CN114891815A

CN114891815A - DNA fragment, expression vector, chimeric antigen receptor T cell and application

Info

Publication number: CN114891815A
Application number: CN202210520393.3A
Authority: CN
Inventors: 张毅; 田永贵
Original assignee: First Affiliated Hospital of Zhengzhou University
Current assignee: Saidete Biopharmaceutical Co ltd
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-08-12
Also published as: CN116515874A

Abstract

The invention relates to a DNA fragment, an expression vector, a chimeric antigen receptor T cell and application, and belongs to the technical field of tumor immunotherapy. The DNA fragment comprises a nucleotide fragment for encoding CCR5, a nucleotide fragment for encoding a chimeric antigen receptor and a nucleotide fragment for encoding IL-12. The DNA fragment of the invention recombines the expressible nucleotide fragment for coding CCR5 and the expressible nucleotide fragment for coding IL-12 in the expressed DNA fragment of the chimeric antigen receptor, can improve the function of the chimeric antigen receptor T cell and the ability of infiltrating tumor, and simultaneously weakens the inhibition effect of the tumor microenvironment on the CART cell, thereby enhancing the anti-tumor effect of the CAR T cell.

Description

DNA fragment, expression vector, chimeric antigen receptor T cell and application

Technical Field

The invention relates to a DNA fragment, an expression vector, a chimeric antigen receptor T cell and application, and belongs to the technical field of tumor immunotherapy.

Background

Among the immunotherapy of tumors, CAR T (Chimeric Antigen Receptor T) immunotherapy shows good clinical efficacy in the treatment of hematologic malignancies, where CD 19-targeted CAR T cells have been approved for the treatment of CD19 positive lymphomas or leukemias. CAR T cell therapy for solid tumors is hampered by a number of challenges and has not achieved the desired therapeutic effect. Among them, the inability of CAR T cells to effectively home to the tumor site, CAR T cell status in the tumor microenvironment, immunosuppressive tumor microenvironment, etc. are key factors affecting CAR T cells in the treatment of malignancies, particularly solid tumors. Therefore, how to increase CAR T cell infiltration, improve CAR T cell function, improve immunosuppressive tumor microenvironment conditions in solid tumors is an important condition to address the application of CAR T cell therapy in solid tumors.

Disclosure of Invention

The invention aims to provide a DNA fragment which can improve the tumor infiltration capacity of chimeric antigen receptor T cells after being integrated into the chimeric antigen receptor T cells, and simultaneously weaken the inhibition effect of a tumor microenvironment on CAR T cells, so that the function of the CAR T cells is improved, and the anti-tumor effect of the CAR T cells is further enhanced.

The invention also provides an expression vector containing the DNA segment and a chimeric antigen receptor T cell containing the DNA segment or the expression vector.

The invention also provides an application of the DNA fragment, the expression vector or the chimeric antigen receptor T cell in preparing a tumor treatment medicament.

In order to achieve the above purpose, the technical scheme adopted by the DNA fragment of the invention is as follows:

a DNA fragment comprising a nucleotide fragment encoding CCR5, a nucleotide fragment encoding a chimeric antigen receptor, and a nucleotide fragment encoding IL-12.

The DNA fragment of the invention recombines an expressible nucleotide fragment coding CCR5 and an expressible nucleotide fragment coding IL-12 in the expressed DNA fragment of the chimeric antigen receptor, can code CCR5, the chimeric antigen receptor and IL-12 at the same time, can improve the function of the chimeric antigen receptor T cell and the ability of infiltrating tumor, and simultaneously weakens the inhibition effect of the tumor microenvironment on the CAR T cell, thereby enhancing the anti-tumor effect of the CAR T cell.

Since chemokines and their receptors play a crucial role in the migration of T cells. The expression of chemokines at the tumor site is closely related to T cell infiltration, tumor control and patient prognosis. Such as the chemokine CCL5 and CD8 in solid tumors ⁺ T cell infiltration is closely related and the expression of CCL5 is correlated with tumor patient survival. As another example CCL4 and CCL5 were highly expressed in tumor tissues of patients with esophageal cancer. According to the invention, after the nucleotide fragment for encoding CCR5 is expressed in a DNA fragment for expressing a chimeric antigen receptor in a recombination way, the CAR T cells over-expressing CCR5(CCL4 and CCL5 receptors) can increase the infiltration capacity of the CAR T cells to tumor tissues so as to eliminate tumors.

The solid tumor microenvironment has a large number of immunosuppressive cells and factors, which affect the function and proliferation of CAR T cells and limit the therapeutic effect of CAR T cells. Tumor Associated Macrophages (TAMs) are important immunoregulatory cells in the microenvironment. TAMs are largely divided into 2 classes, M1 and M2 cells. Among them, M1 cells exert tumor-inhibiting action, while M2 cells accelerate tumor progression by inhibiting T cell killing, promoting angiogenesis and metastasis, and the like. The DNA fragment can convert CAR T cells into IL-12 secretory cells, improve the IL-12 concentration in a tumor microenvironment and promote the conversion of M2 into M1 cells, so that the inhibition effect of the tumor microenvironment on the CAR T cells is relieved, the functions of the CAR T cells are enhanced, and a better tumor removal effect is achieved; in addition, high concentration of IL-12 can further promote T cell proliferation and cytotoxicity, Th1 cell differentiation and NK cell activation to enhance antitumor response.

Further, the chimeric antigen receptor comprises a signal peptide sequence, a single-chain antibody, a hinge region, a transmembrane region, an intracellular costimulatory region and an intracellular signal domain in sequence from the N-terminus to the C-terminus. Wherein the signal peptide can direct the nascent chimeric antigen receptor into the endoplasmic reticulum of the cell. The single-chain antibody in the chimeric antigen receptor can specifically bind to a tumor cell membrane antigen and can be selected according to the tumor type, for example, the single-chain antibody in the chimeric antigen receptor is a mesothelin single-chain antibody. The reason why the single-chain antibody is selected as the mesothelin single-chain antibody in the present invention is that mesothelin is highly expressed in mesothelioma, lung cancer, esophageal cancer, pancreatic cancer, ovarian cancer and other cancers, and the mesothelin-targeted CAR has the potential to treat various solid malignant tumors.

In order to effectively avoid immunological rejection and realize high-efficiency recognition of target cells, the mesothelin single-chain antibody is a human mesothelin single-chain antibody, and the coding gene sequence is shown as SEQ ID NO. 5. The coding gene sequence of the hinge region is shown as SEQ ID NO. 6. The signal peptide is a human CD8a molecular signal peptide, and the coding gene sequence is shown in SEQ ID No. 4. The transmembrane region is a human CD28 molecular transmembrane region, and the coding gene sequence is shown as SEQ ID NO. 7. The intracellular co-stimulation region is a human CD28 intracellular region and/or a human 41BB intracellular region, the coding gene sequence of the human CD28 intracellular region is shown as SEQ ID NO.8, and the coding gene sequence of the human 41BB intracellular region is shown as SEQ ID NO. 9. The intracellular signal domain is the intracellular domain of a human CD3z molecule, and the coding gene sequence is shown in SEQ ID NO. 10.

