CN117736335A

CN117736335A - Double-targeting CAR-T cell targeting mesothelin and NKG2D ligand and application thereof

Info

Publication number: CN117736335A
Application number: CN202211144259.4A
Authority: CN
Inventors: 万晓春; 刘茂玄; 陈有海; 王琪; 许晨光; 刘骏晨
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-09-20
Filing date: 2022-09-20
Publication date: 2024-03-22
Also published as: WO2024060422A1

Abstract

The invention discloses a double-targeting CAR-T cell targeting mesothelin and NKG2D ligand and application thereof, in particular discloses a chimeric antigen receptor, which comprises a nano-antibody targeting mesothelin and a protein targeting NKG2D ligand, wherein the nano-antibody targeting mesothelin and the protein targeting NKG2D ligand are connected by a connecting peptide, and the mesothelin and the NKG2D ligand are expressed on a human killer T cell by utilizing a modern genetic engineering technology to prepare the double-targeting CAR-T cell targeting mesothelin and the NKG2D ligand. The double-targeting CAR-T cell of the targeting mesothelin and the NKG2D ligand can avoid the problems of antigen escape and the like of tumor cells, reduce the recurrence rate of the CAR-T cell for treating tumors, kill cancer cells efficiently and specifically, and can be used for improving the treatment effect of the current immune cell therapy on the tumors.

Description

Double-targeting CAR-T cell targeting mesothelin and NKG2D ligand and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a double-targeting CAR-T cell targeting mesothelin and NKG2D ligand and application thereof.

Background

Many solid tumors, such as triple negative breast cancer, have the characteristics of high invasiveness, easy transfer and recurrence, poor prognosis and the like. The current surgical treatment has a large limitation, and only the target tumor block is resected, so that the healing effect is achieved. However, for the spread tumor, the surgical excision does not solve the problem well, and the best curative effect is possible only by combining other methods for the treatment. More tumor treatments rely on chemoradiotherapy, and no effective therapeutic targets or therapeutic schemes are available at present. However, no suitable targeted drug treatment exists in the prior solid tumor cancer, and only systemic chemotherapy can be adopted. Docetaxel (DTX), for example, belongs to taxane antitumor drugs, and can inhibit the mitosis process of tumor cells by promoting microtubule polymerization in M phase, thereby achieving the purpose of antitumor. However, the ubiquitous resistance of tumor cells to chemotherapeutic agents, and the failure of chemotherapy to produce primary or secondary DTX resistance in tumor cells can affect patient prognosis. The other reported anthracyclines, mTOR inhibitors, src amino acid kinase inhibitors and vascular endothelial growth factor inhibitors all report a certain curative effect on some solid tumors, but have no conclusion on the long-term survival effect of patients. Currently, many solid tumors still lack definite therapeutic methods and effective drugs.

The CAR-T targeting anti-tumor cell immune technology is characterized in that chimeric antigen receptors comprising an antibody for recognizing a specific target of cancer, a hinge region, a transmembrane region, an intracellular signal region (immune receptor tyrosine activation motif, ITAM) and a co-stimulatory molecule CD28 and CD137 (4-1 BB) conduction domain of the intracellular signal region are expressed in a lentiviral vector, and the vector is transfected into autologous T cells, so that the modified CAR-T cells have targeting, can specifically recognize and kill cells expressing the specific antigen, and can proliferate and activate in vivo. There are a number of CAR-T studies currently in use in the treatment of solid tumors. For example, li et al published a report that their in vitro experiments demonstrated that EphA 2-targeted CAR-T cells could lyse EphA 2-positive lung cancer cells. Louis et al treated 19 neuroblastomas with GD 2-targeted CAR-T cells, with complete remission obtained in 3 patients and no serious adverse effects. Beatty et al in 2018 also reported that some refractory metastatic pancreatic cancer patients received mesothelin-targeted CAR-T cell therapy. Recently, gianpiretro led team successfully screened a suitable target for CAR-T cells in solid tumor therapy-B7-H3, the team successfully constructed B7-H3.CAR-T cells co-activated with 4-1BB, and the construction was demonstrated in mouse experiments to increase the activity of T cells, and the efficacy of tumor immunotherapy; in addition, researchers find that the T cell can also reduce the expression quantity of PD-1 in the cell, and has better effect of treating tumors positive to PD- (L) 1. No side effect is detected in the subsequent mouse experiments, and the safety is guaranteed to a certain extent. In addition, there are a variety of CAR-T that are clinically treated for triple negative breast cancers, such as CAR-T targeting MUC1, c-Met, ROR1, mesothelin, NKG2D, and the like. The NKG2D CAR-T has been reported to be used in the study of the triple-negative breast cancer, and the data show that the ligand of the NKG2D is widely expressed on the triple-negative breast cancer cell line, and the NKG2D CAR-T has good killing effect on the triple-negative breast cancer cell line in vitro experiments and in-vivo experiments of mice. In addition, the Mesothelin target is specifically and highly expressed in triple negative breast cancer, and researchers report that the Mesothelin-targeted CAR-T cells have a certain curative effect on the triple negative breast cancer. Although CAR-T cell therapy has achieved great success in the treatment of hematological cancers, it has not been effective for solid tumors. One of the main reasons is the antigenic heterogeneity of solid tumors, which leads to the tendency of single-target CAR-T to cause antigen escape, making the efficacy less than ideal.

