CN109824783B

CN109824783B - Chimeric antigen receptor expressed on surface of T lymphocyte and application thereof

Info

Publication number: CN109824783B
Application number: CN201910065215.4A
Authority: CN
Inventors: 刘未斌; 刘曲波; 李琼书; 胡源
Original assignee: Shenzhen Jiezhihe Biological Technology Co ltd
Current assignee: Shenzhen Jiezhihe Biological Technology Co ltd
Priority date: 2019-01-23
Filing date: 2019-01-23
Publication date: 2022-03-18
Anticipated expiration: 2039-01-23
Also published as: CN109824783A

Abstract

The invention provides a chimeric antigen receptor expressed on the surface of a T lymphocyte and application thereof, wherein the chimeric antigen receptor comprises: an extracellular binding region, a hinge region and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes MUC1 and NKG2D ligands. The chimeric antigen receptor is expressed on the surface of a T lymphocyte and has the effect of specifically recognizing proteins of MUC1 and NKG2D ligands at the same time.

Description

Chimeric antigen receptor expressed on surface of T lymphocyte and application thereof

Technical Field

The invention relates to the technical field of gene modified cells and tumor treatment, in particular to a chimeric antigen receptor expressed on the surface of a T lymphocyte and application thereof.

Background

CAR-T therapy, collectively known as Chimeric Antigen Receptor T-Cell Immunotherapy, Chimeric Antigen Receptor T-Cell Immunotherapy. The main principle is that immune T cells are separated from a cancer patient, a chimeric antibody capable of recognizing tumor cells and activating the T cells is introduced into the T cells by utilizing a genetic engineering technology, namely CAR-T cells are prepared, the expanded CAR-T cells are infused back into the patient, and the cancer cells are resisted through immunotherapy. A Chimeric Antigen Receptor (CAR) is mainly composed of two parts, one end of which is positioned outside cells and can specifically recognize a certain Antigen on the surface of cancer cells; the other end is located in the cell and contains a signal activation element (such as a Zeta chain of a T cell receptor) which plays a role in transmitting signals to activate T cells. Thus, CAR-expressing T lymphocytes (CAR-T cells) can avoid the restriction of T cell receptors to recognize target cells, thereby acting as a killing effect targeting cancer cells. Currently, CAR-T cell CTL019 by nova directed to the treatment of B cell precursor acute lymphoblastic leukemia is on the market and benefits many patients, but many patients have gone due to leukemia relapse and refractory. Zah reports that the novel CAR-T is designed by taking CD19 and CD20 as bidirectional targets, the killing effect is improved, and the possibility of relapse is reduced. In addition, CAR-T therapy suffers from lack of sufficient persistence, antigen escape leading to relapse, etc., which requires researchers to develop more potent CAR-T cells.

Mucin 1 is a member of the membrane-bound Mucin family, abbreviated MUC 1. Under normal conditions, MUC1 is expressed at a low level in the abluminal surface of epithelial cells of the mammary gland, pancreas, gastrointestinal tract, respiratory tract and genitourinary tract, and cannot be recognized by the immune system of the body, and the expression characteristics are polar expression, apical distribution and rich glycosylation. MUC1 is overexpressed and depolarised in tumor cells, becoming tumor-associated MUC1, redistributed on the cell surface and to the cytoplasm. Numerous studies have shown that the transcriptional regulation of many genes involved in tumor proliferation, metastasis, invasion, angiogenesis, apoptosis, drug resistance and immune regulation is closely related to the expression of MUC 1. Therefore, MUC1 can regulate cellular effects by regulating various signal pathways, and is an important oncogene for promoting tumorigenesis and development. A recent survey showed that, of the 75 tumor associated antigens that developed cancer vaccines, MUC1 was the second potential target. Due to the fact that MUC1 is abnormally over-expressed, is not fully glycosylated and loses polarity in most adenocarcinoma and hematologic malignancy, and is distributed on the whole cell surface, the MUC1 becomes a tumor marker and can be recognized by an immune system, and therefore, the MUC1 protein can be used as an ideal target point for CAR-T cell anti-tumor treatment.

