Plasmid for expressing chimeric antigen receptor, gamma delta-T cell and application

Abstract

The invention provides a plasmid for expressing a chimeric antigen receptor (CAR), a gamma delta-T cell and application, and relates to the technical field of biology. According to the plasmid for expressing the CAR, the expression efficiency of various promoters in gamma delta-T cells is compared, the promoter most suitable for the cell type is screened out, and the result shows that the SFFV promoter has the optimal expression performance. Compared with EF1 alpha and NMD, the promoter provided by the invention has higher transduction efficiency. The invention provides a structurally optimized CAR-gamma delta-T cell and a construction method thereof. The CAR structure comprises a combination of an antigen recognition structural domain driven by an SFFV promoter to express, a CD28 transmembrane structural domain and a DAP10 costimulatory signal structural domain. According to the structural design, the expression efficiency of the CAR in the gamma delta-T cells can be remarkably improved, and the recognition capability of the gamma delta-T cells on specific antigens and the tumor cell killing activity are enhanced, so that the maximization of the anti-tumor efficiency is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a recombinant plasmid for expressing chimeric antigen receptor (CAR), the gamma delta-T cell modified by the plasmid and the application thereof in immunotherapy such as anti-tumor, anti-virus and anti-aging. BACKGROUND

[0002] Chimeric antigen receptor (CAR) T cell therapy is one of the most breakthrough progress in the field of translational medicine in recent years, which has shown significant efficacy in the treatment of cancer and autoimmune diseases. In recent years, gamma delta-T cells have gradually become an ideal alternative due to their unique characteristics. Gamma delta-T cells not only have MHC (major histocompatibility complex) non-restricted killing mechanism, but also can play a more extensive immune surveillance role, and due to its lower risk of immune rejection, it becomes a potential candidate for developing universal cell therapy drugs. Compared with traditional alpha beta T cells, the recognition mode of gamma delta-T cells is not dependent on the expression of MHC molecules, which makes them more effective in recognizing and attacking tumor cells or infected cells. Therefore, CAR-gamma delta-T cells (chimeric antigen receptor gamma delta-T cells) as a new type of cell therapy strategy, combining gamma delta-T cells with CAR, can overcome some limitations of traditional CAR-T cells, providing more extensive targeting ability, faster immune response speed, lower immune escape risk and smaller side effects. Studies have shown that CAR-gamma delta-T cells exhibit stronger anti-tumor activity and longer persistence than traditional CAR-alpha beta-T cells in vitro and preclinical models.

[0003] However, although CAR-gamma delta-T cells have significant advantages in theory, they still face many challenges in practical application. Virus transduction (such as lentivirus, retrovirus, etc.) is still one of the main methods for constructing CAR-gamma delta-T cells, but this process has some bottlenecks, especially the problems of low virus transduction efficiency and insufficient cell functional activity, which directly affect the application effect of CAR-gamma delta-T cells in clinical treatment. Low-efficiency virus transduction not only limits the expansion and therapeutic effect of CAR-gamma delta-T cells, but also increases the risk and complexity of the treatment process. Therefore, how to improve the virus transduction efficiency and functional activity of CAR-gamma delta-T cells has become a great challenge for their transformation into clinical treatment.

[0004] The design of CAR is one of the key factors affecting the therapeutic effect of CAR-γδ-T cells. CAR molecule is a modular synthetic receptor, which is usually composed of four parts: extracellular target antigen-binding domain, hinge region, transmembrane domain and one or more intracellular signaling domains. The extracellular part is responsible for recognizing the target antigen, the transmembrane domain acts to anchor the CAR on the cell membrane, and the intracellular signaling domain activates the immune response of T cells after the receptor binds to the antigen. After the assembly of CAR elements, it is loaded into a viral vector, and its expression is driven by the regulatory elements of the promoter.

[0005] The existing researches are all about the influence of different combinations of transmembrane domains and costimulatory signaling molecules (intracellular signaling domains) on the functional therapeutic effect of CAR-γδ-T cells. However, the promoter plays a key role in viral transduction efficiency and gene expression, therefore, selecting a suitable promoter is an important link to improve the therapeutic effect of CAR-γδ-T cells. SUMMARY

[0006] The purpose of the present application is to provide a recombinant plasmid expressing a chimeric antigen receptor (CAR), the γδ-T cells modified by the plasmid and its application in immunotherapy such as anti-tumor, anti-virus and anti-aging, aiming to solve the technical problem of poor therapeutic effect caused by the lack of optimized vector structure combination in the existing CAR-γδ-T cells.