Disclosure of Invention

In order to solve the defects of single antigen target CAR-T in the prior art in tumor treatment, the invention aims to provide double-targeting CAR-T cells targeting mesothelin and NKG2D ligand and application thereof.

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 a target point of endogenous immune response in pleural mesothelioma, ovarian cancer and pancreatic cancer, and the target point is better in immunogenicity. Clinical trials targeting mesothelin-overexpressed tumors using antibody-based strategies have shown preliminary safety and potential efficacy, with only serositis, a toxicity of the targeted extra-tumor antigen determined to be dose-dependent, this target safety is excellent.

NKG2D protein is a C-type lectin-like glycoprotein, widely expressed in human CD8 ⁺ T cells, NK cells, gamma delta T cells and CD4 ⁺ T cell subpopulation surface. NKG2D is an activated receptor and plays an important role in the activation of NK cells. However, it cannot directly activate T cells, but only provides a co-stimulatory signal to participate in T cell activation. The NKG2D receptor can recognize ligand proteins with various structures, and is mainly divided into MHC-I molecule related molecules A/B, namely MICA/MICB ligand proteins and MHC-I molecule related molecules 6 UL16 binding proteins, namely ULBP1-6 ligand proteins. Since two major classes of ligands for NKG2D are generally expressed little or very poorly on normal cells, but expression regulation is mainly elevated at the surface of various tumor cells or at the time of viral infection, it is considered to be an expression of stress response in the body. When the NKG2D protein on the immune cells in vivo is combined with the NKG2D ligand cell expressing the relevant NKG2D, the immune cells are activated rapidly to trigger a series of effect reactions, thereby exerting cytotoxin effect and finally killing the cells.

In order to reduce the recurrence rate of the CAR-T cell for treating the tumor and improve the treatment effect, the invention combines the double-target chimeric antigen receptor of the tumor targeting Mesothelin (Mesothelin) and NKG2D, finds a new treatment method and an optimal treatment target for the tumor, and improves the curative effect of the current CAR-T cell therapy on the tumor.

The specific technical scheme of the invention is as follows:

the invention provides a chimeric antigen receptor targeting mesothelin and an NKG2D ligand, which comprises a nano-antibody targeting mesothelin and a protein targeting NKG2D ligand, wherein the nano-antibody targeting mesothelin and the protein targeting NKG2D ligand are connected by a connecting peptide.

Further, the mesothelin-targeting nanobody comprises an amino acid sequence shown as SEQ ID NO. 1;

the protein targeting the NKG2D ligand comprises an amino acid sequence shown as SEQ ID NO. 3.

Further, the chimeric antigen receptor further comprises a leader peptide, an extracellular hinge region, a transmembrane region, an intracellular signaling region;

the amino acid sequence of the chimeric antigen receptor targeting the mesothelin and the NKG2D ligand comprises a leader peptide, a nano-antibody targeting the mesothelin, a connecting peptide, a protein targeting the NKG2D ligand, an extracellular hinge region, a transmembrane region and an intracellular signal region which are sequentially connected from the amino end to the carboxyl end;

or, the amino acid sequence of the chimeric antigen receptor targeting the mesothelin and the NKG2D ligand comprises a leader peptide, a protein targeting the NKG2D ligand, a connecting peptide, a nano antibody targeting the mesothelin, an extracellular hinge region, a transmembrane region and an intracellular signal region which are connected in sequence from the amino end to the carboxyl end.