NKG2D is a type II transmembrane glycoprotein expressed on the cell surface in the form of a homodimer formed by disulfide bonds, and the extracellular region contains 1C-type lectin-like region, and is therefore commonly referred to as a C-type lectin-like receptor homodimer. Almost all Natural Killer (NK) cell surfaces, including human and murine, express NKG 2D. Human CD8+ T cells naturally express NKG2D, and NKG2D is also expressed in a subset of natural killer T cells (NKTs), γ δ T cells, CD4+ T cells. Ligands for human NKG2D include mainly histocompatibility complex class I chain-related protein A/B (MICA/B) and human cytomegalovirus UL16 binding protein (HCMV UL16-binding protein, ULBP). It is generally accepted that NKG2D ligand expression is an indicator of "stress" in cells, such as viral infections and malignant transformation, but is rarely present on the surface of healthy cells. NKG2D ligand is expressed to varying degrees on tumor cells of almost all tissue origins in humans. Many cancer cells of epithelial origin, malignant melanoma cells and many types of leukemia cells express MICA. In addition, MICA and MICB are expressed in primary hepatocellular carcinoma and are associated with the sensitivity of hepatoma cells to NK cells. Tumor cells of primary leukemia, malignant melanoma, and glioma origin express ULBP molecules. Because NKG2D can identify the corresponding ligand on the tumor cell, and the expression of the NKG2D ligand is used as a specific change on the tumor cell when the organism generates the tumor, the NKG2D ligand provides a more accurate target for the immunotherapy of the tumor, and also provides a direction and an idea for the development and application of a new immunotherapy method for the tumor. In recent years, CAR-modified T cells expressing NKG2D were designed to specifically recognize NKG2D ligand-positive tumor cells, induce tumor lysis, and obtain a better prognosis. The CAR of the chimeric NKG2D is formed by fusing an activating receptor NKG2D and CD3 zeta and is expressed on the surface of a T lymphocyte; CD3 zeta of its cytoplasmic domain is capable of binding to the adaptor protein DAP10 for signaling. After the CAR modified T cell of the chimeric NKG2D is activated, tumor cells positive to NKG2D ligand are dissolved, proinflammatory cytokines are secreted, and the powerful cytotoxic effect is exerted.

There are currently no CAR-T cells that can simultaneously target MUC1 and NKG2D ligands.

Disclosure of Invention

The invention provides a chimeric antigen receptor expressed on the surface of a T lymphocyte, which can specifically recognize MUC1 and NKG2D ligands.

The invention also provides nucleic acids encoding the chimeric antigen receptors, expression vectors comprising the nucleic acids, and viruses comprising the expression vectors.

The invention also provides the application of the nucleic acid, the expression vector or the virus, and is used for preparing the genetically modified T lymphocyte targeting MUC1 and NKG2D ligand.

The invention also provides a genetically modified T lymphocyte that expresses the chimeric antigen receptor on its surface such that the T lymphocyte is capable of targeting MUC1 and NKG2D ligands.

The invention also provides application of the genetically modified T lymphocyte in preparing a medicament for inhibiting tumors, wherein the tumors are MUC1 and NKG2D ligand positive tumors.

The invention provides a chimeric antigen receptor expressed on the surface of a T lymphocyte, which comprises the following components connected in sequence: an extracellular binding region, a hinge region and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes MUC1 and NKG2D ligands.

Further, the extracellular binding region is SCFV specifically recognizing MUC1 protein and NKG2D specifically recognizing NKG2D ligand, namely SCFV-NKG2D, the hinge region is IgD, and the intracellular signal region is CD 28-4-1 BB-CD 3 zeta.

Further, the chimeric antigen receptor has the amino acid sequence of SEQ ID NO. 1.

The invention also provides nucleic acids encoding the chimeric antigen receptors.

In one embodiment of the invention, the nucleic acid has the nucleotide sequence set forth in SEQ ID NO. 2.

In one embodiment of the invention, the expression vector is derived from a lentiviral plasmid, such as pGreen puro.

The invention provides a genetically modified T lymphocyte which is transduced with the nucleic acid, the expression vector or the virus.

The surface of the genetically modified T lymphocyte provided by the invention expresses a chimeric antigen receptor, and the chimeric antigen receptor has an amino acid sequence shown in SEQ ID NO. 1.

The scheme of the invention has the following advantages:

1) the genetically modified T lymphocyte provided by the invention can specifically identify and efficiently kill MUC1 and NKG2D ligand positive tumors.