[0007] To achieve the above-mentioned purpose, the present application provides a plasmid expressing a chimeric antigen receptor, which includes an antigen-binding domain, a hinge domain, a transmembrane domain and an intracellular signaling domain connected in sequence: The transmembrane domain includes CD28; The intracellular signaling domain includes a costimulatory signaling molecule, which includes DAP10; The plasmid includes a promoter that initiates the expression of the gene of the chimeric antigen receptor, and the promoter is SFFV.

[0008] In some embodiments, the nucleotide sequence of the promoter SFFV is SEQ ID NO. 2.

[0009] In some embodiments, the nucleotide sequence of the costimulatory signaling molecule DAP10 is SEQ ID NO. 16, and the amino acid sequence is SEQ ID NO. 17.

[0010] In some embodiments, the nucleotide sequence of the transmembrane domain CD28 is SEQ ID NO. 10, and the amino acid sequence is SEQ ID NO. 11.

[0011] In some embodiments, the nucleotide sequence of the antigen binding domain is SEQ ID NO. 4, and the amino acid sequence is SEQ ID NO. 5.

[0012] In some embodiments, the nucleotide sequence of the hinge domain is SEQ ID NO. 6, and the amino acid sequence is SEQ ID NO. 7.

[0013] In some embodiments, the nucleotide sequence of the intracellular signal transduction domain further comprises CD3 zeta, and the amino acid sequence is SEQ ID NO. 19.

[0014] The application also provides a gamma delta-T cell expressing a chimeric antigen receptor, comprising the plasmid expressing a chimeric antigen receptor described above.

[0015] The application also provides the use of the plasmid expressing a chimeric antigen receptor described above or the gamma delta-T cell expressing a chimeric antigen receptor described above in the preparation of a cell drug for preventing and / or treating tumors and derivative products, including but not limited to exosomes expressed by the gamma delta-T cell expressing a chimeric antigen receptor.

[0016] In some embodiments, the tumor comprises non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, triple-negative breast cancer, bladder cancer, Hodgkin's lymphoma.

[0017] Compared with the prior art, the application has the following beneficial effects: The plasmid expressing a chimeric antigen receptor provided by the application screens out the SFFV promoter most suitable for expression in human gamma delta-T cells, which has higher transduction efficiency than EF1 alpha and MND, and is a very promising gene modification system for clinical application. In order to achieve stronger functional activity, the application optimizes the combination of the promoter and the transmembrane domain and the costimulatory signal molecule of the chimeric antigen receptor (CAR), and the CAR gene modified gamma delta-T cells by the promoter SFFV start vector, the CD28 transmembrane domain molecule and the DAP10 costimulatory signal molecule combination can specifically recognize tumor cells expressing specific antigens, and can efficiently and specifically kill tumors, thereby maximizing the anti-tumor effect. BRIEF DESCRIPTION OF DRAWINGS

[0018] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0019] Figure 1 A schematic diagram of flow cytometry results for CAR-γδ-T cell transfection efficiency; Figure 2 A schematic diagram showing the statistical results of CAR-γδ-T cell transfection efficiency; Figure 3 A schematic diagram showing the results of PDL-1 expression levels on the surface of tumor cell lines; Figure 4 The left side shows the in vitro killing results of ordinary γδ-T cells and CAR γδ-T cells with 8 different vectors listed in Table 2 against the non-small cell lung cancer cell line A549 at 3 donors; the middle side shows the in vitro killing results of ordinary γδ-T cells and CAR γδ-T cells with 8 different vectors listed in Table 2 against the non-small cell lung cancer cell line PC-9 at 3 donors; the right side shows the in vitro killing results of ordinary γδ-T cells and CAR γδ-T cells with 8 different vectors listed in Table 2 against the non-small cell lung cancer cell line HCC827 at 3 donors. Figure 5 This diagram illustrates the release of cytokines TNF-α (left), IFN-γ (middle), and GranzymeB (right) after co-incubation of ordinary γδ-T cells and CAR γδ-T cells with eight different vectors listed in Table 2 with the PDL-1-overexpressing HCC827 tumor cell line. Detailed Implementation

