CN115304679B

CN115304679B - Restricted CAR (CAR) for promoting T cell differentiation and application thereof

Info

Publication number: CN115304679B
Application number: CN202210072840.3A
Authority: CN
Inventors: 张根豪; 张凯
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: First Affiliated Hospital of Zhengzhou University
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2024-07-16
Anticipated expiration: 2042-01-21
Also published as: CN115304679A

Abstract

The application belongs to the technical field of tumor cell immunotherapy, and particularly relates to a restricted type CAR for promoting T cell differentiation and application of the restricted type CAR. The chimeric antigen receptor is an amino acid sequence connected with a plurality of protein fragments, and specifically is connected with each other in sequence: human CD8a molecular signal peptide CD8a, human NKG2D extracellular region, human CD8 molecular transmembrane region and 41BB molecular intracellular region 41BB, human CD3z molecular intracellular region CD3z, CPT1a CDS region sequence CPT1a. In the application, based on the function and application of CPT1a in energy metabolism, the inventor takes CPT1a as a 'target point', and combines the application characteristics of CAR-T technology, and the survival period of T cells in tumor tissues is improved by designing a targeted CAR, so that the technical defects of rapid depletion and apoptosis of the T cells in the application of solid tumor treatment of the existing CAR-T cells are overcome.

Description

Restricted CAR (CAR) for promoting T cell differentiation and application thereof

Technical Field

The application belongs to the technical field of tumor cell immunotherapy, and particularly relates to a restricted CAR (CHIMERIC ANTIGEN Receptor ) for promoting T cell differentiation and application thereof.

Background

In tumor prevention and treatment, CAR-T (CHIMERIC ANTIGEN Receptor T-Cell, chimeric antigen Receptor T Cell) treatment methods based on immune systems have been studied and developed more recently due to technical advantages such as small side effects and wide application.

In clinic, CD19 is taken as a target point, and the directional removal of CD19 ⁺ tumor cells by utilizing CAR-T cells has shown a better control effect in the treatment of Acute Lymphoblastic Leukemia (ALL). However, the application of the technology in solid tumors is not satisfactory at present, and a plurality of problems need to be solved, wherein one important problem is the problem of rapid death and apoptosis of CAR-T cells in tumor tissues.

Studies have shown that fatty acids are transported in an organized, controlled manner into mitochondria for oxidation, with mitochondrial fatty acid beta oxidation being a major pathway of fatty acid catabolism, playing an important role in maintaining cellular energy homeostasis. In this metabolic pathway, carnitine palmitoyl transferase I (CARNITINE PALMITOYL TRANSFERASE I, CPT1 a), located on the outer mitochondrial membrane, is a key rate-limiting enzyme for the oxidation of fatty acids, converting acyl-CoA esters into acyl-carnitine esters by catalyzing the reversible transfer of acyl between CoA and l-carnitine. It has been demonstrated that inhibition of CPT1a expression in T cells significantly reduces fatty acid beta oxidation by T cells and results in a significant reduction in T cell persistence. Therefore, based on the important role of energy metabolism in T cell survival, designing a relevant CAR based on CPT1a metabolic pathway, and thereby improving the persistence of CAR-T cell survival in tumor tissue, is of great technical significance for improving the application of CAR-T technology in solid tumors and further improving the therapeutic effect of tumors.

Disclosure of Invention

Based on CPT1a speed-limiting enzyme action and CAR-T technical characteristics, the application aims to provide a limited CAR, so that a certain technical foundation can be laid for the application of the CAR-T technology in solid tumors.

The technical scheme adopted by the application is detailed as follows.

A constrained CAR that promotes T cell differentiation, the chimeric antigen receptor being an amino acid sequence linked by a number of protein fragments, designated: CD8a-NKG2D-41BB-CD3z-CPT1a; the concrete is that connect gradually: human CD8a molecular signal peptide (CD 8 a), human NKG2D extracellular region, human CD8 molecular transmembrane region and 41BB molecular intracellular region (41 BB), human CD3z molecular intracellular region (CD 3Zeta, CD3 z), CPT1a CDS region sequence (CPT 1 a);

wherein the CPT1a structure is used to promote T cell subtype differentiation;

Further, the connection between the intracellular domain of the human CD3z molecule (CD 3 Zeta) and the CPT1a CDs domain sequence (CPT 1 a) is made by using the PA2 linker sequence (P2A), i.e. the overall structure can be expressed as: CD8 a-NKG 2D-41 BB-CD 3 _Z -P2A-CPT 1a;

The human CD8a molecular signal peptide (CD 8 a) comprises 21 amino acids, and specifically comprises the following components:

MALPVTALLLPLALLLHAARP；

The humanized NKG2D extracellular region comprises 144 amino acids, specifically:

IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV;

The human CD8 molecular transmembrane region and 41BB molecular intracellular region (41 BB) comprise two parts of the human CD8 molecular transmembrane region and the 41BB molecular intracellular region, wherein:

the transmembrane region of human CD8 molecule comprises 69 amino acids, specifically:

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC；

41BB molecule intracellular region, comprising 42 amino acids, specifically:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；

The human CD3z molecule intracellular region (CD 3 _Z) comprises 112 amino acids, specifically:

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR;

the PA 2-linked sequence (P2A) comprises 22 amino acids, specifically:

GSGATNFSLLKQAGDVEENPGP；

the CPT1a CDS region sequence CPT1a comprises 773 amino acids, specifically:

MAEAHQAVAFQFTVTPDGIDLRLSHEALRQIYLSGLHSWKKKFIRFKNGIITGVYPASPSSWLIVVVGVMTTMYAKIDPSLGIIAKINRTLETANCMSSQTKNVVSGVLFGTGLWVALIVTMRYSLKVLLSYHGWMFTEHGKMSRATKIWMGMVKIFSGRKPMLYSFQTSLPRLPVPAVKDTVNRYLQSVRPLMKEEDFKRMTALAQDFAVGLGPRLQWYLKLKSWWATNYVSDWWEEYIYLRGRGPLMVNSNYYAMDLLYILPTHIQAARAGNAIHAILLYRRKLDREEIKPIRLLGSTIPLCSAQWERMFNTSRIPGEETDTIQHMRDSKHIVVYHRGRYFKVWLYHDGRLLKPREMEQQMQRILDNTSEPQPGEARLAALTAGDRVPWARCRQAYFGRGKNKQSLDAVEKAAFFVTLDETEEGYRSEDPDTSMDSYAKSLLHGRCYDRWFDKSFTFVVFKNGKMGLNAEHSWADAPIVAHLWEYVMSIDSLQLGYAEDGHCKGDINPNIPYPTRLQWDIPGECQEVIETSLNTANLLANDVDFHSFPFVAFGKGIIKKCRTSPDAFVQLALQLAHYKDMGKFCLTYEASMTRLFREGRTETVRSCTTESCDFVRAMVDPAQTVEQRLKLFKLASEKHQHMYRLAMTGSGIDRHLFCLYVVSKYLAVESPFLKEVLSEPWRLSTSQTPQQQVELFDLENNPEYVSSGGGFGPVADDGYGVSYILVGENLINFHISSKFSCPETDSHRFGRHLKEAMTDIITLFGLSSNSKK;

Thus, the CAR structural sequence of a specific CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT1a comprises 1183 amino acids, the sequence is shown as SEQ ID NO.1, specifically:

MALPVTALLLPLALLLHAARPIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMAEAHQAVAFQFTVTPDGIDLRLSHEALRQIYLSGLHSWKKKFIRFKNGIITGVYPASPSSWLIVVVGVMTTMYAKIDPSLGIIAKINRTLETANCMSSQTKNVVSGVLFGTGLWVALIVTMRYSLKVLLSYHGWMFTEHGKMSRATKIWMGMVKIFSGRKPMLYSFQTSLPRLPVPAVKDTVNRYLQSVRPLMKEEDFKRMTALAQDFAVGLGPRLQWYLKLKSWWATNYVSDWWEEYIYLRGRGPLMVNSNYYAMDLLYILPTHIQAARAGNAIHAILLYRRKLDREEIKPIRLLGSTIPLCSAQWERMFNTSRIPGEETDTIQHMRDSKHIVVYHRGRYFKVWLYHDGRLLKPREMEQQMQRILDNTSEPQPGEARLAALTAGDRVPWARCRQAYFGRGKNKQSLDAVEKAAFFVTLDETEEGYRSEDPDTSMDSYAKSLLHGRCYDRWFDKSFTFVVFKNGKMGLNAEHSWADAPIVAHLWEYVMSIDSLQLGYAEDGHCKGDINPNIPYPTRLQWDIPGECQEVIETSLNTANLLANDVDFHSFPFVAFGKGIIKKCRTSPDAFVQLALQLAHYKDMGKFCLTYEASMTRLFREGRTETVRSCTTESCDFVRAMVDPAQTVEQRLKLFKLASEKHQHMYRLAMTGSGIDRHLFCLYVVSKYLAVESPFLKEVLSEPWRLSTSQTPQQQVELFDLENNPEYVSSGGGFGPVADDGYGVSYILVGENLINFHISSKFSCPETDSHRFGRHLKEAMTDIITLFGLSSNSKK.

The coding DNA sequence of the CAR structure of the CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT1a is shown as SEQ ID No. 2.

The application of the T cell differentiation promoting restricted CAR in preparing an anti-tumor medicament comprises the steps of preparing a lentiviral expression plasmid by using the T cell differentiation promoting restricted CAR, transfecting T cells with the lentiviral expression plasmid, and then reinfusion of the transfected T cells.

The lentiviral expression plasmid of the restricted CAR for promoting T cell differentiation is prepared by the following steps:

(1) Obtaining the coding sequence DNA of the CAR by adopting the prior art;

(2) Taking pCDH-EF1-conGFP plasmid as an expression vector, carrying out EcoR I digestion on the plasmid, and then utilizing In-Fusion HD Cloning Kits to integrate and recombine the DNA of the coding sequence In the step (1) into pCDH-EF1-conGFP plasmid;

(3) Transforming STABL competent cells with the connection product in the step (2), screening, amplifying and culturing, and further extracting plasmids to obtain recombinant lentiviral expression plasmids capable of expressing CPT1 a: pCDH-EF1-CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT1a.

The lentivirus expression plasmid pCDH-EF1-CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT1a is applied to the preparation of an anti-tumor medicament, wherein the tumor is specifically NSCLC cancer (non-small cell lung cancer, including squamous cell carcinoma, adenocarcinoma and large cell carcinoma) related tumor, and further is cancer corresponding to cell lines such as human non-small cell lung cancer H460; when the method is applied, the lentivirus expression plasmid is transfected to prepare the CAR-T cells for application, and the CAR-T cells are used for tumor cells or tumor stem cells;

the specific application steps comprise the following steps:

(1) Packaging lentiviruses; reference may be made in particular to the following operations:

taking 293T cells as target cells to be transfected, firstly performing plating incubation for 24 hours;

Then mixing the constructed lentivirus expression plasmid with a packaging plasmid, and transfecting a target cell 293T cell to be transfected for 48 hours by using a liposome transfection reagent;

After transfection, the supernatant (i.e., the packaged lentiviral particles) is collected for later use in preparation for infection of T cells;

(2) Preparation of purified T cells

First, mononuclear cells are isolated from human peripheral blood (specifically, for example, by a density gradient centrifugation method);

Then, the purified CD3 ⁺ T cells are obtained by separation (specifically, for example, the T cell separation magnetic beads are used for separation and purification),

Finally, adding a proper amount of CD3/CD28 magnetic beads for activation for 2 days for standby;

(3) T cell infection

Adding the virus supernatant collected in step (1) to the cells after 2 days of activation in step (2), and incubating overnight with polybrene (polybrene, also known as hexadimonium bromide) for infection;

(4) Expansion of T cells

For ease of application, after incubation of infected T cells in step (3), centrifugation washing (typically not less than 3 times), RPMI1640 medium containing 1000U IL-2 and 5% fetal bovine serum is added to further expand T cells;

The amplified T cells can be used for detecting and judging the expression condition of the CAR on the surface of the T cells or the proliferation of the cells by utilizing a flow cytometry, and the T cells can be used for killing tumor cells after further feedback according to the detection result.