Drawings

Figure 1 shows a schematic design of a chimeric antigen receptor recognizing MUC1, and a chimeric antigen receptor recognizing MUC1 and NKG2D ligands.

FIG. 2 shows the killing effect of the genetically modified T lymphocytes provided by the present invention on the breast cancer cell line MCF-7 in vitro.

FIG. 3 shows the killing effect of the genetically modified T lymphocytes of the present invention on the pancreatic cancer cell line BxPC-3 in vitro.

FIG. 4 shows the effect of the genetically modified T lymphocytes of the invention in treating tumors in vivo.

FIG. 5 shows a schematic structure of the lentiviral expression vector pGreen puro.

Detailed Description

Example 1 preparation of T lymphocytes targeting MUC1 and NKG2D ligands, T lymphocytes targeting MUC1

1. Synthesis and amplification

1) The nucleotide sequence (SEQ ID NO:2) encoding the chimeric antigen receptor targeting MUC1 and NKG2D ligands of the present invention and the nucleotide sequence (SEQ ID NO:3) of the chimeric antigen receptor 1 (SCFV-IgD-CD 28-4-1 BB-CD 3 ζ) targeting MUC1 only were synthesized by Suzhou Hongsn Biotech Limited;

2) after amplifying SEQ ID NO 2 and SEQ ID NO 3, respectively cloning the amplified products into a lentivirus expression vector pGreen puro (purchased from Edgene (Add gene)) with a structural schematic diagram as shown in FIG. 5, connecting the SEQ ID NO 2 and the SEQ ID NO 3 into the vector through enzyme cutting sites BamHI and EcoRI to respectively obtain a recombinant lentivirus vector 1 and a recombinant lentivirus vector 2, and carrying out enzyme cutting identification to obtain the recombinant vectors. The amplification and cloning steps in step 2) can be performed using routine experimental conditions in the art, which are within the reach of the skilled person.

2. Packaging of lentiviruses

1) Culturing 293T cells in 1640 medium containing 10% FBS; 293T cells were then plated at 3X 10⁵/cm²The cell confluency is transferred to a culture dish with the diameter of 15cm for culturing for 20 hours, and the cell confluency is ensured to be 80-90% during transfection; replacing the culture medium with 1640 medium without serum for later use;

2) taking two EP tubes, and respectively adding 1ml of 1640 culture medium into the two EP tubes; the recombinant lentiviral vector pGreen puro (i.e., recombinant lentiviral vector 1 or recombinant lentiviral vector 2) was mixed with pMDLg PRRE, pRSV-Rev and pmd2.g plasmids in a molar ratio of 1: 1: 1: 1, uniformly mixing in an EP tube 1, adding 150ul Lipo2000 into an EP tube 2, uniformly mixing and standing for 5 min; adding 1640 culture medium mixed with lipo2000 in EP tube 2 into EP tube 1 containing plasmid, mixing to obtain mixed solution, and standing at room temperature for 20 min;

3) the mixture was then added dropwise to the 293T cell culture dish prepared above, and after further culturing for 4 hours, the culture solution was replaced with 1640 medium containing 10% FBS, and after further culturing for 48 hours, the supernatant of the 293T cells was collected for virus purification.

3. Lentivirus purification

Filtering the supernatant of 293T cells with 0.22 μm filter membrane, collecting the filtrate in 50ml ultrafiltration tube, and centrifuging at 3000g/min for 45 min; transferring the residual concentrated solution to an EP tube, and storing at-80 deg.C for use to obtain concentrated solution containing recombinant lentivirus 1 (containing SEQ ID NO:2) or recombinant lentivirus 2 (containing SEQ ID NO: 3).

Isolation of CD8+ T lymphocytes

A. Human whole blood was poured into a 50ml centrifuge tube and centrifuged at 700g for 20 minutes at room temperature (ordinary centrifugation).

B. The supernatant was discarded and DPBS was added to the pellet to 50 ml.

C: 25ml of each of the above liquids was added to 20ml of a human lymphocyte separation medium (purchased from Beijing Solebao technologies, Ltd.), and the tube was centrifuged at room temperature for 800g for 15 minutes.

D: taking the white membranous layer cells, adding DPBS and making up to 50 ml.

E: centrifuging 600g for 10 minutes, and discarding the supernatant to obtain peripheral blood mononuclear cells PBMC.