[0020] As used in this article: "Prepared from" is synonymous with "comprising". The terms "comprising", "including", "having", "containing", or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0021] The conjunction "composed of..." excludes any unspecified elements, steps, or components. If used in a claim, this phrase makes the claim closed, excluding materials other than those described, except for associated conventional impurities. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following it, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0022] When expressing ranges of values or parameters, the endpoints are included unless specifically stated otherwise. When a range of values is expressed, it is meant to include all values (and sub-ranges) encompassed therein as well as the range itself. For example, expressing a range as "1 to 5" is meant to include all individual numbers from 1 to 5, and sub-ranges such as from 1-3, 2-4, or 3-5, etc. When a value is expressed as an approximation by use of the antecedent "about," it will be understood that the value of the specified quantitative parameter is "within 10% of and preferably within 5% of and more preferably within 1% of" the value so modified.

[0023] In these examples, the parts and percentages are by mass unless otherwise indicated.

[0024] "Mass parts" refers to a basic unit of measurement that represents the proportional relationship of the mass of multiple components, 1 part can represent any unit mass, such as 1 g, 2.689 g, etc. If we say that the mass parts of component A is a parts, and the mass parts of component B is b parts, it means that the ratio of the mass of component A to the mass of component B is a:b. Or, it means that the mass of component A is aK, and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike mass parts, the sum of the mass parts of all components is not limited to 100 parts.

[0025] "and / or" is used to indicate that one or both of the described situations can occur, for example, A and / or B includes (A and B) and (A or B).

[0026] The application provides a plasmid expressing a chimeric antigen receptor, which includes an antigen binding domain, a hinge domain, a transmembrane domain and an intracellular signal transduction domain connected in sequence; The transmembrane domain includes CD28; The intracellular signal transduction domain includes a costimulatory signal molecule, which includes DAP10; The plasmid includes a promoter that initiates the expression of the gene of the chimeric antigen receptor, and the promoter is SFFV.

[0027] In some embodiments, the nucleotide sequence of the promoter SFFV is SEQ ID NO. 2.

[0028] In some embodiments, the nucleotide sequence of the costimulatory signal molecule DAP10 is SEQ ID NO. 16, and the amino acid sequence is SEQ ID NO. 17.

[0029] In some embodiments, the nucleotide sequence of the transmembrane domain CD28 is SEQ ID NO. 10, and the amino acid sequence is SEQ ID NO. 11.

[0030] In some embodiments, the nucleotide sequence of the antigen binding domain is SEQ ID NO. 4, and the amino acid sequence is SEQ ID NO. 5.

[0031] In some embodiments, the nucleotide sequence of the hinge domain is SEQ ID NO. 6, and the amino acid sequence is SEQ ID NO. 7.

[0032] In some embodiments, the nucleotide sequence of the intracellular signal transduction domain further comprises CD3 zeta, and the amino acid sequence is SEQ ID NO. 19.

[0033] The application also provides a gamma delta-T cell expressing a chimeric antigen receptor, comprising the plasmid expressing a chimeric antigen receptor described above.

[0034] The application also provides the use of the plasmid expressing a chimeric antigen receptor described above or the gamma delta-T cell expressing a chimeric antigen receptor described above in the preparation of a cell drug for preventing and / or treating tumors and derivative products, including but not limited to exosomes expressed by the gamma delta-T cell expressing a chimeric antigen receptor.

[0035] In some embodiments, the tumor comprises non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, triple-negative breast cancer, bladder cancer, Hodgkin's lymphoma.

[0036] The plasmid expressing a chimeric antigen receptor provided by the application screens out the SFFV promoter most suitable for expression in human gamma delta-T cells, which has higher transduction efficiency than EF1 alpha and MND, and is a very promising gene modification system for clinical application. In order to achieve stronger functional activity, the application optimizes the combination of the promoter and the transmembrane domain and the costimulatory signal molecule of the chimeric antigen receptor (CAR), and the CAR gene modified gamma delta-T cells by the promoter SFFV start vector, the CD28 transmembrane domain molecule and the DAP10 costimulatory signal molecule combination can specifically recognize tumor cells expressing specific antigens, and can efficiently and specifically kill tumors, thereby maximizing the anti-tumor effect.

[0037] The embodiments of the present application will be described in detail below with specific examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions are not specified in the examples, which are carried out under conventional conditions or manufacturer's recommended conditions. The reagents or instruments used are not specified by the manufacturer, which are conventional products that can be purchased on the market.