In the prior art, exogenous genes are introduced based on lentiviral vectors as a carrier tool, so that the exogenous genes can exert corresponding prevention and control sexual functions in a targeted manner, and the method is an ideal targeted prevention and control strategy and is widely researched and applied. However, in practical applications, the "targeting" property of the exogenous gene has a fundamental decision on the relevant control results. Thus, selecting an appropriate "targeting" site, and thus a rational design of CAR, is a fundamental precondition for CAR-T technology application.

In the application, based on the function and application of CPT1a in energy metabolism, the inventor takes CPT1a as a 'target point', and combines the application characteristics of CAR-T technology, and the survival period of T cells in tumor tissues is improved by designing a targeted CAR, so that the technical defects of rapid depletion and apoptosis of the T cells in the application of solid tumor treatment of the existing CAR-T cells are overcome. Preliminary test results show that the survival period of the CAR-T cells in tumor tissues can be effectively improved based on the CAR designed by the application, a good technical foundation can be laid for improving the application effect of the CAR-T cells, and the CAR-T cells have good application potential and practical value.

Drawings

FIG. 1 is a schematic diagram of the structure of the designed CAR molecules NKG2D-41BB-CD3Z (upper panel), CD8a-NKG2D-41BB-CD3Z-P2A-CPT1a (lower panel); wherein LTR is a framework plasmid long terminal repeat sequence, EF1 is a framework plasmid promoter sequence, and GFP is a framework plasmid fluorescent tag sequence;

FIG. 2 is a schematic diagram showing the structure of recombinant plasmid pCDH-EF 1-CD 8a-NKG2D-41BB-CD3Z-P2A-CPT1 a;

FIG. 3 shows the result of the electrophoresis identification of the plasmid pCDH-EF 1-CD 8a-NKG2D-41BB-CD3Z-P2A-CPT1a obtained by construction;

FIG. 4 is a graph of the transfection efficiency of flow cytometry in detecting CAR-T;

FIG. 5 is a graph of differentiation results after 5 days of activation of different CAR-T cells; in the figure, specific parting cells are: primary T cells (Na [ ve ] T cells, TN), central memory T cells (Central memory T cells, TCM), effector T cells (RECENTLY ACTIVATED effector memory or effector T cells, TEM), depleting T cells (exhausted T cells, TEX);

FIG. 6 is a graph showing the results of killing efficiency of CAR-T cells against different tumor cells (H460, H322);

FIG. 7 is a graph showing PD-1 expression results after co-incubation of different CAR-T cells with tumor cells (H460);

FIG. 8 mice subcutaneous engraftment model to examine the therapeutic effects of different T cell treatments; the left panel is a fluorescence photograph, and the right panel is a fluorescence statistical image.

Detailed Description

The application is further illustrated by the following examples. Before describing the specific embodiments, the following description will briefly explain the experimental background conditions such as part of experimental materials in the following embodiments.

Experimental materials:

Non-obese diabetic/severe combined immunodeficiency (nod/scid) mice, 6-8 week female mice purchased from beijing velutinin limited (beijing, china), bred in a sterile environment;

human non-small cell lung cancer related cell lines: h460, H322 (NKG 2DL negative), purchased from ATCC;

The relevant cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS, hyClone, chicago, IL, USA) and 100U/ml penicillin, 100 μg/ml streptomycin (Invitrogen, carlsbad, calif., USA);

Fluorescent cell lines were obtained: before the start of the experiment, tumor cells were transfected with viral supernatant containing luciferase-GFP, and GFP channels were sorted using FACS ARIATM cell sorter (BD Biosciences, san Jose, calif., USA) to finally obtain cell lines stably expressing luciferase-Green Fluorescent Protein (GFP).

Example 1

In view of the importance of energy metabolism in the cell survival cycle, the inventors have designed a new CAR structure (structural schematic diagram is shown in fig. 1) with CPT1a as the "target", combined with CAR technical features, and based on the early-stage CAR (CD 8a-NKG2D-41BB-CD3 z) structural design of the inventors: CD8a-NKG2D-41BB-CD3z-CPT1a; the chimeric antigen receptor structure is sequentially connected: human CD8a molecular signal peptide (CD 8 a), human NKG2D extracellular region, human CD8 molecular transmembrane region and 41BB molecular intracellular region (41 BB), human CD3z molecular intracellular region (CD 3Zeta, CD3 z), CPT1a CDS region sequence (CPT 1 a);

Wherein the human CD3z molecule intracellular region (CD 3 Zeta) is linked to the CPT1a CDs region sequence (CPT 1 a) by using the PA2 linker sequence (P2A), i.e. the overall structure is expressed as: CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT 1a.

As a control, the inventors have designed the CAR structure at the same time: CD8a-NKG2D-41BB-CD3z.

MALPVTALLLPLALLLHAARP；

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC；

41BB molecule intracellular region, comprising 42 amino acids, specifically:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；

the PA 2-linked sequence (P2A) comprises 22 amino acids, specifically:

GSGATNFSLLKQAGDVEENPGP；

the CPT1a CDS region sequence CPT1a comprises 773 amino acids, specifically:

the coding DNA sequence (3549 bp) of the CAR structure of the CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT1a is shown as SEQ ID No.2, and is specifically as follows:

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGCATGCAAAGGACTGTGACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCGGCAGCGGGGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCAGGGCCCATGGCAGAAGCTCACCAAGCTGTGGCCTTTCAGTTCACGGTCACTCCGGACGGGATTGACCTGCGGCTGAGCCATGAAGCTCTTAGACAAATCTATCTCTCTGGACTTCATTCCTGGAAAAAGAAGTTCATCAGATTCAAGAACGGCATCATCACTGGCGTGTACCCGGCAAGCCCCTCCAGTTGGCTTATCGTGGTGGTGGGCGTGATGACAACGATGTACGCCAAGATCGACCCCTCGTTAGGAATAATTGCAAAAATCAATCGGACTCTGGAAACGGCCAACTGCATGTCCAGCCAGACGAAGAACGTGGTCAGCGGCGTGCTGTTTGGCACCGGCCTGTGGGTGGCCCTCATCGTCACCATGCGCTACTCCCTGAAAGTGCTGCTCTCCTACCACGGGTGGATGTTCACTGAGCACGGCAAGATGAGTCGTGCCACCAAGATCTGGATGGGTATGGTCAAGATCTTTTCAGGCCGAAAACCCATGTTGTACAGCTTCCAGACATCGCTGCCTCGCCTGCCGGTCCCGGCTGTCAAAGACACTGTGAACAGGTATCTACAGTCGGTGAGGCCTCTTATGAAGGAAGAAGACTTCAAACGGATGACAGCACTTGCTCAAGATTTTGCTGTCGGTCTTGGACCAAGATTACAGTGGTATTTGAAGTTAAAATCCTGGTGGGCTACAAATTACGTGAGCGACTGGTGGGAGGAGTACATCTACCTCCGAGGACGAGGGCCGCTCATGGTGAACAGCAACTATTATGCCATGGATCTGCTGTATATCCTTCCAACTCACATTCAGGCAGCAAGAGCCGGCAACGCCATCCATGCCATCCTGCTTTACAGGCGCAAACTGGACCGGGAGGAAATCAAACCAATTCGTCTTTTGGGATCCACGATTCCACTCTGCTCCGCTCAGTGGGAGCGGATGTTTAATACTTCCCGGATCCCAGGAGAGGAGACAGACACCATCCAGCACATGAGAGACAGCAAGCACATCGTCGTGTACCATCGAGGACGCTACTTCAAGGTCTGGCTCTACCATGATGGGCGGCTGCTGAAGCCCCGGGAGATGGAGCAGCAGATGCAGAGGATCCTGGACAATACCTCGGAGCCTCAGCCCGGGGAGGCCAGGCTGGCAGCCCTCACCGCAGGAGACAGAGTTCCCTGGGCCAGGTGTCGTCAGGCCTATTTTGGACGTGGGAAAAATAAGCAGTCTCTTGATGCTGTGGAGAAAGCAGCGTTCTTTGTGACGTTAGATGAAACTGAAGAAGGATACAGAAGTGAAGACCCGGATACGTCAATGGACAGCTACGCCAAATCTCTACTACACGGCCGATGTTACGACAGGTGGTTTGACAAGTCGTTCACGTTTGTTGTCTTCAAAAACGGGAAGATGGGCCTCAACGCTGAACACTCCTGGGCAGATGCGCCGATCGTGGCCCACCTTTGGGAGTACGTCATGTCCATTGACAGCCTCCAGCTGGGCTATGCGGAGGATGGGCACTGCAAAGGCGACATCAATCCGAACATTCCGTACCCCACCAGGCTGCAGTGGGACATCCCGGGGGAATGTCAAGAGGTTATAGAGACCTCCCTGAACACCGCAAATCTTCTGGCAAACGACGTGGATTTCCATTCCTTCCCATTCGTAGCCTTTGGTAAAGGAATCATCAAGAAATGTCGCACGAGCCCAGACGCCTTTGTGCAGCTGGCCCTCCAGCTGGCGCACTACAAGGACATGGGCAAGTTTTGCCTCACATACGAGGCCTCCATGACCCGGCTCTTCCGAGAGGGGAGGACGGAGACCGTGCGCTCCTGCACCACTGAGTCATGCGACTTCGTGCGGGCCATGGTGGACCCGGCCCAGACGGTGGAACAGAGGCTGAAGTTGTTCAAGTTGGCGTCTGAGAAGCATCAGCATATGTATCGCCTCGCCATGACCGGCTCTGGGATCGATCGTCACCTCTTCTGCCTTTACGTGGTGTCTAAATATCTCGCTGTGGAGTCCCCTTTCCTTAAGGAAGTTTTATCTGAGCCTTGGAGATTATCAACAAGCCAGACCCCTCAGCAGCAAGTGGAGCTGTTTGACTTGGAGAATAACCCAGAGTACGTGTCCAGCGGAGGGGGCTTTGGACCGGTTGCTGATGACGGCTATGGTGTGTCGTACATCCTTGTGGGAGAGAACCTCATCAATTTCCACATTTCTTCCAAGTTCTCTTGCCCTGAGACGGATTCTCATCGCTTTGGAAGGCACCTGAAAGAAGCAATGACTGACATCATCACTTTGTTTGGTCTCAGTTCTAATTCCAAAAAG.

Example 2

Based on the technical characteristics of CAR-T in example 1, the inventor takes pCDH-EF1-conGFP plasmid as an expression vector to further construct a recombinant lentiviral expression plasmid vector, so that the recombinant lentiviral expression plasmid vector is convenient to be further used for infecting T cells to prepare CAR-T, and the construction process of the specific lentiviral expression vector is introduced as follows.

(1) The coding sequence DNA of the CAR (CD 8a-NKG2D-41BB-CD3Z-P2A-CPT1 a) is obtained by adopting the prior art and synthesizing or combining a PCR amplification technology for standby.

(2) The pCDH-EF1-conGFP plasmid is taken as an expression vector, and restriction enzyme EcoR I (NEB) is used for enzyme digestion at 37 ℃; subsequently, the coding sequence DNA In step (1) was integrated and recombined into the pCDH-EF1-conGFP plasmid using In-Fusion HD Cloning Kits (Bao Bio Inc.) with reference to the specification.

(3) Transforming STABL competent cells with the connection product in the step (2), screening, amplifying and culturing, and further extracting plasmids to obtain recombinant lentivirus expression plasmids capable of expressing CPT1 a: pCDH-EF1-CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT1a.

Synchronously, the inventors prepared a control recombinant plasmid: pCDH-EF1-CD8a-NKG2D-41BB-CD3 _Z.

The structure schematic diagram of the constructed recombinant expression plasmid vector pCDH-EF 1-CD 8a-NKG2D-41BB-CD3Z-P2A-CPT1a is shown in figure 2. Related operations in the construction process are only needed by referring to conventional operations in the prior art, and are not repeated. The result of the electrophoresis identification of the constructed recombinant plasmid expression vector is shown in figure 3. It can be seen that the relevant identification result meets the expectation, which indicates that the relevant recombinant plasmid expression vector is successfully obtained.

Example 3

Based on example 2, the inventors prepared CAR-T cells from the constructed lentiviral expression plasmid by transfection and carried out further preliminary cell experiments. The specific experimental procedure is briefly described below.