It is understood that the arrangement order of the nucleotide fragment encoding CCR5, the nucleotide fragment encoding the chimeric antigen receptor and the nucleotide fragment encoding IL-12 in the DNA fragment is not limited as long as the chimeric antigen receptor T cell containing the DNA fragment can express CCR5 and IL-12 at the same time as the expression of the chimeric antigen receptor protein. For example, the nucleotide fragment encoding CCR5 is located upstream of the nucleotide fragment encoding the chimeric antigen receptor and the nucleotide fragment encoding IL-12 is located downstream of the nucleotide fragment encoding the chimeric antigen receptor.

It is understood that the DNA fragment includes a promoter for controlling the expression of a nucleotide fragment encoding CCR5, a promoter for controlling the expression of a nucleotide fragment encoding a chimeric antigen receptor, and a promoter for controlling the expression of a nucleotide fragment encoding IL-12. Any two or three of the nucleotide fragment coding for CCR5, the nucleotide fragment coding for the chimeric antigen receptor and the nucleotide fragment coding for IL-12 can share one promoter to control the expression. The promoter is operably linked to a promoter that expresses a fragment of a nucleotide encoding CCR5, a fragment of a nucleotide encoding a chimeric antigen receptor, and a fragment of a nucleotide encoding IL-12. For example, the DNA fragment includes CMV promoter and EF 1-alpha promoter, and the CMV promoter-nucleotide fragment encoding CCR 5-EF 1-alpha promoter-nucleotide fragment encoding chimeric antigen receptor-gene encoding self-cleaving 2A connecting peptide-nucleotide fragment encoding IL-12 are sequentially linked. Further, the self-cleaving 2A linker peptide is a P2A linker peptide. It can be understood that: when the nucleotide fragment encoding the chimeric antigen receptor and the nucleotide fragment encoding IL-12 are linked by a gene encoding a self-cleaving 2A linker, the DNA fragment further includes a gene encoding a self-cleaving 2A linker. In addition to the CMV promoter and EF 1-alpha promoter described above, other promoters which can promote the corresponding genes may be used in the DNA fragment of the present invention.

Further, the nucleotide sequence of the DNA fragment is shown as SEQ ID NO. 14.

The technical scheme adopted by the expression vector of the invention is as follows:

an expression vector comprising the above DNA fragment.

The expression vector contains the DNA fragment, can effectively increase CAR T cell infiltration and tumor killing capacity, and simultaneously reduces immunosuppression of tumor microenvironment.

Further, the expression vector is a lentivirus plasmid recombined with the DNA segment. The lentiviral plasmid is preferably a pCDH-EF1a-MCS-T2A-GFP plasmid recombined with the DNA fragment.

The technical scheme adopted by the chimeric antigen receptor T cell is as follows:

a chimeric antigen receptor T cell comprising the above DNA fragment or comprising the above expression vector.

Compared with the conventional CAR T cells, the chimeric antigen receptor T cells (CART5-12 cells) can obviously increase the CAR T cell infiltration at the tumor site due to the expression of CCR 5; the expression of IL-12 enables CART5-12 cells to have stronger cytokine secretion and cytotoxicity capacity, and weakens the immunosuppression of tumor microenvironment; therefore, the CART5-12 cells of the invention show excellent anti-tumor activity, and experiments prove that (taking an esophageal cancer model as an example) immunotherapy adopting the CART5-12 cells of the invention can slow down the growth of tumors and prolong the survival rate of mice.

The technical scheme adopted by the application of the invention is as follows:

the application of the DNA fragment or the expression vector or the chimeric antigen receptor T cell in preparing a tumor treatment drug. The application of the invention can improve the anti-tumor activity of the tumor treatment medicine and delay the growth of tumors.

Further, the tumor is a solid tumor.

Drawings

FIG. 1 is a schematic representation of the engineered CAR structure in the lentiviral expression plasmids of example 2 and experimental examples, transfection efficiency, expression of CCR5 and IL-12, and the structure of the lentiviral expression plasmid prepared in example 2, wherein: FIG. 1A is a schematic representation of the CAR structure in a recombinant lentiviral expression plasmid expressing mesoCAR and a recombinant lentiviral expression plasmid expressing mesoCAR-CCR 5-IL-12; figure 1B is a graph of the transfection efficiency of flow cytometry assay CAR T cells;

FIGS. 1C and 1D are schematic diagrams of flow cytometry detection of CCR5 and IL-12 expression in CAR T cells, respectively, and FIG. 1E is a schematic structural diagram of the lentiviral expression plasmid prepared in example 2;

figure 2 is the expression profile of functional molecules of CAR T cells, wherein: detecting the expression of the effector cytokine in the CAR T cell by flow cytometry;

FIG. 3 is a graph showing the results of the killing efficiency of CAR T cells against tumor cells (KYSE-510 cells);

figure 4 is the transwell results of CAR T cells;

figure 5 is a graph of the effect of CAR T cell supernatant on macrophage polarization, wherein: FIG. 5A is a schematic drawing of the ratios of M1 and M2 in macrophages after supernatant treatment for flow cytometry detection of CAR T cells; FIGS. 5B and 5C are schematic diagrams of qPCR detection of expression of M1 and M2 related genes in macrophages, respectively; FIG. 5D is a schematic representation of immunofluorescence detecting CD163 expression in macrophages following supernatant treatment of CAR T cells;

figure 6 is a mouse esophageal cancer model testing the anti-tumor effect of different CAR T cells, wherein: FIG. 6A is a fluorescence image of tumors from mice at various time points; FIG. 6B is a fluorescence histogram.

Detailed Description

The term "single-chain antibody" (scFv) as used herein refers to an antibody fragment having the ability to bind to an antigen, which is formed by the amino acid sequence of the light chain variable region (VL region) and the amino acid sequence of the heavy chain variable region (VH region) being linked by a hinge. In certain embodiments, the single chain antibody (scFv) of interest is from an antibody of interest. The antibody of interest can be a human antibody, including human murine chimeric antibodies and humanized antibodies. The antibody may be secreted or membrane anchored.

The "coding gene" of the invention is defined herein as the part of the nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR, monoclonal antibody, hinge region and transmembrane region). The boundaries of the coding sequence are generally determined by a ribosome binding site immediately upstream of the 5 'open reading frame of the mRNA (for prokaryotic cells) and a transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.