F: CD8+ T cells were sorted using CD8+ T cell magnetic bead sorting kit (purchased from american day and whirlpool, germany).

5. Preparation of T lymphocytes targeting MUC1 and NKG2D ligands, and preparation of T lymphocytes targeting MUC1 only

a. Recombinant lentivirus 1 infected CD8+ T lymphocytes:

CD8+ T cells were cultured in RPMI1640 complete medium (RPMI1640 was purchased from Gibco-BRL, and complete RPMI1640 medium was obtained after addition of fetal bovine serum) containing 10% fetal bovine serum (purchased from Gibco-BRL).

The first day: adding anti-CD 3 monoclonal antibody (purchased from Hitachi bioscience, Inc. of Beijing Hokkaiyuan) to activate CD8+ T cells;

and on the third day: the activated CD8+ T cells were divided into experimental and control groups

1) Experimental group, at 4X10⁶150MOI of recombinant lentivirus 1 (containing the gene encoding SCFV-NKG 2D-IgD-CD 28-4-1 BB-CD 3 ζ, SEQ ID NO:2) was added to each activated CD8+ T cell;

2) control group, no treatment;

after 16h, the medium was replaced with RPMI1640 complete medium containing 50IU/ml recombinant human IL-2 (IL-2 purchased from Hippocampus Biotech Co., Ltd., Beijing) and the culture was continued for 10-20 days, followed by observation of the growth of T lymphocytes. The results show that: cells are capable of forming a typical proliferating clonal colony upon infection with a virus.

On the fourteenth day, the experimental group of CD8+ T cells was centrifuged at 600g for 10min, and the supernatant was discarded to collect CD8+ T cell pellet; then, the product is processedResuspend CD8+ T cells in PBS solution and adjust CD8+ T cells to a density of 1X 10⁷Per ml; mu.l of CD8+ T cells were placed in an EP tube, and 10. mu.l of FITC-labeled mouse anti-human CD8 monoclonal antibody (purchased from BD Co.) and 10. mu.l of PE-labeled goat anti-mouse F (ab)2 antibody (purchased from Coulter Co.) were added; incubation at 4 ℃ for 30min, washing 2 times with PBS solution, and detecting the correct expression of the chimeric antigen receptor SCFV-NKG 2D-IgD-CD 28-4-1 BB-CD 3 ζ that specifically recognizes MUC1 and NKG2D ligands on an up-flow cytometer, indicate that T lymphocytes targeting MUC1 and NKG2D ligands (i.e., Anti MUC1/NKG2D ligand CAR-T cells) are obtained.

b. Recombinant lentivirus 2 infected CD8+ T lymphocytes:

the experimental procedure was the same as for recombinant lentivirus 1, except that on day three: experimental group 4X10⁶150MOI of recombinant lentivirus 2 (containing the gene encoding SCFV-IgD-CD 28-4-1 BB-CD 3 ζ, SEQ ID NO:3) was added to each activated CD8+ T cell.

On the fourteenth day, correct expression of only the chimeric antigen receptor 1 SCFV-IgD-CD 28-4-1 BB-CD 3 ζ that specifically recognizes MUC1 was detected by an up-flow cytometer, indicating that T lymphocytes targeting MUC1 (i.e., Anti MUC1CAR-T cells) were obtained.

Example 2 the present invention provides demonstration of the efficacy of genetically modified CD8+ T lymphocytes in the treatment of tumors in vitro

First, the breast cancer cell line MCF-7 stably expressing MUC1 and NKG2D ligands was used as a target cell (purchased from ATCC), and was divided into 3 groups.

CD8+ T lymphocytes targeting MUC1 and NKG2D ligands prepared in example 1 were used as effector cells 1; the CD8+ T lymphocytes targeted to MUC1 prepared in example 1 were used as effector cells 2; CD8+ T cells not infected with virus were also present as effector cells 3;

1) experimental group (Anti MUC1/NKG2D ligand CAR-T cells/MCF-7), effector cells 1 were added to the first group of target cells at a 5:1, 10:1, 20:1 effective target ratio;

2) control group 1(Anti MUC1CAR-T cells/MCF-7), effector cells 2 were added to the second group of target cells at a 5:1, 10:1, 20:1 effector-target ratio;

3) control 2(T cells/MCF-7), effector cells 3 were added to the third set of target cells at a 5:1, 10:1, 20:1 effector-target ratio;

specifically, the target cells were applied at a density of 1X 10⁵Each/ml was inoculated into a 96-well plate at 100. mu.l/well, effector cells were added as described above in 1) -3) and placed in 5% CO₂And culturing in an incubator at 37 ℃ for 4 hours.