Lentiviral packaging

293T cells are used as primary target cells to be transfected, specifically:

first, 293T cells were plated and incubated for 24h (37 ℃ C., DMEM complete medium) using six well plates;

subsequently, the medium was changed to an OPTI-MEM medium, 1.5g of the lentiviral expression plasmid constructed in example 2, 1.5g of the packaging plasmid psPAX, and 2.G 1g of Pmd, and 8 μl of the liposome transfection reagent were added and mixed uniformly to transfect the target cell 293T; after 12 hours of transfection, the medium was replaced with normal DMEM medium;

Finally, after culturing for 48 hours, collecting the virus supernatant (namely the packaged virus-like particles), centrifuging at 1500rpm for 10 minutes, and taking the supernatant to be preserved at-80 ℃ for later use for infecting T cells.

(II) preparation of purified CD3 ⁺ T cells

Firstly, separating from human peripheral blood to obtain mononuclear cells (specifically adopting a density gradient centrifugation method);

Then, separating and obtaining purified CD3 ⁺ T cells (specifically, separating and purifying by using T cell separation magnetic beads);

then, the purified T cells were cultured in 24-well cell culture plates with RPMI-1640 containing IL-2 (100 IU/ml, peproTech, suzhou, jiangsu, china) and L-glutamine (2 mM, gibco/Life Technologies/Thermo FISHER SCIENTIFIC, waltham, mass., USA);

Finally, T cells were stimulated with CD3/CD28 activating antibody (1 μl/10 ⁷ cells) for 2 days prior to use.

The specific operation mode is only required by referring to the prior art, and is not repeated.

(III) T cell infection

Using a 24-well cell culture plate, adding T cells (10 ⁶/well) activated for 2 days in step (2) per well while adding 1mL of the virus supernatant collected in step (1) per well with polybrene (8 μg/mL), and incubating overnight for infection;

After 24 hours, the culture was performed with a normal RPMI-1640 containing IL-2 (100 IU/ml) and L-glutamine (2 mM).

(IV) T cell expansion

For the convenience of subsequent experimental application, further, after incubating the infected T cells in step (three), after washing by centrifugation 3 times, RPMI1640 medium containing 1000U IL-2 and 5% fetal bovine serum (medium was changed every 2-3 days) was added to further expand T cells, and the prepared cells were respectively noted as:

NKG 2D-T (containing the control lentiviral vector plasmid pCDH-EF 1-CD 8a-NKG2D-41BB-CD 3Z),

NKG 2D-CPT 1 a-T (containing lentiviral vector plasmid pCDH-EF1-CD8a-NKG2D-41BB-CD3Z-P2A-CPT1 a).

In addition, as a blank, referring to the procedure described above, the inventors packaged pCDH-EF1 empty plasmid containing GFP into lentiviruses and then directly infected T cells, and the prepared cells were noted as: GFP-T (corresponds to a blank T cell group expressing GFP alone).

(V) specific Experimental detection

(1) Virus packaging condition detection and T cell proliferation condition evaluation

And (3) detecting the expression condition of the CAR on the surface of the T cells by using flow cytometry after the T cells are infected for 5 days in the step (four). The results are shown in FIG. 4.

It can be seen that: the positive rate of CAR expression reaches 50% -60%, and the result shows that the constructed CAR expression plasmid is packaged into lentiviral particles, then T cells are successfully infected, and better expression is obtained, so that the CAR expression plasmid can be used for subsequent experiments.

(2) Differentiation assay for different T cells

In the above-described evaluation of the proliferation of T cells, the inventors further examined the differentiation of T cells.

Collecting T cells after 5 days of infection in the step (IV), re-suspending the cell sediment by PBS, and then adding 1 μl of CD3 anti-ibody, 1 μl of CD45 anti-ibody and 1 μl of CD62L anti-ibody, and incubating for 15 minutes in a light-resistant environment at 4 ℃; the analysis was performed using a C6 flow cytometer (Becton Dickinson) and the data was analyzed using FlowJo software (FlowJo, LLC, ashland, covington, KY, USA).

The results are shown in FIG. 5. Analysis can be seen: the proportion of different cell types in the NKG2D-CPT1a-T group cells was significantly changed in the presence of CPT1a compared with the control GFP-T, NKG 2D-T group cells, and the proportion of central memory T cells (Central memory T cells, TCM) was significantly higher than that of the other control groups. In other words, CPT1a promotes the differentiation of T cells further towards the central memory subtype TCM, while a high proportion of the central memory subtype TCM can enable the T cells to better survive in the tumor microenvironment, so that the CAR-T cells can better play an anti-tumor function, and the treatment effect is improved.

(3) Detection of apoptosis of different tumor cells

The technical effect of the constructed CAR was detected and evaluated by tumor cell apoptosis with tumor cells highly expressing NKG2DL as the subject and CAR-T cells as the experimental "agent". The detailed experimental procedure is outlined below.

Human non-small cell lung cancer cell line H460 and NKG2DL negative human non-small cell lung cancer cell line H322, which are respectively used for high expression of NKG2DL, are used as target cells, T cells treated differently are used as effector cells (GFP-T, NKG2D-T, NKG D-CPT1 a-T), the T cells and the target cells are incubated for 6 hours in 96-well plates according to different effective target ratios (1:1, 5:1 and 20:1), and three compound wells are arranged in each group.

After the incubation, cell supernatants after co-incubation were collected, cell pellet was resuspended with an Annexin V-binding buffer (bioleged, san Diego, CA, USA), followed by addition of 1 μl CD326 anti body and 1 μl l Annexin V antibody (bioleged), and incubation in a dark environment at 4 ℃ for 15 min; propidium (Propidium iodide, sigma) was added before the machine was started. The analysis was performed using a C6 flow cytometer (Becton Dickinson) and the data was analyzed using FlowJo software (FlowJo, LLC, ashland, covington, KY, USA).

The results are shown in FIG. 6. It can be seen that: the NKG2D-T, NKG2D-CPT1 a-T group specifically killed the NKG2 DL-expressing positive H460 tumor cells relative to the control group, while the NKG2D-CPT1 a-T cells were equally effective in killing the NKG2 DL-positive tumor cells at the same ratio, which demonstrated that: the killing effect of the CAR-T cells on NKG2DL antigen expressing positive tumor cells is not affected in the presence of CPT1 a.

(4) Detection of depletion of different T cells

In the above "(3) detection of apoptosis of different tumor cells" evaluation procedure, the inventors further performed detection analysis of depletion of T cells. The detailed experimental procedure is outlined below.