The "chimeric antigen receptor" (CAR) of the present invention is an artificially engineered receptor that is capable of anchoring a specific molecule (e.g., an antibody) that recognizes a tumor cell surface antigen to an immune cell (e.g., a T cell) so that the immune cell recognizes the tumor antigen or a viral antigen and kills the tumor cell or a virally infected cell. The CAR typically comprises, in order, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signal region. In general, polypeptides that bind to tumor cell membrane antigens are capable of binding with moderate affinity to membrane antigens that are widely expressed by tumor cells. The polypeptide binding to the tumor cell membrane antigen may be a natural polypeptide or an artificially synthesized polypeptide.

The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, which results in expression of the latter. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence.

The technical solution of the present invention will be further described with reference to the following embodiments. The information concerning the biological material, the test reagents and the test apparatus in the examples and experimental examples is as follows:

biological material:

pCDH-EF1a-MCS-T2A-GFP plasmid was purchased from System Biosciences (site of System Biosciences: https:// www.systembio.com /);

human esophageal cancer cell line (KYSE-510 cells) and human embryonic kidney cell line 293T were obtained from Shanghai cell bank of Chinese academy of sciences;

during the experiment, all cell lines were cultured in DMEM or RPMI-1640 medium (Gibco) containing 10% fetal bovine serum supplemented with 100U/mL penicillin and 100. mu.g/mL streptomycin.

Experimental reagent:

CD3/CD28 activated beads were purchased from Miltenyi Biotec, Inc.;

flow antibodies were purchased from BioLegend;

transfection reagent jetPRIME was purchased from

A company;

5 μm Transwell plates were purchased from Corning;

qPCR fluorescent dye was purchased from BCS company;

female NOD/SCID mice, 5 weeks old, were obtained from Wintolite, Beijing.

An experimental instrument:

BD flow cytometer (BD, BD FACSCAnto II), C6 flow cytometer (BD, BD)

) Instruments such as BIO-RAD temperature gradient PCR instrument (BIO-RAD, C1000Touch), electrophoresis instrument (Beijing, six Biotechnologies Co., Ltd., DYY-6D), blue light gel cutting instrument (Sangon Biotech, G500312EQU312), and fluorescence microscope (OLYMPUS, IX73) are all common equipments in the field, and are not described in detail.

Example 1

The nucleotide sequence of the DNA fragment of this example is shown in SEQ ID NO.14, which was named: mesoCAR-CCR5-IL-12 (abbreviated as CAR 5-12). The specific structure of the DNA fragment is as follows: the nucleotide fragment (CDS region sequence) encoding CCR5 is linked and promoted by a CMV promoter, the gene encoding a signal peptide of a human CD8a molecule, the gene encoding scFv (meso) derived from a human mesothelin antibody, the hinge region, the gene encoding a transmembrane region of a human CD28 molecule, the gene encoding an intracellular region of a CD28 molecule, the gene encoding an intracellular region of a human 41BB molecule, the gene encoding an intracellular region of a human CD3Zeta molecule, the gene encoding a P2A linking peptide, and the nucleotide fragment (CDS region sequence) encoding human IL-12 are sequentially linked and promoted after the connection of an EF1 alpha promoter, namely the nucleotide molecules of the embodiment are sequentially from upstream to downstream: CMV promoter-nucleotide fragment encoding CCR 5-gene encoding EF1 α promoter-gene encoding signal peptide of CD8 a-scfv (meso) encoding gene-hinge region-gene encoding transmembrane region of CD 28-gene encoding intracellular region of CD 28-gene encoding BB encoding gene of CD3 Zeta-nucleotide fragment encoding human IL-12-encoding gene of P2A linker peptide; wherein, the coding gene sequence of CMV promoter is shown as SEQ ID NO.1, the coding gene sequence of nucleotide fragment (CDS region) for coding human CCR5 is shown as SEQ ID NO.2, the coding gene sequence of EF1 alpha promoter is shown as SEQ ID NO.3, the coding gene sequence of human CD8a molecular signal peptide (CD8a) is shown as SEQ ID NO.4, the coding gene sequence of scFv (meso) from human mesothelin antibody is shown as SEQ ID NO.5, the coding gene sequence of hinge region is shown as SEQ ID NO.6, the coding gene sequence of transmembrane region of human CD28 molecule is shown as SEQ ID NO.7, the coding gene sequence of intracellular region of CD28 molecule (CD28) is shown as SEQ ID NO.8, the coding gene sequence of intracellular region of human 41BB molecule is shown as SEQ ID NO.9, the coding gene sequence of intracellular region (CD3Zeta) of human CD3z molecule is shown as SEQ ID NO.10, the coding gene sequence of P2A connecting peptide is shown as SEQ ID NO.11, the nucleotide fragment (CDS region) sequence encoding human IL-12 is shown in SEQ ID NO. 12.

Example 2

The chimeric antigen receptor T cell of this example was prepared by a method comprising the steps of:

1) shanghai Sangon Biotech In China was entrusted to synthesize a nucleotide fragment (nucleotide sequence is shown In SEQ ID NO. 2) encoding CCR5, a nucleotide fragment (nucleotide sequence is shown In SEQ ID NO. 13) encoding a chimeric antigen and a nucleotide fragment (nucleotide sequence is shown In SEQ ID NO. 12) encoding IL-12, respectively, and the plasmids were digested at 37 ℃ with pCDH-EF1a-MCS-T2A-GFP plasmid (purchased from System biosciences) as expression vectors using the restriction enzyme EcoR I (NEB), and then the synthesized DNA coding sequences were integrated and recombined into pCDH-EF1a-MCS-T2A-GFP plasmid using In-Fusion HD Cloning Kits (Takara Bio). And finally, transforming the ligation product into competent cells, screening, carrying out amplification culture, and then further extracting plasmids to obtain recombinant lentiviral expression plasmids capable of expressing mesoCAR-CCR5-IL-12 (the related operation refers to conventional operation in the prior art and is not described in detail), wherein the CAR structure is shown in fig. 1A, and the CAR structure is the DNA fragment of example 1.

2) Lentivirus packaging was performed with 293T cells as the primary cells to be transfected, specifically:

six-well plate was plated and incubated at 37 ℃ with 5% CO ₂ Incubate in incubator to culture 293T cells (DMEM medium) for 24 h; then 1 μ g of the recombinant lentivirus expression plasmid capable of expressing mesoCAR-CCR5-IL-12 constructed in step 1), 0.6 μ g of the packaging plasmid psPAX2, 0.4 μ g of the plasmid PMD2.G, and

6 mu L of transfection reagent and 200 mu L of transfection buffer are mixed to transfect the 293T of the target cell; replacing the medium with a normal DMEM medium after transfection for 4 hours;

after culturing for another 48 hours, collecting the virus supernatant (i.e. the packaged virus-like particles), centrifuging at 1500rpm for 10 minutes, and storing the supernatant at-80 ℃ for later use to infect T cells.