[0038] Example 1 Joint optimization design of CAR promoter, transmembrane domain and costimulatory signaling molecule In this embodiment, the single-chain antibody (scFV) of the anti-PDL-1 antibody is used as the antigen binding region, combined with the CD8α signal peptide, the CD8 hinge region, the CD8 or CD28 transmembrane domain, the CD28, 4-1BB or DAP10 costimulatory signaling molecule and the CD3ζ intracellular signal transduction domain, to construct a second-generation CAR targeting PDL-1, which is driven by the vector promoters EF1-α, SFFV or MND for expression. The CAR lentiviral recombinant plasmid used in this embodiment is provided by Yunzhou Biotech (Guangzhou) Co., Ltd.

[0039] I. Construction of PDL-1 CAR lentiviral recombinant plasmid Specifically, the CAR structure is used to genetically modify γδ-T cells, and the CAR structure sequentially from the amino terminus to the carboxyl terminus is: scFV (PDL-1)-Hinge (CD8)-TM (CD8 / CD28)-CD28 / 4-1BB / DAP10-CD3ζ, i.e., sequentially from the amino acid to the carboxyl terminus is: single-chain variable region derived from PDL-1 monoclonal antibody, CD8 hinge region, (CD8 or CD28) transmembrane region, (CD28, 4-1BB or DAP10) costimulatory signaling molecule and CD3ζ chain intracellular region, the promoter is EF1-α, SFFV or MND, the nucleotide sequence and amino acid sequence of each structure are numbered in the sequence listing as shown in Table 1. The CAR lentiviral recombinant plasmid used in this embodiment is provided by Yunzhou Biotech (Guangzhou) Co., Ltd., and the combination obtains twelve kinds of PDL-1 CAR plasmids as shown in Table 2.

[0040] Table 1 Numbering of nucleotide sequence and amino acid sequence of each structure

[0041] Table 2 Combination of different PDL-1 CAR plasmids

[0042] II. Packaging of recombinant lentivirus The above obtained 8 recombinant CAR lentivirus plasmids are packaged to obtain 8 recombinant lentivirus particles respectively. The present application uses Lenti-Pac HIV lentivirus packaging kit (GeneCopoeia, LT002) to package the lentivirus, and the specific steps are as follows: Take 293FT in the logarithmic growth phase, digest with 0.25% trypsin (containing EDTA), prepare a cell suspension, inoculate 2.5x10 6 6 cells and 10 mL of inactivated DMEM medium containing 10% FBS in a 10 cm cell culture dish, gently shake crosswise, and then place in a 37℃, 5% CO2 cell incubator.

[0043] The next day, prepare the following reagents: opti-MEM medium, 2.5 μg CAR lentivirus plasmid and 5 μg LentiPac mixed packaging plasmid, mix evenly, stand at room temperature for 20 min, then add 15 μl ectin Lenti transfection reagent, mix gently, stand at room temperature for 10 min, obtain DNA-EndoFectin transfection complex. Take out the cells, evenly drop the DNA-EndoFectin transfection complex into each dish, shake gently, and place in a 37℃, 5% CO2 cell incubator for continuous culture. After 16 h of culture, take out the cells, remove the old medium containing the transfection complex, add fresh medium 10 mL, and place in a 37℃, 5% CO2 cell incubator for continuous culture.

[0044] Collect the culture supernatant at 48 and 72 h respectively, centrifuge at 500xg for 10 min to remove cell debris, and obtain crude virus liquid of lentivirus; filter using 0.45 μm low protein binding PES filter membrane to obtain purified virus stock solution; centrifuge at 18500xg at 4℃ for 2h, discard the supernatant, add PBS solution to resuspend the virus particles, and obtain lentivirus concentrate.

[0045] Take 10 μl of lentivirus concentrate for virus titer detection, and store the remaining virus in a -80℃ refrigerator.

[0046] III. Lentivirus titer determination The present application uses HIV lentivirus detection kit (GeneCopoeia, LT006) for titer detection, and the specific steps are as follows: Take 0.25 ml RNAzol RT RNA extraction reagent into the 1.5 ml EP tube containing 10 μl purified concentrated lentivirus solution, mix the solution by inverting the centrifuge tube up and down for 10 times, then stand still at room temperature for 10 min. Add 90 μl water to each 1.5 ml EP tube, centrifuge at 20℃, 18000xg for 10 min.