Human non-small cell lung cancer cell line H460 and NKG2DL negative human non-small cell lung cancer cell line H322, which are respectively used for high expression of NKG2DL, are used as target cells, T cells treated differently are used as effector cells (GFP-T, NKG2D-T, NKG D-CPT 1 a-T), the T cells and the target cells are incubated for 6 hours in 96-well plates according to different effective target ratios (1:1, 5:1 and 20:1), and three compound wells are arranged in each group.

After incubation, collecting cell supernatant after co-incubation, re-suspending cell sediment by PBS, then adding 1 μl of CD3 anti body, 1 μl of PD-1 antibody (BioLegend), and incubating in a light-resistant environment at 4deg.C for 15 minutes; the analysis was performed using a C6 flow cytometer (Becton Dickinson) and the data was analyzed using FlowJo software (FlowJo, LLC, ashland, covington, KY, USA).

The results are shown in FIG. 7. It can be seen that: compared to NKG2D-T cells, NKG2D-CPT1a-T cells had lower expression of the depletion indicator PD-1 after co-incubation with NKG2DL positive tumor cells. By combining the grouping of the T cells and the expression condition of PD-1, the method further proves that under the condition of CPT1a, the T cell exhaustion caused by tumor cells can be effectively reduced, and the CAR-T cells can better play an anti-tumor function, so that the treatment effect is improved.

(5) Mouse tumor growth detection

Based on the above experiments, the inventors further conducted mouse animal experiments, and the specific procedures are briefly described below.

Using NOD/SCID immunodeficient mice, 100ul (i.e., 10 ⁷/ml) of PBS resuspended 10H 460-fluc cells were inoculated subcutaneously, a mouse subcutaneous engrafting tumor model was constructed, and after 10 days, 1X 10 ⁶ different groups of T cells (Mock-T, NKG2D-T, NKG2D-CPT1a-T, 10 ⁷/ml, injection amount 100 ul) were each injected via the tail vein for treatment (3 per group).

Fluorescence changes in tumor expression were detected during the experiment using a small animal in vivo imaging device (bioluminescence photographs taken every 7 days).

When detecting the fluorescence change of tumor expression by using a living animal imaging device, firstly, 3% isoflurane (RWD life science, shenzhen, china) is used for inducing indoor anesthesia of mice; each mouse was then intraperitoneally injected with 100ul of d-fluorescein solution (0.15 mg/ml, yeasen Biotech Co., ltd., shanghai, china) using a syringe, fluorescence was detected 10 minutes later using an in vivo animal imaging device IVIS Lumina, series III spectrometer (Caliper Life Science), and finally analyzed using LIVE IMAGE.4.3.1 software (Perkinelmer, waltham, mass., USA).

The experimental results are shown in FIG. 8. The statistics and analysis of the results can be seen:

In the Mock-T treated group, the tumor growth of the mice is faster, the tumor fluorescence of the mice treated by NKG2D-T cells is reduced to the level of 10 ⁷, the tumor fluorescence of the mice treated by NKG2D.CPT1a-T cells is reduced to the level of 10 ⁵, and the fluorescence of the NKG2D-CPT 1a-T cells is further reduced by two orders of magnitude compared with that of the NKG2D-T cells treated group, so that the difference is obvious.

From the experimental results, the invention can further improve the control effect of related tumors by further improving the existing NKG2D targeting CAR structure and further promoting the subtype differentiation of T cells by utilizing CPT1a, thereby improving the condition of T cell exhaustion.

SEQUENCE LISTING

<110> Zhengzhou university first affiliated hospital

<120> A restricted CAR for promoting T cell differentiation and application thereof