3) Preparation of CD3 ⁺ T cells, in particular:

separating mononuclear cells from human peripheral blood by density gradient centrifugation, and separating with T-cell separation magnetic beads to obtain purified CD3 ⁺ A T cell; purifying the obtained CD3 ⁺ T cells were plated in 24-well cell culture plates with RPMI-1640 containing 100IU/mL IL-2Culturing;

before use, CD3 is mixed ⁺ T cells were activated with beads (1. mu.L/10) using CD3/CD28 ⁷ cells) were activated for 2 days.

4) Infecting the virus collected in step 2) with T cells, specifically:

using 24-well cell culture plates, CD3 was added 2 days after activation per well ⁺ T cell (10) ⁶ Per well) while adding 1mL of collected viral supernatant and polybrene (final concentration 8 μ g/mL) per well, and incubating overnight for infection; after 24 hours, the cells were replaced with normal RPMI-1640 containing 100IU/mLIL-2 and cultured to obtain chimeric antigen receptor T cells.

Fifth day after virus infection in step 4) of example 2, the transfection efficiency was analyzed by detecting Green Fluorescent Protein (GFP) by flow cytometry, and the results are shown in fig. 1B.

As a control, the inventors also transduced non-transduced CD3 ⁺ The related experiments were performed in synchronization with T cells, CARTmeso cells (same as the CARTmeso cells of the experimental examples below) and CART19 (CAR T cells targeting CD19, same as the CAR T19 cells of the experimental examples below) cells, and the transfection efficiency was analyzed under the same conditions, and the results are shown in fig. 1B.

Statistically relevant transfection efficiencies were calculated by measuring T cell GFP expression and the results indicated that: the transfection efficiency of the CAR T cells targeting CD19 (CART19 cells) was 41.7%, that of the mesothelin-targeting CAR T cells (CART meso cells) was 44.1%, and that of the CCR5 and IL-12 modified CAR T cells (CART5-12) was 47.7%, which indicates that the constructed CAR expression plasmids can successfully infect T cells after being packaged into lentiviral particles and are well expressed, and thus can be used in subsequent experiments.

Examples of the experiments

Using the chimeric antigen receptor T cells prepared in example 2 (hereinafter referred to as CART5-12 cells), the inventors further performed relevant cell experiments and animal experiment analyses while using CART eso cells and CART19 (CD 19-targeted CAR T cells) cells as controls.

Wherein, the CARTmeso cell is obtained by replacing the recombinant lentivirus expression plasmid capable of expressing mesoCAR-CCR5-IL-12 in the step 2) of the example 2 with the recombinant lentivirus expression plasmid capable of expressing mesoCAR-CAR at the time of preparation, and the content is not completely the same as the example 2; the recombinant lentiviral expression plasmid expressing meso-CAR differs from the recombinant lentiviral expression plasmid capable of expressing meso-CCR 5-IL-12 in example 2 only in that the DNA fragment used is replaced by the meso-CAR in preparation, and the CAR structure in the recombinant lentiviral expression plasmid is shown in FIG. 1A.

The CART19 cell is obtained by replacing the recombinant lentivirus expression plasmid capable of expressing mesoCAR-CCR5-IL-12 in step 2) of example 2 with a recombinant lentivirus expression plasmid capable of expressing CD19-CAR at the time of preparation, and the content is not completely the same as that of example 2; wherein the recombinant lentiviral expression plasmid expressing CD19-CAR differs from the recombinant lentiviral expression plasmid capable of expressing mesoCAR-CCR5-IL-12 in example 2 only in that the DNA fragment used was replaced with CD19-CAR at the time of preparation.

The following description and analysis are provided with respect to relevant experimental items and experimental results.

1) Expression analysis of CAR T cells CCR5 and IL-12

First, T cells at 5 days after virus infection in the preparation of CART5-12 cells, CART19 cells and CART meso cells were collected, and the expression of CCR5 and IL-12 in CAR T cells was examined by flow cytometry.

The results are shown in FIGS. 1C and 1D, and analysis can see that: the expression ratio of CCR5 and IL-12 in CART19 and CART meso cells is 10-20%, while the expression ratio of CCR5 and IL-12 in CART5-12 cells is 30-60%, which indicates that the modified CART5-12 cells successfully over-express CCR5 and IL-12 and can be used for subsequent experiments.

2) CAR T cell functional assays

Detecting the expression of effector cytokines in CAR T cells, in particular: KYSE-510 cells (high expressing mesothelin) were mixed with CAR T cells in a targeted ratio of 1: 1 co-incubation for 24 hours; the CAR T cells collected were washed with PBS containing 2% serum, surface stained by adding 1 μ L of CD3 flow antibody to the cells, and incubated for 20 minutes at 4 ℃ protected from light; then, incubating the cells with 4% paraformaldehyde and a membrane breaking agent respectively for 30 minutes at 4 ℃; cells were then stained with 1 μ L cytokine antibody (BioLegend) (IFN- γ, Perforin and granzyme b); all samples were analyzed using a flow cytometer.

The results are shown in fig. 2, and analysis can see that:

after co-incubation with tumor cells, mesothelin-targeted CART meso cells had increased ability to express functional molecules compared to CART19 cells, whereas CART5-12 cells had higher expression of functional molecules than CART meso cells, suggesting that after CCR5 and IL-12 modification, the effector function of CART5-12 cells was increased. Specific examples thereof include: after 24 hours of co-incubation with tumor cells, 10-17% of CART19 expressing IFN-gamma, 10-20% of Perforin, 10-20% of granzyme B, 20-30% of CARTmeso cell expressing IFN-gamma, 10-38% of Perforin, 15-40% of granzyme B, increased expression of CART5-12 cells, 34-50% of IFN-gamma, 40-60% of Perforin and 45-70% of granzyme B. (P <0.05, P <0.01, P <0.00 in FIG. 2.)

3) Cytotoxicity assays for CAR T cells

Tumor cells with different mesothelin expression conditions are taken as experimental objects, CAR T cells are taken as experimental 'medicaments', and the technical effect of the constructed CAR is detected and evaluated through tumor cell apoptosis. The specific experimental procedures are briefly described as follows.

Respectively using human esophageal cancer cell line KYSE-510 cells highly expressing mesothelin as target cells, using different treated CART cells as effector cells (CART19, CARTmeso and CART5-12), and co-incubating the CAR T cells and the target cells in a 96-well plate for 6 hours according to different effective-to-target ratios (1: 1, 5: 1 and 10: 1), wherein each group is provided with three multiple wells.