[0047] Transfer the supernatant after centrifugation to a new 1.5 ml EP tube, add linear polyacrylamide with a final concentration of 10 μg / ml. Add the same volume of 100% isopropanol, mix thoroughly by inverting up and down, then place at -20℃ for 4 h. Take out each EP tube, centrifuge at 10℃, 18000xg for 20 min, discard the supernatant to obtain the RNA precipitate. Add 0.5 ml 75% ethanol solution to each EP tube to wash the RNA precipitate, centrifuge at 10℃, 18000xg for 5 min, discard the supernatant, and repeat the washing once more.

[0048] After the second washing, discard the supernatant, invert the EP tube, and dry the RNA precipitate at room temperature for 5 min, then add 50 μl DEPC water to each EP tube to dissolve the RNA precipitate.

[0049] Take out a new EP tube, add 1.5 μl DEPC water, 20 μl of the above prepared lentivirus RNA solution, 2.5 μl 10 x DNase I buffer and 1 μl DNase I to each tube, incubate at 75℃ for 10 min to inactivate the DNase I.

[0050] Add the above 10 μl of RNA and 5 μl of cDNA synthesis primer with a concentration of 1 μM to a new EP tube, incubate at 70℃ for 5 min, then cool on ice. Continue to add 2 μl 10 x Reverse Transcription Buffer, 1 μl 25 mM dNTP, 1 μl RNase Inhibitor and 1 μl Reverse Transcription Enzyme to each EP tube, incubate at 90℃ for 10 min.

[0051] The product obtained in the previous step was subjected to qPCR reaction. The specific steps were as follows: a standard curve was prepared by serially diluting the positive standard 10-fold. 2 μl of cDNA sample, 10 μl of 2×All-in-One qPCR Mix, 2 μl of 2.5 μM qPCR Primer Mix, and 6 μl of ddH2O were added to each tube. The samples were mixed thoroughly, and the following program was followed: Denaturation: 95℃, 10 min, 1 cycle; Denaturation: 95℃, 10 s; Annealing: 60℃, 20 s; Extension: 72℃, 15 s, 40 cycles.

[0052] The Ct value of each standard was taken, and a standard curve was generated using LOG (copy number) and Ct value. The Ct value of each sample was substituted into the generated standard curve to calculate the copy number. Then, the copy number was multiplied by the dilution factor to obtain the copy number of the original sample. Multiplying the copy number of the original sample by 1 / 2 gives the viral titer. The lentivirus titer detection results are shown in Table 3.

[0053] Table 3. Lentiviral titer detection results

[0054] Example 2: Preparation of CAR γδ-T cells 1. Preparation of CAR γδ-T cells Peripheral blood was collected from healthy donors, and PBMCs were isolated using Ficoll lymphocyte separation medium. Cells were counted using a hemocytometer at a resolution of 3.0 × 10⁻⁶. 6 PBMCs were inoculated at a density of cells / mL in RIPM 1640 medium containing 10% FBS, 1% P / S, 5µM zoledronic acid (ZOL), and 400 IU / mL IL-2, and recorded as Day 0.

[0055] Day 3: Collect cells, centrifuge at 600 rpm for 5 min, discard the supernatant, and count cells using a hemocytometer at a value of 3.0 × 10⁻⁶. 6 Cells were seeded at a density of cells / mL in RIPM 1640 medium containing 10% FBS, 1% P / S, 5µM zoledronic acid (ZOL), and 100 IU / mL IL-2.

[0056] Day 5: Collect cells, centrifuge at 800 rpm for 5 min, discard supernatant, and sort γδ-T cells using the TCRγ / δ+T Cell Iso Kit (Meticlan). Resuspend cells in RIPM 1640 medium containing 10% FBS, 1% P / S, 100 IU / mL IL-2, and 5 μg / mL polybrene at a concentration of 1.0 × 10⁻⁶. 6The cells were inoculated in 24-well plates at 1.0×105 / mL / well, and the lentiviral vectors in Table 2 were added for transduction at an MOI of 10, mixed, centrifuged at 1000xg for 90 min at 32℃, and then incubated in a CO2incubator.