<130> none

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1183

<212> PRT

<213> Artificial design

<400> 1

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe

20 25 30

Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys

35 40 45

Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp

50 55 60

Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn

65 70 75 80

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

85 90 95

Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn

100 105 110

Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu

115 120 125

Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser

130 135 140

Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys

145 150 155 160

Met Gln Arg Thr Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro

165 170 175

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

180 185 190

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

195 200 205

Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu

210 215 220

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

225 230 235 240

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

245 250 255

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

260 265 270

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

275 280 285

Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

290 295 300

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

305 310 315 320

Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

325 330 335

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

340 345 350

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

355 360 365

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

370 375 380

Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln

385 390 395 400

Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Glu Ala His Gln

405 410 415

Ala Val Ala Phe Gln Phe Thr Val Thr Pro Asp Gly Ile Asp Leu Arg

420 425 430

Leu Ser His Glu Ala Leu Arg Gln Ile Tyr Leu Ser Gly Leu His Ser

435 440 445

Trp Lys Lys Lys Phe Ile Arg Phe Lys Asn Gly Ile Ile Thr Gly Val

450 455 460

Tyr Pro Ala Ser Pro Ser Ser Trp Leu Ile Val Val Val Gly Val Met

465 470 475 480

Thr Thr Met Tyr Ala Lys Ile Asp Pro Ser Leu Gly Ile Ile Ala Lys

485 490 495

Ile Asn Arg Thr Leu Glu Thr Ala Asn Cys Met Ser Ser Gln Thr Lys

500 505 510

Asn Val Val Ser Gly Val Leu Phe Gly Thr Gly Leu Trp Val Ala Leu

515 520 525

Ile Val Thr Met Arg Tyr Ser Leu Lys Val Leu Leu Ser Tyr His Gly

530 535 540

Trp Met Phe Thr Glu His Gly Lys Met Ser Arg Ala Thr Lys Ile Trp

545 550 555 560

Met Gly Met Val Lys Ile Phe Ser Gly Arg Lys Pro Met Leu Tyr Ser

565 570 575

Phe Gln Thr Ser Leu Pro Arg Leu Pro Val Pro Ala Val Lys Asp Thr

580 585 590

Val Asn Arg Tyr Leu Gln Ser Val Arg Pro Leu Met Lys Glu Glu Asp

595 600 605

Phe Lys Arg Met Thr Ala Leu Ala Gln Asp Phe Ala Val Gly Leu Gly

610 615 620

Pro Arg Leu Gln Trp Tyr Leu Lys Leu Lys Ser Trp Trp Ala Thr Asn

625 630 635 640

Tyr Val Ser Asp Trp Trp Glu Glu Tyr Ile Tyr Leu Arg Gly Arg Gly

645 650 655

Pro Leu Met Val Asn Ser Asn Tyr Tyr Ala Met Asp Leu Leu Tyr Ile

660 665 670

Leu Pro Thr His Ile Gln Ala Ala Arg Ala Gly Asn Ala Ile His Ala

675 680 685

Ile Leu Leu Tyr Arg Arg Lys Leu Asp Arg Glu Glu Ile Lys Pro Ile

690 695 700

Arg Leu Leu Gly Ser Thr Ile Pro Leu Cys Ser Ala Gln Trp Glu Arg

705 710 715 720

Met Phe Asn Thr Ser Arg Ile Pro Gly Glu Glu Thr Asp Thr Ile Gln

725 730 735

His Met Arg Asp Ser Lys His Ile Val Val Tyr His Arg Gly Arg Tyr

740 745 750

Phe Lys Val Trp Leu Tyr His Asp Gly Arg Leu Leu Lys Pro Arg Glu

755 760 765

Met Glu Gln Gln Met Gln Arg Ile Leu Asp Asn Thr Ser Glu Pro Gln

770 775 780

Pro Gly Glu Ala Arg Leu Ala Ala Leu Thr Ala Gly Asp Arg Val Pro

785 790 795 800

Trp Ala Arg Cys Arg Gln Ala Tyr Phe Gly Arg Gly Lys Asn Lys Gln

805 810 815

Ser Leu Asp Ala Val Glu Lys Ala Ala Phe Phe Val Thr Leu Asp Glu

820 825 830

Thr Glu Glu Gly Tyr Arg Ser Glu Asp Pro Asp Thr Ser Met Asp Ser

835 840 845

Tyr Ala Lys Ser Leu Leu His Gly Arg Cys Tyr Asp Arg Trp Phe Asp

850 855 860

Lys Ser Phe Thr Phe Val Val Phe Lys Asn Gly Lys Met Gly Leu Asn

865 870 875 880

Ala Glu His Ser Trp Ala Asp Ala Pro Ile Val Ala His Leu Trp Glu

885 890 895

Tyr Val Met Ser Ile Asp Ser Leu Gln Leu Gly Tyr Ala Glu Asp Gly

900 905 910

His Cys Lys Gly Asp Ile Asn Pro Asn Ile Pro Tyr Pro Thr Arg Leu

915 920 925

Gln Trp Asp Ile Pro Gly Glu Cys Gln Glu Val Ile Glu Thr Ser Leu

930 935 940

Asn Thr Ala Asn Leu Leu Ala Asn Asp Val Asp Phe His Ser Phe Pro

945 950 955 960

Phe Val Ala Phe Gly Lys Gly Ile Ile Lys Lys Cys Arg Thr Ser Pro

965 970 975

Asp Ala Phe Val Gln Leu Ala Leu Gln Leu Ala His Tyr Lys Asp Met

980 985 990

Gly Lys Phe Cys Leu Thr Tyr Glu Ala Ser Met Thr Arg Leu Phe Arg

995 1000 1005

Glu Gly Arg Thr Glu Thr Val Arg Ser Cys Thr Thr Glu Ser Cys

1010 1015 1020

Asp Phe Val Arg Ala Met Val Asp Pro Ala Gln Thr Val Glu Gln

1025 1030 1035

Arg Leu Lys Leu Phe Lys Leu Ala Ser Glu Lys His Gln His Met

1040 1045 1050

Tyr Arg Leu Ala Met Thr Gly Ser Gly Ile Asp Arg His Leu Phe

1055 1060 1065

Cys Leu Tyr Val Val Ser Lys Tyr Leu Ala Val Glu Ser Pro Phe

1070 1075 1080

Leu Lys Glu Val Leu Ser Glu Pro Trp Arg Leu Ser Thr Ser Gln

1085 1090 1095

Thr Pro Gln Gln Gln Val Glu Leu Phe Asp Leu Glu Asn Asn Pro

1100 1105 1110

Glu Tyr Val Ser Ser Gly Gly Gly Phe Gly Pro Val Ala Asp Asp

1115 1120 1125

Gly Tyr Gly Val Ser Tyr Ile Leu Val Gly Glu Asn Leu Ile Asn

1130 1135 1140

Phe His Ile Ser Ser Lys Phe Ser Cys Pro Glu Thr Asp Ser His

1145 1150 1155

Arg Phe Gly Arg His Leu Lys Glu Ala Met Thr Asp Ile Ile Thr

1160 1165 1170

Leu Phe Gly Leu Ser Ser Asn Ser Lys Lys

1175 1180

<210> 2

<211> 3549

<212> DNA

<213> Artificial design

<400> 2

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgatatgga gtgctgtatt cctaaactca ttattcaacc aagaagttca aattcccttg 120