WST-1 is adopted to detect cell viability, killing efficiency is calculated, results show that T lymphocytes targeting MUC1 and T lymphocytes targeting MUC1 and NKG2D ligands have strong killing effect on MUC1 and NKG2D ligand positive tumor cells (MCF-7), and the anti-tumor effect of the T lymphocytes targeting MUC1 and NKG2D ligands is stronger than that of the T lymphocytes targeting MUC1, and experimental results are shown in figure 2.

Secondly, pancreatic cancer cell line BxPC-3 stably expressing MUC1 and NKG2D ligand (purchased from ATCC) was used as a target cell and divided into 3 groups.

CD8+ T lymphocytes targeting MUC1 and NKG2D ligands prepared in example 1 were used as effector cells 1; CD8+ T lymphocytes targeted to MUC1 as effector cells 2; CD8+ T cells not infected with virus were also present as effector cells 3;

1) experimental group (Anti MUC1/NKG2D ligand CAR-T cells/BxPC-3), effector cells 1 were added to the first group of target cells at a 5:1, 10:1, 20:1 effective target ratio;

2) control group 1(Anti MUC1CAR-T cells/BxPC-3), effector cells 2 were added to the second group of target cells at a 5:1, 10:1, 20:1 effector-target ratio;

3) control group 2(T cells/BxPC-3), effector cells 3 were added to the third group of target cells at a 5:1, 10:1, 20:1 effective target ratio;

WST-1 is adopted to detect cell viability, killing efficiency is calculated, results show that T lymphocytes targeting MUC1 and T lymphocytes targeting MUC1 and NKG2D ligands have strong killing effect on MUC1 and NKG2D ligand positive tumor cells (BxPC-3), and the anti-tumor effect of the T lymphocytes targeting MUC1 and NKG2D ligands is stronger than that of the T lymphocytes targeting MUC1, and experimental results are shown in figure 3.

Example 3 demonstration of the Effect of genetically modified T lymphocytes on in vivo tumor treatment

Injecting 5x 10 subcutaneous injection into the right axilla of 20 nude mice (6 weeks old, 18-20 g in weight, purchased from Guangdong province medical experiment animal center)⁶MCF-7 Breast cancer cells (purchased from ATCC) and mice were observed for the presence of tumors until they grew to 60mm³Size, it was randomly divided into 4 groups.

1) Control group (control/MCF-7), tail vein injected with normal saline 200 ul/time, 2 times per week;

2) t cell treatment group (T cell/MCF-7) with tail vein injection of 1X 10 CD8+ T cells without viral infection, respectively⁷2 times per week;

3) MUC 1-targeted T lymphocyte therapy group (Anti MUC1CAR-T cell/MCF-7) into which MUC 1-targeted T lymphocytes prepared in example 1 were administered by tail vein injection at 1X 10⁷2 times per week;

4) t lymphocyte therapy group targeting MUC1 and NKG2D ligands (Anti MUC1/NKG2D ligand CAR-T cells/MCF-7): t lymphocytes targeting MUC1 and NKG2D ligands prepared in example 1 were injected into tail vein at 1X 10⁷One/time, 2 times per week.

The survival state of the mice within 100 days is counted, a survival rate curve is made, and the experimental result is shown in figure 4. The T lymphocytes targeting MUC1 and the T lymphocytes targeting MUC1 and NKG2D ligands can prolong the survival time of a mouse model of the breast cancer transplantation tumor with high expression of MUC1 and NKG2D, and the effect of the T lymphocytes targeting MUC1 and NKG2D ligands is better than that of the T lymphocytes targeting MUC 1.