[0057] After 24 h of lentiviral transduction, fresh RPMI 1640 medium containing 10% FBS, 1% P / S, and 100 IU / mL IL-2 was added for culture, and the viable cell density was adjusted to 1.0×105 / mL. 6 Fresh complete medium was replaced every 2-3 days, and the culture was continued for 10-15 days.

[0058] 2. Detection of CAR γδ-T cell transduction efficiency 1.0×105transduced γδ-T cells were washed twice with normal saline, and the FITC fluorescence signal was detected by flow cytometry to measure the ratio of FITC positive cells, reflecting the proportion of CAR γδ-T cells in the total cells. 6 The results of transduction efficiency detection of the lentiviral vectors listed in Table 2 are shown in Table 4. Figure 1 and Figure 2 Table 4 shows that CAR γδ-T cells were successfully prepared, and the transduction efficiency of lentiviral vectors numbered 5-12 is better, and the expression efficiency of CAR of SFFV-CD28-DAP10 lentiviral vector is significantly higher than that of other vectors. This result shows that the use of different promoters, transmembrane domains, and costimulatory molecules has different effects on the expression of chimeric antigen receptors in γδ-T cells, and the optimized combination of SFFV promoter, CD28 transmembrane domain, and DAP10 costimulatory molecule significantly improves the expression of chimeric antigen receptors in γδ-T cells.

[0059] Table 4. Results of CAR γδ-T cell transduction efficiency detection

[0060] Example 3. In vitro function detection of CAR γδ-T cells 1. Detection of PDL-1 expression level on the surface of tumor cells An appropriate amount of non-small cell lung cancer cell lines A549, PC-9, and HCC827 were used to digest the cells with trypsin-EDTA (0.25%), prepare a cell suspension, wash twice with normal saline, then add an appropriate amount of PDL-1-FITC antibody, incubate at 4℃ in the dark for 15 min, and detect the FITC fluorescence signal by flow cytometry to measure the ratio of FITC positive cells. The PDL-1 expression level is shown in Figure 3 The results show that the PDL-1 expression levels of the three non-small cell lung cancer cell lines are in the order of HCC827>PC-9>A549.

[0061] 2. In vitro tumor killing function detection In vitro tumor killing function detection was performed by LDH (Promega, Cat: G1780) method.

[0062] Target cells were A549, PC-9 and HCC827 non-small cell lung cancer cell lines. The target cells were taken and 1 x 10 4 cells / 100μL, and normal γδ-T cells and CAR γδ-T cells of 8 different vectors (Nos. 5-12) with better transduction efficiency listed in Table 4 were added at an effector-to-target ratio of 10:1, centrifuged at 250 x g for 5 min to allow the effector and target cells to fully contact, and incubated in a cell incubator for 10 h. After incubation, the 96-well plate was taken out, centrifuged at 250 x g for 5 min, and 50 μl of supernatant was taken from each well to a new flat-bottom 96-well plate using a syringe, 50 μl of Assay buffer was added, and incubated at room temperature for 30 min in the dark. After incubation, 50 μl of stop solution was added to each well, and the absorbance value was detected at 490 nm wavelength, and the percentage of cell lysis was calculated according to the spontaneous release control and the maximum release control.

[0063] The in vitro tumor killing function results of normal γδ-T cells and CAR γδ-T cells of 8 different vectors listed in Table 4 on PDL-1 low-expressing non-small cell lung cancer cell line A549 are shown in Figure 4 (left), the in vitro tumor killing function results of PDL-1 moderately-expressing non-small cell lung cancer cell line PC-9 are shown in Figure 4 (middle), and the in vitro tumor killing function results of PDL-1 moderately-expressing non-small cell lung cancer cell line HCC827 are shown in Figure 4 (right), the results show that the target killing ability of CAR γδ-T cells of 8 different vectors is improved compared with normal γδ-T cells, and the killing ability on HCC827 cells with high PDL-1 expression is the strongest, indicating that they have in vitro killing and targeting functions on A549, PC-9 and HCC827 cell lines. And in the co-incubation system with A549, PC-9 and HCC827 cell lines, the targeting lysis ability of SFFV-CD28-DAP10 CAR γδ-T cells is significantly higher than that of the other seven CAR vectors. From the above in vitro tumor killing results, it can be known that the CAR γδ-T cells constructed by the preferred SFFV-CD28-DAP10 vector are used for treating tumors.