Preferably, the leader peptide is selected from the group consisting of one or more of a CD8 a signal peptide, GM-CSF signal peptide, CD28 signal peptide, CD4 signal peptide, CD5 signal peptide, CD134 signal peptide and CD137 signal peptide;

preferably, the extracellular hinge region is selected from the group consisting of one or more of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, and an ICOS hinge region;

preferably, the transmembrane region is selected from the group consisting of one or more of a CD3 transmembrane region, a CD4 transmembrane region, a CD8 transmembrane region and a CD28 transmembrane region;

preferably, the intracellular signaling region is selected from the group consisting of one or more of a 4-1BB signaling region, a CD3 zeta signaling region, an ICOS signaling region, a CD27 signaling region, an OX40 signaling region, a CD28 signaling region, an IL1R1 signaling region, a CD70 signaling region, and a TNFRSF19L signaling region.

The invention also provides nucleotide sequences encoding the chimeric antigen receptor targeting mesothelin and NKG2D ligands.

In another aspect, the invention provides an expression vector comprising said nucleotide sequence.

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

In another aspect, the invention provides a dual targeting immune cell that targets mesothelin and NKG2D ligand, said cell being an immune cell that expresses a chimeric antigen receptor that targets mesothelin and NKG2D ligand.

Further, the immune cells are selected from T cells, NK cells or NKT cells;

preferably, the immune cells are selected from T cells.

In another aspect, the invention provides a pharmaceutical composition comprising said expression vector or said dual targeting immune cell targeting mesothelin and NKG2D ligand.

The invention also provides application of the targeting mesothelin and NKG2D ligand double-targeting immune cells in preparing tumor therapeutic drugs.

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

preferably, the tumor is a solid tumor;

preferably, the tumor comprises a pleural mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, breast cancer.

The beneficial effects of the invention are as follows:

the invention provides a double-target chimeric antigen receptor targeting Mesothelin (Mesothelin) and NKG2D ligand (NKG 2 DL), which comprises a VHH nanobody specifically recognizing the Mesothelin and an antibody specifically recognizing the NKG2D, wherein the Mesothelin VHH and the NKG2D are expressed on human killer T cells by utilizing a modern genetic engineering technology, so that the double-target CAR-T cells of the Mesothelin and the NKG2D ligand are prepared, besides the tumor cells, the NKG2D CAR can further recognize tumor vascular endothelial cells in the tumor microenvironment, the constructed cells are returned to a patient, the problems of antigen escape and the like of the tumor cells can be avoided, the recurrence rate of the CAR-T cells for treating tumors can be reduced, and the double-target CAR-T cells can be efficiently and specifically killed, and can be used for improving the treatment effect of the current immune cell therapy on the tumors.

Drawings

Fig. 1: the construction of lentiviral plasmids is schematically shown.

Fig. 2: two double-target CAR-T cell preparation processes.

Fig. 3: positive rate of two double-target CAR-T cells.

Fig. 4: schematic of two dual-target CAR-T targeted killing tumors.

Fig. 5: two double-target CAR-T cells have in-vitro tumor killing effect. After about 36h of killing, E: t=5: 1, a step of; (B) after about 36 hours of killing, E: t=2: 1.

Detailed Description

For a clearer understanding of the present invention, the present invention will now be further described with reference to the following examples and drawings. The examples are for illustration only and are not intended to limit the invention in any way. In the examples, each of the starting reagent materials is commercially available, and the experimental methods without specifying the specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

Example 1

1. Structure of chimeric antigen receptor targeting mesothelin and NKG2D ligand

This example provides a chimeric antigen receptor targeting mesothelin and NKG2D ligand comprising a mesothelin-targeting nanobody, a NKG2D ligand-targeting protein, the mesothelin-targeting nanobody and the NKG2D ligand-targeting protein being linked by a connecting peptide. Further, the chimeric antigen receptor further comprises a leader peptide, an extracellular hinge region, a transmembrane region, an intracellular co-stimulatory region, and an intracellular signaling region.

