Application of EF1 alpha promoter in construction of reporter gene for indicating killing function of CAR-T cells

Abstract

The invention discloses an application of an EF1 alpha promoter in construction of a reporter gene for indicating a CAR-T cell killing function, and it is found through contrast experiments for the first time that the EF1 alpha promoter is more suitable for construction of a Luc reporter gene for indicating the CAR-T cell killing function, and not all promoters are effective. The invention provides a favorable reference for constructing a method for determining the killing activity of the CAR-T cells, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to the use of an EF1α promoter in constructing a reporter gene for indicating the killing function of CAR-T cells. Background Art

[0002] Detecting the cytotoxic activity of effector cells against target cells is a common technique for evaluating the efficacy and specificity of engineered immune cell therapies and is crucial for product development and finished product quality control. The US FDA's industry guidance for developing CAR-T cell products requires that in preclinical trials of CAR-T cells, the efficacy and specificity of CAR-T cells against target cells be verified in vitro using specific, sensitive, and reproducible assays. However, there are currently no guidelines that provide unified regulations for specific assay methods. Therefore, in practice, the evaluation system for verifying the ability of CAR-T cells to kill target cells is often established independently by the experimenter.

[0003] Construct target cells that overexpress luciferase, co-culture the target cells with effector cells, add substrate, detect the fluorescence signal generated by the enzyme-catalyzed substrate, and convert the number of surviving cells according to the signal intensity. This method is simple to operate and easy to implement in the laboratory, and has become one of the commonly used methods to evaluate the killing function of engineered immune cell drugs on target cells. Target cells expressing luciferase are usually constructed by researchers themselves based on indications, targets, and other aspects. There may be a long-ignored link in this. Different researchers have different choices of promoters to drive luciferase expression when constructing cells that overexpress luciferase, but no study has systematically compared the effects of different promoters on the accuracy of the test results, that is, whether the measured fluorescence value can accurately reflect the survival of target cells, that is, the killing function of engineered immune cell drugs. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the art with the use of the EF1α promoter in constructing a reporter gene for indicating the killing function of CAR-T cells.

[0005] The present invention uses target cells constructed with a Luc reporter gene tag expressed by a CMV promoter to evaluate the killing function of CAR-T cells, but unexpectedly discovered the abnormal phenomenon of increased Luc measurement values ​​when CAR-T cells kill target cells. The unavailability of the CMV promoter in this system indicates that the choice of promoter may affect the accurate determination of the killing efficiency of CAR-T cells, and also suggests the necessity of screening the promoter. Therefore, the inventors of the present invention detected the CAR-T cell killing efficiency measured by the Luc method and the absolute target cell counting method based on flow cytometry under the same CAR-T cell attack conditions and performed statistical analysis. The five promoters commonly used to construct target cell Luc tags - EF1α, SFFV, hPGK, CAG and SV40 - were evaluated for the accuracy of the CAR-T cell killing efficiency measurement and the optimal selection was given, which provides a favorable reference for the method of constructing a target cell line for measuring the killing activity of CAR-T cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides the use of an EF1α promoter in constructing a reporter gene for indicating the killing function of CAR-T cells. The sequence of the EF1α promoter is shown in SEQ ID NO: 1.

[0008] Furthermore, the reporter gene is Luciferase, GFP, eGFP, mGFP, RFP, mCherry, tdTomato or Luciferase-GFP;

[0009] Optionally, the reporter gene is Luciferase.

[0010] In some embodiments, the reporter gene of the present invention is not limited to: Luciferase, GFP, eGFP, mGFP, RFP, mCherry, tdTomato or Luciferase-GFP. Those skilled in the art can make routine selections based on actual needs. In a specific embodiment of the present invention, the reporter gene is Luciferase.

[0011] In the present invention, the Luciferase is an enzyme that catalyzes bioluminescent reactions and is widely used in molecular biology and medical research as a reporter gene for detecting gene expression, cell activity, or biomolecular interactions. In a specific embodiment of the present invention, tumor cells are labeled with Luciferase and co-cultured with the CAR-T, and the killing efficiency is calculated by the reduction of luminescence (such as the killing of Her2 breast cancer cells by Her2-CAR-T).

[0012] Furthermore, the application is achieved by constructing a lentiviral expression vector in which the EF1α promoter drives the expression of the Luciferase gene, transducing the vector into target cells, and detecting the function of CAR-T cells in killing target cells.

[0013] Furthermore, the lentiviral expression vector in which the EF1α promoter drives the expression of the Luciferase gene is a lentiviral vector expressing the pCDH-EF1α-Luciferase plasmid.

