NKG2D ligand expression) to the E-Plate provided with the instrument and culture it in the E-Plate wells. About 24 hours later, add corresponding numbers of BiTE-secreting CAR-T cells, non-BiTE-secreting CAR-T cells and UTD cells (only stimulated by magnetic beads, without virus infection) according to different effective-to-target ratios (E:T). T cell control), followed by co-culture for a period of time (>24 hours). The cell killing effect was analyzed based on the CI value of the real-time cell analyzer. The killing results of MDA-231 cells are shown in Figure 5. CAR-T cells secreting BiTE have very strong killing ability against MDA-231 cells, indicating that BiTE is successfully secreted and promotes the killing function of T cells.

Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (10)

1. A chimeric antigen receptor that secretes BiTE, characterized in that the chimeric antigen receptor includes a leader peptide connected sequentially from the amino terminus to the carboxyl terminus, a nanobody targeting mesothelin, and an extracellular hinge region, transmembrane region, intracellular signal region, P2A connecting peptide, Igκ leader peptide, protein targeting NKG2D ligand, connecting peptide, single chain antibody targeting CD3. 2. The chimeric antigen receptor according to claim 1, wherein the mesothelin-targeting Nanobody comprises the amino acid sequence shown in SEQ ID NO.1; The protein targeting NKG2D ligand includes the amino acid sequence shown in SEQ ID NO.3; The CD3-targeting single-chain antibody includes the amino acid sequence shown in SEQ ID NO. 5. 3. The chimeric antigen receptor according to claim 1, wherein the leader peptide is selected from the group consisting of CD8α signal peptide, GM-CSF signal peptide, CD28 signal peptide, CD4 signal peptide, CD5 signal peptide, and CD134 signal peptide. and a combination of one or more of the CD137 signal peptides; The extracellular hinge region is selected from one or more combinations of CD8α hinge region, CD28 hinge region, CD4 hinge region, CD5 hinge region, CD134 hinge region, CD137 hinge region and ICOS hinge region; The transmembrane region is selected from one or a combination of more of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region and CD28 transmembrane region; The intracellular signal region is selected from one of the 4-1BB signal region, CD3ζ signal region, ICOS signal region, CD27 signal region, OX40 signal region, CD28 signal region, IL1R1 signal region, CD70 signal region and TNFRSF19L signal region, or Various combinations; Preferably, the leader peptide is selected from CD8α signal peptide; Preferably, the extracellular hinge region is selected from the CD8α hinge region; Preferably, the transmembrane region is selected from the CD8 transmembrane region; Preferably, the intracellular signal region is selected from 4-1BB and CD3ζ, and the 4-1BB and CD3ζ are connected in sequence. 4. A nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 1 to 3. 5. An expression vector containing the nucleotide sequence of claim 4. 6. The expression vector according to claim 5, characterized in that the expression vector is selected from a lentiviral expression vector, a retroviral expression vector or an adenoviral expression vector. 7. A CAR-T cell secreting BiTE, characterized in that the cell is a T cell expressing the chimeric antigen receptor according to any one of claims 1-3. 8. A pharmaceutical composition, characterized in that it contains the expression vector of claim 5 or the BiTE-secreting CAR-T cell of claim 7. 9. Use of the BiTE-secreting CAR-T cells according to claim 7 in the preparation of tumor therapeutic drugs. 10. The application according to claim 9, characterized in that the tumor is a tumor with high mesothelin expression; Preferably, the tumor is a solid tumor; Preferably, the tumor includes pleural mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, and breast cancer. Preferably, the tumor is triple negative breast cancer.