In a specific embodiment, the chimeric antigen receptor, designated as NKG2D-MSLN CAR, is linked in sequence from amino-terminus to carboxy-terminus by a Leader, a protein targeting the NKG2D ligand (NKG 2D), a linker, a nanobody targeting mesothelin (MSLN VHH), an extracellular hinge region (hinge), a transmembrane region (TM), an intracellular co-stimulatory region (4-1 BB), and an intracellular signal transduction region (cd3ζ).

In another specific embodiment, the chimeric antigen receptor, designated as MSLN-NKG2D CAR, may also be linked in sequence from amino-terminus to carboxy-terminus by a Leader, a nanobody targeting mesothelin (MSLN VHH), a Linker, a protein targeting the NKG2D ligand (NKG 2D), an extracellular hinge region (hinge), a transmembrane region (TM), an intracellular co-stimulatory region (4-1 BB) and an intracellular signal transduction region (CD 3 zeta).

Upon binding of CAR-NKG2D-MSLN and CAR-MSLN-NKG2D to Mesothelin or NKG2D, the cd3ζ and 41BB signaling regions are activated, thereby promoting expansion of T cells and killing in the patient.

The amino acid sequences and nucleotide sequences of the coding genes involved in the CAR-NKG2D-MSLN and the CAR-MSLN-NKG2D are as follows:

mesothelin-targeting nanobody (MSLN VHH) (amino acid sequence) SEQ ID No.1:

QVQLVQSGGGLVHPGGSLRLSCAASGIDLSLYRMRWYRQAPGKERDLVALITDDGTSYYEDSVKGRFTITRDNPSNKVFLQMNSLKPEDTAVYYCNAETPLSPVNYWGQGTQVTVS

mesothelin-targeting nanobody (MSLN VHH) (nucleotide sequence) SEQ ID No.2:

CAAGTACAACTCGTGCAAAGTGGAGGCGGATTGGTGCATCCAGGAGGGAGCCTCAGACTGTCATGCGCTGCCAGCGGCATAGATCTTTCTTTGTACCGGATGAGATGGTACAGGCAGGCGCCAGGAAAGGAGAGAGATCTCGTCGCACTGATCACCGACGATGGGACCAGCTACTACGAAGACAGTGTCAAGGGCCGGTTCACAATCACCAGAGACAACCCCAGCAACAAGGTGTTTCTCCAAATGAACAGCCTTAAACCAGAGGACACCGCCGTGTATTATTGCAACGCAGAGACACCTCTGTCTCCTGTAAACTACTGGGGGCAGGGAACTCAGGTGACCGTGAGC

protein targeting NKG2D ligand (NKG 2D) (amino acid sequence) SEQ ID No.3:

VTRQMCIYTNPTSCNEIYGKFSSAYLACDGKQMEIITLLNPSLISGDEWQWSGNTPIHVLGMWHYSKVLKLLDQDEKSYVKLLSANQSMCSAQSEYWNKSEDFFQYCNNKYCIWNKPCPGCYSETLPIQVEQNFLSNLFVASWI

protein targeting NKG2D ligand (NKG 2D) (nucleotide sequence) SEQ ID No.4:

GTGACCAGGCAGATGTGTATTTATACAAACCCTACTTCATGCAATGAGATTTACGGAAAATTCAGTAGCGCCTATCTCGCCTGCGATGGCAAGCAAATGGAGATCATTACCCTCTTGAACCCAAGTCTGATTTCCGGGGATGAATGGCAATGGTCAGGAAATACCCCCATTCACGTGCTGGGCATGTGGCATTACTCCAAGGTGCTTAAGCTTCTGGATCAGGACGAGAAAAGCTACGTAAAACTCTTGTCTGCTAATCAATCTATGTGTAGCGCTCAGAGCGAGTACTGGAATAAATCTGAAGATTTCTTCCAGTATTGTAACAATAAGTACTGTATTTGGAACAAACCTTGTCCCGGCTGTTATTCTGAGACCTTGCCTATTCAGGTGGAGCAGAACTTCTTGTCCAACCTGTTCGTGGCCTCCTGGATT

leader (nucleotide sequence) SEQ ID No.5:

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC

a linker peptide (Link) (nucleotide sequence) SEQ ID No.6:

GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT

extracellular hinge region (hinge) (nucleotide sequence) SEQ ID No.7:

ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGATATC

transmembrane region (TM) (nucleotide sequence) SEQ ID No.8:

TACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGT

intracellular costimulatory region (4-1 BB) (nucleotide sequence) SEQ ID NO.9:

CGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG

intracellular signal transduction region (cd3ζ) (nucleotide sequence) SEQ ID No.10:

CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG

construction of CAR-NKG2D-MSLN and CAR-MSLN-NKG2D lentiviral plasmids

This example further provides a method for constructing CAR-NKG2D-MSLN and CAR-MSLN-NKG2D lentiviral plasmids, the construction of which is schematically shown in fig. 1, the vectors of CAR-NKG2D-MSLN and CAR-MSLN-NKG2D lentiviral plasmids being derived from NL4-3 clones of HIV. The internal structure of HIV has been disrupted to the greatest extent to remove its pathogenicity, so the vector retains only a portion of the conserved regions of HIV. NKG2D-MSLN, MSLN-NKG2D recognition domain structure was incorporated into the SIN vector, plus elongation factor 1α (EF 1 α) as promoter. In addition, vesicular stomatitis virus glycoprotein (VSV-G) capsids are employed for viral packaging. To ensure safety, VSV-G DNA and vector DNA are co-transferred into HEK293T cells only when viral vectors are produced using different plasmids. VSV-G capsids assist in the adhesion of lentiviral vectors to cell membranes and maintain lentiviral infectivity. The method comprises the following specific steps:

(1) Construction of pCDH recombinant plasmid containing CAR coding Gene

The coding gene of the CAR was inserted into pCDH vector and located after elongation factor 1α (EF 1 α) of the vector. When the coding gene of the CAR is inserted into the pCDH vector, a start codon (such as ATG) can be added to the 5 'end of the coding gene of the CAR-and a stop codon (such as TAA) can be added to the 3' end. Then transferring into competent cells DH5 alpha of the escherichia coli, and carrying out positive clone PCR identification and sequencing identification. And (3) detecting and sequencing by using a PCR product gel electrophoresis to identify the size and sequence of the fragment meeting the purpose, and successfully constructing the pCDH recombinant plasmid, namely the recombinant expression plasmid.

(2) Recombinant lentivirus construction

The pCDH-CAR recombinant plasmid obtained above, envelope plasmid pMD2G and packaging plasmid (pMDLg/pRRE and pRSV-REV) are co-transfected into cultured HEK293T cells through liposome transfection reagent Lipofectamine 3000, and the recombinant lentivirus is obtained after 48 hours by centrifugation.

Preparation method of NKG2D-MSLN CAR-T, MSLN-NKG2D CAR-T cell

Preparation of NKG2D-MSLN CAR-T, MSLN-NKG2D CAR-T cells can be transfected with lentiviral vectors, retroviral vectors, adenoviral vectors, etc. expressing chimeric antigen receptor. When the lentiviral vector, the retrovirus vector and the adenovirus vector expressing the chimeric antigen receptor are prepared, transfection methods such as liposome and calcium phosphate can be adopted. The preparation of NKG2D-MSLN CAR-T, MSLN-NKG2D CAR-T cells by using the recombinant lentivirus constructed above is described below, the preparation flow is shown in FIG. 2, and the preparation method specifically comprises the following steps:

a) Isolation of PBMC (peripheral blood mononuclear cells)

PBMCs are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood, and the like. Preferably from fresh peripheral blood or bone marrow collected one month after surgery and one month after chemoradiotherapy of cancer patients.

Drawing the blood of the patient and sending the sample to a blood separation chamber; collecting peripheral blood mononuclear cells, and taking middle-layer cells after Ficoll centrifugal separation; after washing with PBS, PBMC were obtained.

b) Isolation of antigen-specific T lymphocytes by immunomagnetic bead method

Adding a basic culture medium without serum into the PBMC to prepare cell suspension; adding CD3/CD28 immunomagnetic beads according to the ratio of the magnetic beads to the cells being 1:1, and incubating for 1-2 hours at room temperature; screening cells incubated with the magnetic beads by using a magnet; washing with PBS, and removing immunomagnetic beads to obtain CD3 positive T lymphocytes.

c) Method for preparing antigen-specific T lymphocyte by virus transfection method

Co-culturing the CD3 positive T lymphocyte obtained by immunomagnetic bead separation method in step (2) with the recombinant lentivirus, counting cells and changing liquid on the 3 rd day of culture, and adjusting cell concentration to 0.5X10 ⁶ Inoculating and culturing the strain in a single/mL mode; on day 5 of culture, the cell state was observed, and if the cell density was increased, the diluted cell concentration was 0.5X10 ⁶ Cell activity was measured at each mL and culture was continued. Cells were collected after expansion culture to days 9-11, while the expression of the chimeric antigen receptor CAR targeting mesothelin was detected by flow cytometry, and the results are shown in fig. 3.