Sequence listing

<110> Shenzhen Chongzhihe Biotech Limited

<120> chimeric antigen receptor expressed on T lymphocyte surface and uses thereof

<130> CNCNP201811695

<160> 3

<170> PatentIn version 3.5

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<220>

<223> chimeric antigen receptor

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Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asn Tyr

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Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Val

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Ala Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala Glu

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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

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Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr

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Tyr Cys Thr Phe Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr Thr

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

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

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

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Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His

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Leu Phe Thr Gly Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val

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Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr

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Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu

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Trp Tyr Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val

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Leu Gly Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

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Gly Gly Ser Met Gly Trp Ile Arg Gly Arg Arg Ser Arg His Ser Trp

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Glu Met Ser Glu Phe His Asn Tyr Asn Leu Asp Leu Lys Lys Ser Asp

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Phe Ser Thr Arg Trp Gln Lys Gln Arg Cys Pro Val Val Lys Ser Lys

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Cys Arg Glu Asn Ala Ser Pro Phe Phe Phe Cys Cys Phe Ile Ala Val

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Phe Leu Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu

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Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn

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Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala

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Ser Cys Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu

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Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu

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Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile

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Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr

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Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Gly Gly Gly Gly Ser

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Phe Thr Cys Phe Val

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Lys Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser

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

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Val Lys Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala

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Ala Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

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Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg

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Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro

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ggcaattcct tcgcctattg gggccaggga accacagtga cagtgtctag cggcggagga 360

ggatctggag gaggaggaag cggaggagga ggaagcgata tcgtggtgac ccaggagagc 420

gctctgacaa caagcccagg cgagacagtg accctgactt gcagaagcag cacaggagcc 480

gtgaccacca gcaactacgc caattgggtg caggagaagc ccgaccatct gttcaccgga 540

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atcggcgata aggccgctct gaccatcacc ggagcccaga cagaagacga ggccatctac 660

ttctgcgccc tctggtacag caaccattgg gtgttcggcg gcggcacaaa actgacagtg 720

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ggctggatca gaggcaggag aagcagacac tcttgggaga tgagcgagtt ccacaactac 840

aacctggacc tgaagaagag cgacttcagc acccgctggc agaaacagag gtgtccagtc 900

gtgaagagca agtgtcgcga gaacgccagc cccttcttct tctgttgctt catcgccgtg 960

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ctgttcaacc aggaggtgca gatccccctg accgaaagct attgcggccc ttgccccaag 1080

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gagagccagg cctcttgcat gagccagaac gccagcctgc tgaaggtgta cagcaaggag 1200

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acaaacggct cttggcagtg ggaggacgga agcatcctga gccccaacct gctgaccatc 1320

atcgagatgc agaagggcga ttgcgccctg tacgccagca gcttcaaggg ctacatcgag 1380

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ggaggaggag gaagcggagg aggaggctct accttcactt gcttcgtcgt gggcagcgac 1500

ctgaaggacg ctcacctgac ttgggaggtg gccggaaaag tgcctacagg aggagtggag 1560

gaaggactgc tggagagaca cagcaacggc tctcagagcc agcacagcag actgaccctg 1620

cctagaagcc tgtggaacgc cggtacatcc gtgacctgca ccctgaacca cccttctctg 1680

cctccccaga gactgatggc tctgagagag ccagccgctc aggctccagt gaaactgtct 1740

ctgaacctgc tggcctctag cgatcctcca gaagccgcct tttgggtgct ggtggtcgtg 1800

ggaggagtgc tggcttgtta cagcctgctg gtgaccgtgg ccttcatcat cttttgggtc 1860

cggagcaagc ggagcagact gctgcacagc gactacatga acatgacccc caggagaccc 1920

ggccctacca gaaagcacta ccagccctac gcccctccta gagattttgc cgcctaccgg 1980

agcagattca gcgtggtgaa gaggggccgg aagaagctgc tgtacatctt caagcagccc 2040

ttcatgcggc cagtgcagac aacacaggag gaagacggct gcagttgcag gtttccagag 2100

gaggaagagg gcggttgcga gctgagagtg aagttcagcc ggagcgccga acctccagct 2160

tatcagcagg gccagaacca gctgtacaac gagctgaacc tgggcaggag ggaggaatac 2220

gacgtgctgg acaagcggag gggaagagat ccagagatgg gcggcaagcc tagaaggaag 2280

aacccccagg agggcctgta caacgagctg cagaaggaca agatggccga ggcttacagc 2340

gagatcggca tgaagggcga gaggagaaga ggcaaaggcc acgacggact gtatcagggc 2400

ctgagcacag ccaccaagga cacctacgac gctctgcaca tgcaggctct gcctcctaga 2460

<210> 3

<211> 1722

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of chimeric antigen receptor 1 targeting MUC1 only