accgaaagtt actgtggccc atgtcctaaa aactggatat gttacaaaaa taactgctac 180

caattttttg atgagagtaa aaactggtat gagagccagg cttcttgtat gtctcaaaat 240

gccagccttc tgaaagtata cagcaaagag gaccaggatt tacttaaact ggtgaagtca 300

tatcattgga tgggactagt acacattcca acaaatggat cttggcagtg ggaagatggc 360

tccattctct cacccaacct actaacaata attgaaatgc agaagggaga ctgtgcactc 420

tatgcctcga gctttaaagg ctatatagaa aactgttcaa ctccaaatac gtacatctgc 480

atgcaaagga ctgtgaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 540

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 600

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 660

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 720

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 780

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 840

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 900

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 960

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1020

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1080

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1140

cacatgcagg ccctgccccc tcgcggcagc ggggccacca acttcagcct gctgaagcag 1200

gccggcgacg tggaggagaa cccagggccc atggcagaag ctcaccaagc tgtggccttt 1260

cagttcacgg tcactccgga cgggattgac ctgcggctga gccatgaagc tcttagacaa 1320

atctatctct ctggacttca ttcctggaaa aagaagttca tcagattcaa gaacggcatc 1380

atcactggcg tgtacccggc aagcccctcc agttggctta tcgtggtggt gggcgtgatg 1440

acaacgatgt acgccaagat cgacccctcg ttaggaataa ttgcaaaaat caatcggact 1500

ctggaaacgg ccaactgcat gtccagccag acgaagaacg tggtcagcgg cgtgctgttt 1560

ggcaccggcc tgtgggtggc cctcatcgtc accatgcgct actccctgaa agtgctgctc 1620

tcctaccacg ggtggatgtt cactgagcac ggcaagatga gtcgtgccac caagatctgg 1680

atgggtatgg tcaagatctt ttcaggccga aaacccatgt tgtacagctt ccagacatcg 1740

ctgcctcgcc tgccggtccc ggctgtcaaa gacactgtga acaggtatct acagtcggtg 1800

aggcctctta tgaaggaaga agacttcaaa cggatgacag cacttgctca agattttgct 1860

gtcggtcttg gaccaagatt acagtggtat ttgaagttaa aatcctggtg ggctacaaat 1920

tacgtgagcg actggtggga ggagtacatc tacctccgag gacgagggcc gctcatggtg 1980

aacagcaact attatgccat ggatctgctg tatatccttc caactcacat tcaggcagca 2040

agagccggca acgccatcca tgccatcctg ctttacaggc gcaaactgga ccgggaggaa 2100

atcaaaccaa ttcgtctttt gggatccacg attccactct gctccgctca gtgggagcgg 2160

atgtttaata cttcccggat cccaggagag gagacagaca ccatccagca catgagagac 2220

agcaagcaca tcgtcgtgta ccatcgagga cgctacttca aggtctggct ctaccatgat 2280

gggcggctgc tgaagccccg ggagatggag cagcagatgc agaggatcct ggacaatacc 2340

tcggagcctc agcccgggga ggccaggctg gcagccctca ccgcaggaga cagagttccc 2400

tgggccaggt gtcgtcaggc ctattttgga cgtgggaaaa ataagcagtc tcttgatgct 2460

gtggagaaag cagcgttctt tgtgacgtta gatgaaactg aagaaggata cagaagtgaa 2520

gacccggata cgtcaatgga cagctacgcc aaatctctac tacacggccg atgttacgac 2580

aggtggtttg acaagtcgtt cacgtttgtt gtcttcaaaa acgggaagat gggcctcaac 2640

gctgaacact cctgggcaga tgcgccgatc gtggcccacc tttgggagta cgtcatgtcc 2700

attgacagcc tccagctggg ctatgcggag gatgggcact gcaaaggcga catcaatccg 2760

aacattccgt accccaccag gctgcagtgg gacatcccgg gggaatgtca agaggttata 2820

gagacctccc tgaacaccgc aaatcttctg gcaaacgacg tggatttcca ttccttccca 2880

ttcgtagcct ttggtaaagg aatcatcaag aaatgtcgca cgagcccaga cgcctttgtg 2940

cagctggccc tccagctggc gcactacaag gacatgggca agttttgcct cacatacgag 3000

gcctccatga cccggctctt ccgagagggg aggacggaga ccgtgcgctc ctgcaccact 3060

gagtcatgcg acttcgtgcg ggccatggtg gacccggccc agacggtgga acagaggctg 3120

aagttgttca agttggcgtc tgagaagcat cagcatatgt atcgcctcgc catgaccggc 3180

tctgggatcg atcgtcacct cttctgcctt tacgtggtgt ctaaatatct cgctgtggag 3240

tcccctttcc ttaaggaagt tttatctgag ccttggagat tatcaacaag ccagacccct 3300

cagcagcaag tggagctgtt tgacttggag aataacccag agtacgtgtc cagcggaggg 3360

ggctttggac cggttgctga tgacggctat ggtgtgtcgt acatccttgt gggagagaac 3420

ctcatcaatt tccacatttc ttccaagttc tcttgccctg agacggattc tcatcgcttt 3480

ggaaggcacc tgaaagaagc aatgactgac atcatcactt tgtttggtct cagttctaat 3540

tccaaaaag 3549

Claims

1. A constrained CAR that promotes T cell differentiation, characterized in that the chimeric antigen receptor is designated as: CD8a-NKG2D-41BB-CD3z-CPT1a; the concrete is that connect gradually: human CD8a molecular signal peptide CD8a, human NKG2D extracellular region, human CD8 molecular transmembrane region and 41BB molecular intracellular region 41BB, human CD3z molecular intracellular region CD3z, CPT1a CDS region sequence CPT1a;

Wherein, the intracellular region CD3Z of the human CD3Z molecule is connected with CPT1a CDS region sequence CPT1a by adopting a PA2 connection sequence P2A;

the CAR structure sequence of the specific CD8a-NKG2D-41BB-CD3z-CPT1a comprises 1183 amino acids, and the sequence is shown as SEQ ID NO. 1.

2. The restricted CAR that promotes T cell differentiation of claim 1, wherein the DNA sequence encoding the CAR structure of CD8a-NKG2D-41BB-CD3 _Z -P2A-CPT1a is shown as SEQ ID No. 2.

3. The use of a T cell differentiation promoting constrained CAR according to claim 1 for the preparation of an anti-human non-small cell lung cancer agent, wherein the use of the T cell differentiation promoting constrained CAR for the preparation of a lentiviral expression plasmid, transfecting T cells with the lentiviral expression plasmid, and then reinfusion of the transfected T cells.

4. Lentiviral expression plasmid prepared using the restricted CAR promoting T cell differentiation of claim 1, characterized in that it is prepared by the following steps:

(1) Obtaining the coding sequence DNA of the CAR by adopting the prior art;

5. The use of the lentiviral expression plasmid of claim 4 in the preparation of an anti-human non-small cell lung cancer agent, wherein the use is performed by transfecting the lentiviral expression plasmid into a CAR-T cell.

6. The use of the lentiviral expression plasmid of claim 5, wherein the step of applying comprises the steps of:

(1) Packaging lentiviruses;

Then mixing the constructed lentivirus expression plasmid with a packaging plasmid, and transfecting target cells to be transfected by using a liposome transfection reagent to prepare packaged lentivirus particles;

(2) Preparation of purified T cells

First, mononuclear cells are obtained by isolation from human peripheral blood;

then, separating to obtain purified CD3 ⁺ T cells, and activating for later use;

(3) T cell infection

Adding the lentiviral particles collected in step (1) to the cells after activation in step (2), and incubating to perform infection;

(4) Expansion of T cells

After further expansion and detection of the post-infection T cells incubated in step (3), further reinfusion for killing tumor cells.