Example 2

The schematic diagram of NKG2D-MSLN CAR-T, MSLN-NKG2D CAR-T targeting tumor killing is shown in figure 4. After the NKG2D-MSLN CAR-T, MSLN-NKG2D CAR-T cells are amplified in vitro, preliminary efficacy experiments are carried out on the NKG2D-MSLN CAR-T, MSLN-NKG2D CAR-T cells, and the specific method for killing the RTCA is as follows:

cell killing experiments were performed using a real-time cell analyzer (xCElligence RTCA SP). First, 5,000 complete media of MDA-231 cells overexpressing Mesothelin were added to the E-Plate well fitted with the instrument. After about 24 hours, a corresponding number of positive rates were adjusted to be identical for two double-targeted CAR-T cells, two single-target CAR-T and UTD cells (bead-stimulated only, non-virally infected T cell control) were added at different potency target ratios (E: T), followed by co-culture for a period (> 24 hours). And analyzing the cell killing effect according to the CI value of the real-time cell analyzer. The results of killing MDA-231 cells that overexpressed Mesothelin are shown in FIG. 5, and two types of dual-targeted CAR-T cells have very strong killing ability against MDA-231 cells that overexpressed Mesothelin, and are significantly superior to single-target CAR-T (NKG 2D CAR-T or MSLN CAR-T).

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art.

Claims

1. A chimeric antigen receptor targeting mesothelin and NKG2D ligand, characterized in that the chimeric antigen receptor includes a nanobody targeting mesothelin and a protein targeting NKG2D ligand, so The nanobody targeting mesothelin and the protein targeting NKG2D ligand are connected by a connecting peptide.

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.

3. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor further comprises a leader peptide, an extracellular hinge region, a transmembrane region, and an intracellular signal region;

The amino acid sequence of the chimeric antigen receptor targeting mesothelin and NKG2D ligand includes a leader peptide connected sequentially from the amino terminus to the carboxyl terminus, a nanobody targeting mesothelin, a connecting peptide, and a ligand targeting NKG2D. Body proteins, extracellular hinge region, transmembrane region, and intracellular signaling region;

Or, the amino acid sequence of the chimeric antigen receptor targeting mesothelin and NKG2D ligand includes a leader peptide connected sequentially from the amino terminus to the carboxyl terminus, a protein targeting NKG2D ligand, a connecting peptide, and a targeting mesothelin ligand. Nanobodies, extracellular hinge region, transmembrane region, and intracellular signal region of cortin.

Preferably, 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;

Preferably, 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;

Preferably, the transmembrane region is selected from one or more combinations of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region and CD28 transmembrane region;

Preferably, the intracellular signal region is selected from the group consisting of 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. One or a combination of more.

4. Nucleotide sequence encoding the chimeric antigen receptor targeting mesothelin and NKG2D ligand according to any one of claims 1-3.

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 dual-targeted immune cell targeting mesothelin and NKG2D ligand, characterized in that the cell is a chimeric cell expressing the targeting mesothelin and NKG2D ligand of any one of claims 1-3. Immune cells that bind antigen receptors.

8. The dual-targeted immune cell targeting mesothelin and NKG2D ligand according to claim 7, characterized in that the immune cell is selected from T cells, NK cells or NKT cells;

Preferably, the immune cells are selected from T cells.

9. A pharmaceutical composition, characterized in that it contains the expression vector according to claim 5 or the dual-targeted immune cell targeting mesothelin and NKG2D ligand according to claim 7.

10. Application of the dual-targeted immune cells targeting mesothelin and NKG2D ligand according to claim 7 or 8 in the preparation of tumor therapeutic drugs.

11. The application according to claim 10, 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.