After the incubation was completed, the cell supernatant after the co-incubation was collected, and the cell pellet was resuspended in Annexin V-binding buffer (BioLegent), followed by addition of 1. mu.L of Annexin V antibody (BioLegent) and incubation at 4 ℃ in a dark environment for 20 minutes; before the machine, 1. mu.L of Propidium (Propidium iodide, Sigma) was added. And detecting and analyzing by adopting a flow cytometer.

The results are shown in fig. 3, and it can be seen that:

compared with the CART19 cell, the CART meso cell can effectively kill the tumor cells with high expression of mesothelin, while the killing effect of the CART5-12 cell is better than that of the CART meso cell under the same proportion, and the result indicates that: in the presence of CCR5 and IL-12, CART5-12 cells have better effect on eliminating tumor cells. Specific examples thereof include: the effective target ratio is 10: under the condition of 1, the difference effect is more obvious, the removal effect of CARTmeso cells on tumor cells with high mesothelin expression is 30-37%, and the removal effect of CART5-12 cells on tumor cells with high mesothelin expression can reach more than 40%, specifically 40-55%. (p <0.05, p <0.01 in FIG. 3.)

4) Transwell experiment

The migration capacity of CAR T cells was evaluated in 24-well transwell plates (Corning Inc, Corning) with 5 μm pore size polycarbonate membranes. The specific operation is as follows:

600 μ L of serum-free RPMI-1640 medium containing CCL5 protein (PeproTech, final concentration 10 μ g/mL) was added to the lower chamber of the transwell plate, and 200 μ L of CAR T cells (CART19, CARTmeso, CART5-12) resuspended in serum-free RPMI-1640 medium were separately supplemented to the upper chamber (5X 10 per well ⁵ Individual cells); subjecting 24-well plate to 37 deg.C and 5% CO ₂ Culturing for 3 hours in the incubator; cells in the lower chamber were counted by flow cytometry to determine the difference in CAR T cell migration.

The results are shown in fig. 4, where it can be seen that:

when the CART19 and CARTmeso cells are used as upper chamber cells, the cell mobility is 0.5-1.4, and when the CART5-12 cells are used as upper chamber cells, the cell mobility is 1.5-2.5, which shows that the chemokine CCL5 can increase the migration capacity of the CART5-12 cells. (p <0.05, p <0.01 in FIG. 4.)

5) Analysis of the Effect of CAR T cells on macrophage polarization

Detecting the polarization of the supernatant of the CAR T cells to macrophages, and specifically: magnetic sorting method for obtaining CD14 from peripheral blood of healthy people ⁺ Monocytes, induced with M-CSF (final concentration 20ng/mL) for 7 days, M2 cells matured. The collected supernatants of each CAR T cell (CART19, CART eso, CART5-12) were treated for 3 days with M2 cells, which were collected for subsequent macrophage polarization assay.

Firstly, flow cytometry detects the polarization of macrophage cells, and specifically: the collected macrophages were washed with PBS containing 2% serum, surface stained by adding 1 μ L of flow antibodies (7AAD, CD14, CD86, and CD163) to the cells, incubated for 20 minutes at 4 ℃ in the absence of light, and all samples were analyzed using a flow cytometer.

The results are shown in fig. 5A, and analysis can see that:

CD14 in macrophages after treatment of CART19 or CARTmeso cell supernatants ⁺ CD86 ⁺ The proportion of which is 5 to 11 percent, CD14 ⁺ CD163 ⁺ The proportion is 80 to 90 percent, and the CART5-12 cell CD14 ⁺ CD86 ⁺ The proportion of which is 18 to 30 percent, and CD14 ⁺ CD163 ⁺ The ratio is 60% -78%, the ratio of M1 in CART5-12 cells is increased and the ratio of M2 is decreased. (p in FIG. 5A)<0.05,**p<0.01,***P<0.001。)

Secondly, qPCR detects the expression condition of the polarization-related gene of the macrophage, and the specific operation is as follows: adding 1mL of Trizol into each tube of the collected macrophages for resuspension, extracting RNA, and carrying out reverse transcription by using a TaKaRa reverse transcription kit to obtain cDNA; taking 2 mu L of cDNA as a template, adding 10 mu L of qPCR fluorescent dye and 8 mu L of 1 mu M upstream and downstream primer mixture, and obtaining 20 mu L of reaction system with the reaction conditions as follows: 10min at 95 ℃, 10s at 60 ℃, 10s at 72 ℃, 40 cycles, and 10min at 72 ℃. The M1 and M2 related genes (M1: TNF-a and NOS 2; M2: MRC1, CD163 and ARG1) were examined, and the results were expressed as 2 ^-ΔΔCt And (4) showing.

The results are shown in fig. 5B and 5C, and analysis can see that:

3 days after the supernatant of CAR T cells (CART19, CART eso or CART5-12) was treated with M2 cells, the expression of M1-related genes (TNF-a and NOS2) and M2-related genes (MRC1, CD163 and ARG1) were down-regulated in macrophages after CART5-12 cell treatment, relative to CART19 and CART eso cells. Specific examples thereof include: the TNF-alpha expressed by macrophages after CART19 or CARTmeso cell supernatant treatment is 0.8-1.4, NOS2 is 0.5-1.7, MRC1 is 0.9-1.6, CD163 is 0.9-1.3, ARG1 is 1.1-1.5, the TNF-alpha expressed by macrophages after CART5-12 cell supernatant treatment is 1.8-4.0, NOS2 is 5.0-8.5, MRC1 is 0.3-0.6, CD163 is 0.5-0.8, and ARG1 is 0.5-0.8. (p <0.05, p <0.01 in FIGS. 5B and 5℃)

Subsequently, immunofluorescence detects the expression of CD163 by macrophages. The specific operation is as follows: CAR T cell supernatant treated macrophages were washed and fixed with PBS, permeabilized for 10min and blocked for 30min, incubated overnight with CD163 immunofluorescent antibody (abcam), secondary antibody incubated and nucleated with DAPI, and visualized and photographed under a fluorescent microscope (OLYMPUS, IX 73).

The results are shown in fig. 5D, and analysis can see that:

expression of CD163 is reduced following macrophage treatment with CART5-12 cells relative to CART19 and CART teso cells. The overall results in FIG. 5 show that CCR5 and IL-12 engineered CART5-12 can increase M1 polarization in macrophages, decrease M2 polarization, and promote immune responses.

6) Animal experiment of tumor growth in mice

On the basis of the above experiments, the inventors further performed mouse animal experiments, and the specific procedures are briefly described as follows.