<400> 3

gaagtgcagc tgcagcagtc aggaggagga ctggtgcagc caggaggctc tatgaagctg 60

tcttgcgtgg ccagcggctt taccttcagc aactactgga tgaattgggt ccggcagagc 120

ccagagaaag gactcgagtg ggtggcagag atccggctga agagcaacaa ctacgccacc 180

cactacgccg aaagcgtgaa gggcaggttc accatcagca gggacgacag caagagcagc 240

gtgtacctgc agatgaacaa cctgagggcc gaggacaccg gcatctacta ttgcaccttc 300

ggcaattcct tcgcctattg gggccaggga accacagtga cagtgtctag cggcggagga 360

ggatctggag gaggaggaag cggaggagga ggaagcgata tcgtggtgac ccaggagagc 420

gctctgacaa caagcccagg cgagacagtg accctgactt gcagaagcag cacaggagcc 480

gtgaccacca gcaactacgc caattgggtg caggagaagc ccgaccatct gttcaccgga 540

ctgatcggcg gcacaaacaa cagagcccca ggagtgccag ccagattcag cggatctctg 600

atcggcgata aggccgctct gaccatcacc ggagcccaga cagaagacga ggccatctac 660

ttctgcgccc tctggtacag caaccattgg gtgttcggcg gcggcacaaa actgacagtg 720

ctgggaagcg aaaccttcac ttgcttcgtc gtgggcagcg acctgaagga cgctcacctg 780

acttgggagg tggccggaaa agtgcctaca ggaggagtgg aggaaggact gctggagaga 840

cacagcaacg gctctcagag ccagcacagc agactgaccc tgcctagaag cctgtggaac 900

gccggtacat ccgtgacctg caccctgaac cacccttctc tgcctcccca gagactgatg 960

gctctgagag agccagccgc tcaggctcca gtgaaactgt ctctgaacct gctggcctct 1020

agcgatcctc cagaagccgc cttttgggtg ctggtggtcg tgggaggagt gctggcttgt 1080

tacagcctgc tggtgaccgt ggccttcatc atcttttggg tccggagcaa gcggagcaga 1140

ctgctgcaca gcgactacat gaacatgacc cccaggagac ccggccctac cagaaagcac 1200

taccagccct acgcccctcc tagagatttt gccgcctacc ggagcagatt cagcgtggtg 1260

aagaggggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg gccagtgcag 1320

acaacacagg aggaagacgg ctgcagttgc aggtttccag aggaggaaga gggcggttgc 1380

gagctgagag tgaagttcag ccggagcgcc gaacctccag cttatcagca gggccagaac 1440

cagctgtaca acgagctgaa cctgggcagg agggaggaat acgacgtgct ggacaagcgg 1500

aggggaagag atccagagat gggcggcaag cctagaagga agaaccccca ggagggcctg 1560

tacaacgagc tgcagaagga caagatggcc gaggcttaca gcgagatcgg catgaagggc 1620

gagaggagaa gaggcaaagg ccacgacgga ctgtatcagg gcctgagcac agccaccaag 1680

gacacctacg acgctctgca catgcaggct ctgcctccta ga 1722

Claims

1. A chimeric antigen receptor expressed on the surface of T lymphocytes,

the chimeric antigen receptor comprises, in sequential linkage: an extracellular binding region, a hinge region and an intracellular signaling region, wherein the extracellular binding region comprises a protein that specifically recognizes MUC1 and NKG2D ligands;

the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 1.

2. Nucleic acid encoding the chimeric antigen receptor of claim 1, wherein the nucleotide sequence of said nucleic acid is set forth in SEQ ID No. 2.

3. An expression vector comprising the nucleic acid of claim 2.

4. The expression vector of claim 3, wherein the expression vector is a lentiviral plasmid.

5. A virus comprising the expression vector of claim 3 or 4.

6. Use of the nucleic acid of claim 2, or the expression vector of claim 3 or 4, or the virus of claim 5, for the preparation of genetically modified T lymphocytes targeting MUC1 and NKG2D ligands.

7. A genetically modified T lymphocyte transduced with the nucleic acid of claim 2, or the expression vector of claim 3 or 4, or the virus of claim 5.

8. Use of the genetically modified T lymphocyte of claim 7, for the preparation of a medicament for inhibiting tumors that are MUC1 and NKG2D ligand positive.