[0064] 2. In vitro cytokine level detection Take the appropriate amount of target cells (PDL-1 high expression cell line HCC827), add 1 x10 4 cells / 100μL, add ordinary γδ-T cells and CAR γδ-T cells of 8 different vectors (No. 5-12) listed in Table 4 to each well of the round hole 96-well plate at an effector to target ratio of 10:1, centrifuge at 250 x g for 5 min, so that the effector and target cells are in close contact, and incubate in a cell incubator for 10 h. After incubation, remove the 96-well plate, aspirate the supernatant, and measure the concentrations of IFN-γ, TNF-α and Granzyme B in the supernatant using a CBA kit (Biolegend).

[0065] The results of the secretion of IFN-γ, TNF-α and Granzyme B by ordinary γδ-T cells and CAR γδ-T cells of 8 different vectors listed in Table 4 after in vitro incubation with PDL-1 high expression HCC827 tumor cell lines are shown in Figure 5 As shown, consistent with the in vitro tumor killing results, the results of IFN-γ, TNF-α and Granzyme B show that the secretion of FN-γ, TNF-α and Granzyme B by CAR γδ-T cells of 8 different vectors is improved compared with ordinary γδ-T cells, and the secretion of FN-γ, TNF-α and Granzyme B by CAR γδ-T cells constructed by SFFV-CD28-DAP10 vector is the highest, further indicating its in vitro killing function.

[0066] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

[0067] In addition, those skilled in the art will appreciate that although some embodiments herein include certain features included in other embodiments rather than other features, the combination of features of different embodiments means that it is within the scope of the present application and forms different embodiments. For example, in the above claims, any one of the claimed embodiments can be used in any combination. The information disclosed in the BACKGROUND section is only intended to deepen the understanding of the general background of the present application, and should not be considered as acknowledging or implying in any form that the information constitutes prior art known to those skilled in the art.

Claims

1. A plasmid expressing a chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antigen binding domain, a hinge domain, a transmembrane domain and an intracellular signal transduction domain connected in sequence; The transmembrane domain comprises CD28; The intracellular signal transduction domain comprises a costimulatory signal molecule, and the costimulatory signal molecule comprises DAP10; The plasmid comprises a promoter for initiating gene expression of the chimeric antigen receptor, and the promoter is SFFV.

2. The plasmid expressing chimeric antigen receptor according to claim 1, characterized in that, The nucleotide sequence of the promoter SFFV is SEQ ID NO.

2.

3. The plasmid expressing chimeric antigen receptor according to claim 1, characterized in that, The nucleotide sequence of the costimulatory signal molecule DAP10 is SEQ ID NO. 16, and the amino acid sequence is SEQ ID NO.

17.

4. The plasmid expressing chimeric antigen receptor according to claim 1, characterized in that, The nucleotide sequence of the transmembrane domain CD28 is SEQ ID NO. 10, and the amino acid sequence is SEQ ID NO.

11.

5. The plasmid expressing chimeric antigen receptor according to claim 1, characterized in that, The antigen binding domain is a single-chain antibody against PDL-1, the nucleotide sequence is SEQ ID NO. 4, and the amino acid sequence is SEQ ID NO.

5.

6. The plasmid expressing chimeric antigen receptor according to claim 1, characterized in that, The hinge domain is a hinge region of CD8, the nucleotide sequence is SEQ ID NO. 6, and the amino acid sequence is SEQ ID NO.

7.

7. The plasmid expressing chimeric antigen receptor according to claim 1, characterized in that, The intracellular signal transduction domain further comprises CD3ζ, the nucleotide sequence is SEQ ID NO. 18, and the amino acid sequence is SEQ ID NO.

19.

8. A gamma delta-T cell expressing a chimeric antigen receptor, characterized in that, The plasmid for expressing the chimeric antigen receptor comprises the plasmid for expressing the chimeric antigen receptor according to any one of claims 1-7.

9. Use of the plasmid for expressing the chimeric antigen receptor according to any one of claims 1-7 or the γδ-T cell for expressing the chimeric antigen receptor according to claim 8 in the preparation of a cell drug for preventing and / or treating tumors and derivative products, wherein the derivative products include but are not limited to exosomes expressed by the γδ-T cell for expressing the chimeric antigen receptor.

10. Use according to claim 9, characterized in that, The tumors include non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, triple-negative breast cancer, bladder cancer, and Hodgkin's lymphoma.

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