5 weeks old female NOD/SCID immunodeficient mice were injected subcutaneously with 5X 10 resuspended in PBS ⁵ KYSE-510 cell mixture for expressing luciferase over-expressed CCL5 5X 10 ⁵ Constructing a mouse esophageal cancer tumor-bearing model by using the macrophages (induced by M-CSF for 7 days); 7 days later, each mouse was given an intravenous infusion of 5X 10 ⁶ CART19 cells, CARTmeso cells, CART5-12 cells (5 per group) were treated. Fluorescence changes expressed by tumors were detected during the experiment using a small animal in vivo imaging device (bioluminescent photographs taken every 7 days). When detecting the fluorescence change of tumor expression by using a small animal living body imaging device, firstly, anesthetizing a mouse in an induction room by using 3% isoflurane (RWD life science); each mouse was then injected intraperitoneally with a syringe with 100uL of d-fluorescein solution (0.15mg/mL, Yeasen Biotech co., Ltd.), 10 minutes later fluorescence was detected using the animal in vivo imaging device IVIS lumine, Series iii spectrometer (caliper Life science), and finally analyzed using the live image 4.3.1software (PerkinElmer, Waltham, MA, USA).

The results are shown in FIG. 6. The statistics and analysis of the results can show that:

in the CART19 and CARTmeso treatment groups, mice in each group were treated on day 21More than 60% of the mice had died, while the mice treated with CART5-12 cells were still alive all the time at 21 days. The tumor fluorescence of mice treated by CART19 and CARTmeso cells reaches 5 x 10 ⁹ About, the fluorescence of the mouse tumor treated by the CART5-12 cell is controlled at 5X 10 ⁸ Grades and comparison among groups show that the CART5-12 treatment group is obviously different from the former two groups, and fluorescence is obviously reduced, which indicates that the CART5-12 cells show excellent anti-tumor capability. (in FIG. 6B. x.p)<0.01,***P<0.001。)

Taken together, the results suggest that modification of engineered CAR T cells with CCR5 and IL-12 can improve CAR T cell immune function and delay tumor growth. The anti-tumor effect is mainly reflected in the following three aspects: firstly, the modification of CCR5 and IL-12 promotes the secretion of effector factors of CAR T cells, enhancing the killing ability of CAR T cells; secondly, the introduction of chemokine receptors can increase the migration of CAR T cells to the tumor site and exert effector functions; finally, CART5-12 cells promote polarization of M2 to M1 cells, reduce the M2/M1 cell ratio, reduce tumor microenvironment immunosuppression, thereby inhibiting tumor progression and increasing CAR T cell killing activity. Based on the results, a certain technical foundation can be laid for the application of CAR T technology in solid tumors.

<110> first subsidiary Hospital of Zhengzhou university

<120> DNA fragment, expression vector, chimeric antigen receptor T cell, and use

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 391

<212> DNA

<213> Artificial sequence

<400> 1

tcgatactag tattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 60

ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 120

tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 180

tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 240

gacgcaaatg ggcggtaggc gtgtacggtg ggaggtttat ataagcagag ctcgtttagt 300

gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagattcta 360

gagctagcct cgaggaattt aaatcttcga a 391

<210> 2

<211> 1059

<212> DNA

<213> Artificial sequence

<400> 2

atggattatc aagtgtcaag tccaatctat gacatcaatt attatacatc ggagccctgc 60

caaaaaatca atgtgaagca aatcgcagcc cgcctcctgc ctccgctcta ctcactggtg 120

ttcatctttg gttttgtggg caacatgctg gtcatcctca tcctgataaa ctgcaaaagg 180

ctgaagagca tgactgacat ctacctgctc aacctggcca tctctgacct gtttttcctt 240

cttactgtcc ccttctgggc tcactatgct gccgcccagt gggactttgg aaatacaatg 300

tgtcaactct tgacagggct ctattttata ggcttcttct ctggaatctt cttcatcatc 360

ctcctgacaa tcgataggta cctggctgtc gtccatgctg tgtttgcttt aaaagccagg 420

acggtcacct ttggggtggt gacaagtgtg atcacttggg tggtggctgt gtttgcgtct 480

ctcccaggaa tcatctttac cagatctcaa aaagaaggtc ttcattacac ctgcagctct 540

cattttccat acagtcagta tcaattctgg aagaatttcc agacattaaa gatagtcatc 600

ttggggctgg tcctgccgct gcttgtcatg gtcatctgct actcgggaat cctaaaaact 660

ctgcttcggt gtcgaaatga gaagaagagg cacagggctg tgaggcttat cttcaccatc 720

atgattgttt attttctctt ctgggctccc tacaacattg tccttctcct gaacaccttc 780

caggaattct ttggcctgaa taattgcagt agctctaaca ggttggacca agctatgcag 840

gtgacagaga ctcttgggat gacgcactgc tgcatcaacc ccatcatcta tgcctttgtc 900

ggggagaagt tcagaaacta cctcttagtc ttcttccaaa agcacattgc caaacgcttc 960

tgcaaatgct gttctatttt ccagcaagag gctcccgagc gagcaagctc agtttacacc 1020

cgatccactg gggagcagga aatatctgtg ggcttgtga 1059

<210> 3

<211> 546

<212> DNA

<213> Artificial sequence

<400> 3

aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60

cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240

acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300

ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360

tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420

cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480

actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540

gcctac 546

<210> 4

<211> 63

<212> DNA

<213> Artificial sequence

<400> 4

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 5

<211> 738

<212> DNA

<213> Artificial sequence

<400> 5

caggtgcagc tggtgcagtc tggggctgag gtgaagcggc ctggggcctc agtgcaggtt 60

tcctgcaggg catctggata ctccatcaac acctactata tgcaatgggt gcgacaggcc 120

cctggagcag ggcttgagtg gatgggagta atcaacccta gtggtgtcac aagctacgca 180

cagaagttcc agggcagagt caccttgacc aatgacacgt ccacgaacac agtctacatg 240

cagctgaaca gcctgacatc tgcagacacg gccgtgtatt actgtgcgag atgggcattg 300

tggggggatt tcgggatgga cgtctggggc aagggaaccc tggtcaccgt ctcctcaggt 360

ggtggtggtt ctggtggtgg tggttctggt ggtggtggtt ccggtggtgg tggttccgac 420

atccagatga cccagtctcc ttccaccctg tctgcatcta ttggagacag agtcaccatc 480

acctgccggg ccagtgaggg tatttatcac tggttggcct ggtatcagca gaagccaggg 540

aaagccccta aactcctgat ctataaggcc tctagtttag ccagtggggc cccatcaagg 600

ttcagcggca gtggatctgg gacagatttc actctcacca tcagcagcct gcagcctgat 660

gattttgcaa cttattactg ccaacaatat agtaattatc cgctcacttt cggcggaggg 720

accaagctgg agatcaaa 738

<210> 6

<211> 135

<212> DNA

<213> Artificial sequence

<400> 6

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 7

<211> 81

<212> DNA

<213> Artificial sequence

<400> 7

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 8

<211> 123

<212> DNA

<213> Artificial sequence

<400> 8

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 9

<211> 126

<212> DNA

<213> Artificial sequence

<400> 9

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 10

<211> 339

<212> DNA

<213> Artificial sequence

<400> 10

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180

aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240

cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300

acctacgacg cccttcacat gcaggccctg ccccctcgc 339

<210> 11

<211> 66

<212> DNA

<213> Artificial sequence

<400> 11

ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60

ggacct 66

<210> 12

<211> 1608

<212> DNA

<213> Artificial sequence

<400> 12

atgggtcacc agcagttggt catctcttgg ttttccctgg tttttctggc atctcccctc 60

gtggccatat gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatccggat 120

gcccctggag aaatggtggt cctcacctgt gacacccctg aagaagatgg tatcacctgg 180

accttggacc agagcagtga ggtcttaggc tctggcaaaa ccctgaccat ccaagtcaaa 240

gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300

ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaggaccag 360

aaagaaccca aaaataagac ctttctaaga tgcgaggcca agaattattc tggacgtttc 420

acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480

ggctcttctg acccccaagg ggtgacgtgc ggagctgcta cactctctgc agagagagtc 540

agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600

gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660

gaaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac 720

ttgcagctga agccattaaa gaattctcgg caggtggagg tcagctggga gtaccctgac 780

acctggagta ctccacattc ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840

agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900

cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc atcttggagc 960

gaatgggcat ctgtgccctg cagtgttcct ggagtagggg tacctggggt gggcgccaga 1020

aacctccccg tggccactcc agacccagga atgttcccat gccttcacca ctcccaaaac 1080

ctgctgaggg ccgtcagcaa catgctccag aaggccagac aaactctaga attttaccct 1140

tgcacttctg aagagattga tcatgaagat atcacaaaag ataaaaccag cacagtggag 1200

gcctgtttac cattggaatt aaccaagaat gagagttgcc taaattccag agagacctct 1260

ttcataacta atgggagttg cctggcctcc agaaagacct cttttatgat ggccctgtgc 1320

cttagtagta tttatgaaga cttgaagatg taccaggtgg agttcaagac catgaatgca 1380

aagctgctga tggatcctaa gaggcagatc tttctagatc aaaacatgct ggcagttatt 1440

gatgagctga tgcaggccct gaatttcaac agtgagactg tgccacaaaa atcctccctt 1500

gaagaaccgg atttttataa aactaaaatc aagctctgca tacttcttca tgctttcaga 1560

attcgggcag tgactattga tagagtgatg agctatctga atgcttcc 1608

<210> 13

<211> 1605

<212> DNA

<213> Artificial sequence

<400> 13

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgcaggtgc agctggtgca gtctggggct gaggtgaagc ggcctggggc ctcagtgcag 120

gtttcctgca gggcatctgg atactccatc aacacctact atatgcaatg ggtgcgacag 180

gcccctggag cagggcttga gtggatggga gtaatcaacc ctagtggtgt cacaagctac 240

gcacagaagt tccagggcag agtcaccttg accaatgaca cgtccacgaa cacagtctac 300

atgcagctga acagcctgac atctgcagac acggccgtgt attactgtgc gagatgggca 360

ttgtgggggg atttcgggat ggacgtctgg ggcaagggaa ccctggtcac cgtctcctca 420

ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttccggtgg tggtggttcc 480

gacatccaga tgacccagtc tccttccacc ctgtctgcat ctattggaga cagagtcacc 540

atcacctgcc gggccagtga gggtatttat cactggttgg cctggtatca gcagaagcca 600

gggaaagccc ctaaactcct gatctataag gcctctagtt tagccagtgg ggccccatca 660

aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 720

gatgattttg caacttatta ctgccaacaa tatagtaatt atccgctcac tttcggcgga 780

gggaccaagc tggagatcaa aaccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgattttt gggtgctggt ggtggttggt 960

ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1020

agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080

cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1200

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1260

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1320

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1380

cgtggccggg accctgagat ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc 1440

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1500

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1560

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc 1605

<210> 14

<211> 5275

<212> DNA

<213> Artificial sequence

<400> 14

tcgatactag tattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 60

ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 120

tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 180

tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 240

gacgcaaatg ggcggtaggc gtgtacggtg ggaggtttat ataagcagag ctcgtttagt 300

gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagattcta 360

gagctagcct cgaggaattt aaatcttcga aatggattat caagtgtcaa gtccaatcta 420

tgacatcaat tattatacat cggagccctg ccaaaaaatc aatgtgaagc aaatcgcagc 480

ccgcctcctg cctccgctct actcactggt gttcatcttt ggttttgtgg gcaacatgct 540

ggtcatcctc atcctgataa actgcaaaag gctgaagagc atgactgaca tctacctgct 600

caacctggcc atctctgacc tgtttttcct tcttactgtc cccttctggg ctcactatgc 660

tgccgcccag tgggactttg gaaatacaat gtgtcaactc ttgacagggc tctattttat 720

aggcttcttc tctggaatct tcttcatcat cctcctgaca atcgataggt acctggctgt 780

cgtccatgct gtgtttgctt taaaagccag gacggtcacc tttggggtgg tgacaagtgt 840

gatcacttgg gtggtggctg tgtttgcgtc tctcccagga atcatcttta ccagatctca 900

aaaagaaggt cttcattaca cctgcagctc tcattttcca tacagtcagt atcaattctg 960

gaagaatttc cagacattaa agatagtcat cttggggctg gtcctgccgc tgcttgtcat 1020

ggtcatctgc tactcgggaa tcctaaaaac tctgcttcgg tgtcgaaatg agaagaagag 1080

gcacagggct gtgaggctta tcttcaccat catgattgtt tattttctct tctgggctcc 1140

ctacaacatt gtccttctcc tgaacacctt ccaggaattc tttggcctga ataattgcag 1200

tagctctaac aggttggacc aagctatgca ggtgacagag actcttggga tgacgcactg 1260

ctgcatcaac cccatcatct atgcctttgt cggggagaag ttcagaaact acctcttagt 1320

cttcttccaa aagcacattg ccaaacgctt ctgcaaatgc tgttctattt tccagcaaga 1380

ggctcccgag cgagcaagct cagtttacac ccgatccact ggggagcagg aaatatctgt 1440

gggcttgtga aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc 1500

ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt 1560

ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 1620

ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 1680

ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc 1740

ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg 1800

cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt 1860

gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc 1920

tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc 1980

tgtgaccggc gcctacatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct 2040

gctccacgcc gccaggccgc aggtgcagct ggtgcagtct ggggctgagg tgaagcggcc 2100

tggggcctca gtgcaggttt cctgcagggc atctggatac tccatcaaca cctactatat 2160

gcaatgggtg cgacaggccc ctggagcagg gcttgagtgg atgggagtaa tcaaccctag 2220

tggtgtcaca agctacgcac agaagttcca gggcagagtc accttgacca atgacacgtc 2280

cacgaacaca gtctacatgc agctgaacag cctgacatct gcagacacgg ccgtgtatta 2340

ctgtgcgaga tgggcattgt ggggggattt cgggatggac gtctggggca agggaaccct 2400

ggtcaccgtc tcctcaggtg gtggtggttc tggtggtggt ggttctggtg gtggtggttc 2460

cggtggtggt ggttccgaca tccagatgac ccagtctcct tccaccctgt ctgcatctat 2520

tggagacaga gtcaccatca cctgccgggc cagtgagggt atttatcact ggttggcctg 2580

gtatcagcag aagccaggga aagcccctaa actcctgatc tataaggcct ctagtttagc 2640

cagtggggcc ccatcaaggt tcagcggcag tggatctggg acagatttca ctctcaccat 2700

cagcagcctg cagcctgatg attttgcaac ttattactgc caacaatata gtaattatcc 2760

gctcactttc ggcggaggga ccaagctgga gatcaaaacc acgacgccag cgccgcgacc 2820

accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg 2880

gccagcggcg gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atttttgggt 2940

gctggtggtg gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat 3000

tattttctgg gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac 3060

tccccgccgc cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt 3120

cgcagcctat cgctccaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt 3180

tatgagacca gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga 3240

agaagaagga ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta 3300

ccagcagggc cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga 3360

tgttttggac aagagacgtg gccgggaccc tgagatgggg ggaaagccgc agagaaggaa 3420

gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag 3480

tgagattggg atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg 3540

tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg 3600

cggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc 3660

tggacctatg ggtcaccagc agttggtcat ctcttggttt tccctggttt ttctggcatc 3720

tcccctcgtg gccatatggg aactgaagaa agatgtttat gtcgtagaat tggattggta 3780

tccggatgcc cctggagaaa tggtggtcct cacctgtgac acccctgaag aagatggtat 3840

cacctggacc ttggaccaga gcagtgaggt cttaggctct ggcaaaaccc tgaccatcca 3900

agtcaaagag tttggagatg ctggccagta cacctgtcac aaaggaggcg aggttctaag 3960

ccattcgctc ctgctgcttc acaaaaagga agatggaatt tggtccactg atattttaaa 4020

ggaccagaaa gaacccaaaa ataagacctt tctaagatgc gaggccaaga attattctgg 4080

acgtttcacc tgctggtggc tgacgacaat cagtactgat ttgacattca gtgtcaaaag 4140

cagcagaggc tcttctgacc cccaaggggt gacgtgcgga gctgctacac tctctgcaga 4200

gagagtcaga ggggacaaca aggagtatga gtactcagtg gagtgccagg aggacagtgc 4260

ctgcccagct gctgaggaga gtctgcccat tgaggtcatg gtggatgccg ttcacaagct 4320

caagtatgaa aactacacca gcagcttctt catcagggac atcatcaaac ctgacccacc 4380

caagaacttg cagctgaagc cattaaagaa ttctcggcag gtggaggtca gctgggagta 4440

ccctgacacc tggagtactc cacattccta cttctccctg acattctgcg ttcaggtcca 4500

gggcaagagc aagagagaaa agaaagatag agtcttcacg gacaagacct cagccacggt 4560

catctgccgc aaaaatgcca gcattagcgt gcgggcccag gaccgctact atagctcatc 4620

ttggagcgaa tgggcatctg tgccctgcag tgttcctgga gtaggggtac ctggggtggg 4680

cgccagaaac ctccccgtgg ccactccaga cccaggaatg ttcccatgcc ttcaccactc 4740

ccaaaacctg ctgagggccg tcagcaacat gctccagaag gccagacaaa ctctagaatt 4800

ttacccttgc acttctgaag agattgatca tgaagatatc acaaaagata aaaccagcac 4860

agtggaggcc tgtttaccat tggaattaac caagaatgag agttgcctaa attccagaga 4920

gacctctttc ataactaatg ggagttgcct ggcctccaga aagacctctt ttatgatggc 4980

cctgtgcctt agtagtattt atgaagactt gaagatgtac caggtggagt tcaagaccat 5040

gaatgcaaag ctgctgatgg atcctaagag gcagatcttt ctagatcaaa acatgctggc 5100

agttattgat gagctgatgc aggccctgaa tttcaacagt gagactgtgc cacaaaaatc 5160

ctcccttgaa gaaccggatt tttataaaac taaaatcaag ctctgcatac ttcttcatgc 5220

tttcagaatt cgggcagtga ctattgatag agtgatgagc tatctgaatg cttcc 5275

Claims

1. A DNA fragment characterized by: including nucleotide fragments encoding CCR5, chimeric antigen receptor, and IL-12.

2. The DNA fragment of claim 1, wherein: the chimeric antigen receptor sequentially comprises a signal peptide sequence, a single-chain antibody, a hinge region, a transmembrane region, an intracellular costimulatory region and an intracellular signal region from the N end to the C end; the single-chain antibody in the chimeric antigen receptor is a mesothelin single-chain antibody.

3. The DNA fragment of claim 2, characterized in that: the mesothelin single-chain antibody is a human mesothelin single-chain antibody; the signal peptide is a human CD8a molecular signal peptide CD8 a; the transmembrane region is a human CD28 molecular transmembrane region; the intracellular co-stimulation region is a human CD28 intracellular region and/or a human 41BB intracellular region; the intracellular signal domain is the intracellular domain of human CD3z molecule.

4. The DNA fragment of claim 1, wherein: the nucleotide sequence of the DNA fragment is shown as SEQ ID NO. 14.

5. An expression vector, characterized in that: comprising the DNA fragment according to any one of claims 1 to 4.

6. The expression vector of claim 5, wherein: the expression vector is a lentivirus plasmid recombined with the DNA segment.

7. A chimeric antigen receptor T cell, characterized by: comprising a DNA fragment according to any one of claims 1 to 4 or comprising an expression vector according to claim 5 or 6.

8. Use of the DNA fragment of any one of claims 1 to 4 or the expression vector of claim 5 or 6 or the chimeric antigen receptor T cell of claim 7 for the preparation of a medicament for the treatment of tumors.

9. Use according to claim 8, characterized in that: the tumor is a solid tumor.