[0014] Furthermore, the CAR-T cells are PDPN CAR-T cells, CD19 CAR-T cells or Her2 CAR-T cells;

[0015] Optionally, the PDPN CAR in the PDPN CAR-T cell is obtained by sequentially connecting EF1α, RQR8, T2A, PDPN antibody, CD8 transmembrane domain, 4-1BB costimulatory signaling domain, and CD3ε intracellular signaling domain; optionally, the CD19 CAR in the CD19 CAR-T cell is obtained by sequentially connecting EF1α, RQR8, T2A, CD19 antibody, CD8 transmembrane domain, 4-1BB costimulatory signaling domain, and CD3ε intracellular signaling domain; optionally, the Her2 CAR in the Her2 CAR-T cell is obtained by sequentially connecting CD8α signal peptide, Her2 antibody, CD8 hinge region, CD8 transmembrane domain, CD28 costimulatory signaling domain, and CD3ε intracellular signaling domain; optionally, the amino acid sequence of the heavy chain variable region of the PDPN antibody is as shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8; optionally, the amino acid sequence of the heavy chain variable region of the CD19 antibody is as shown in SEQ ID NO: NO: 10, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12; optionally, the amino acid sequence of the heavy chain variable region of the Her2 antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16; optionally, the sequence of EF1α in the PDPN CAR or CD19 CAR is shown in SEQ ID NO: 18, the amino acid sequence of RQR8 is shown in SEQ ID NO: 19, the amino acid sequence of T2A is shown in SEQ ID NO: 21, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO: 23, the amino acid sequence of the 4-1BB costimulatory signaling domain is shown in SEQ ID NO: 25, and the amino acid sequence of the CD3ε intracellular signaling domain is shown in SEQ ID NO: 27; optionally, the amino acid sequence of the CD8α signal peptide in the Her2 CAR is shown in SEQ ID NO: 31, the amino acid sequence of the CD8 hinge region is shown in SEQ ID NO: 33, and the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO: NO:35, the amino acid sequence of the CD28 costimulatory signaling domain is shown in SEQ ID NO:37, and the amino acid sequence of the CD3ε intracellular signaling domain is shown in SEQ ID NO:39.

[0016] Furthermore, the killing function of the CAR-T cells was detected using flow cytometry absolute counting magnetic bead method or luciferase method.

[0017] In the present invention, as long as the EF1α promoter is used to construct a reporter gene indicating the killing function of CAR-T cells, it will fall within the scope of protection of the present invention. The present invention has no special restrictions on the specific CAR-T cell type. Therefore, the use of the EF1α promoter to construct a reporter gene indicating the killing function of any CAR-T cell is within the scope of protection of the present invention.

[0018] The second aspect of the present invention provides a plasmid for driving the expression of the Luciferase gene by an EF1α promoter, wherein the plasmid is a pCDH-EF1α-Luciferase plasmid;

[0019] The sequence of the EF1α promoter is shown in SEQ ID NO: 1.

[0020] In the present invention, the pCDH-EF1α-Luciferase plasmid is a high-efficiency reporter gene plasmid based on a lentiviral system, which utilizes the EF1α promoter to drive the stable expression of the Luciferase gene.

[0021] The third aspect of the present invention provides a lentiviral vector in which an EF1α promoter drives the expression of the Luciferase gene, wherein the lentiviral vector is a lentiviral vector expressing a pCDH-EF1α-Luciferase plasmid;

[0022] The sequence of the EF1α promoter is shown in SEQ ID NO: 1.

[0023] Furthermore, the method comprises the following steps:

[0024] (1) Using the pCDH-EF1α plasmid as a backbone, the Luciferase gene fragment was ligated into the backbone to obtain the plasmid pCDH-EF1α-Luciferase plasmid in which the EF1α promoter drives the expression of the Luciferase gene, which is the target plasmid;

[0025] (2) co-transfecting the target plasmid with psPAX2 and pMD2.G into host cells;

[0026] (3) 48-72 hours after transfection, the virus-containing supernatant is collected. The virus is the lentiviral vector described in the third aspect of the present invention.

[0027] Furthermore, the target plasmid: psPAX2: pMD2.G = 7.5 μg: 5 μg: 2.5 μg;

[0028] Optionally, the host cell is a 293T cell, a HEK293 cell, a 293FT cell, a HT1080 cell or a NIH / 3T3 cell; optionally, the host cell is a 293T cell.

[0029] A fourth aspect of the present invention provides a method for detecting the killing function of CAR-T cells, the method comprising the following steps:

[0030] (1) using the lentiviral vector described in the third aspect of the present invention to construct tumor cells overexpressing EF1α;

[0031] (2) The tumor cells are co-cultured with the CAR-T cells, and the killing function of the CAR-T cells is detected by flow cytometry absolute counting magnetic bead method or luciferase method.

[0032] In some embodiments, the detection principle of the flow cytometry absolute counting magnetic bead method is: target cells (such as tumor cells) are labeled with magnetic beads, and after co-incubation with CAR-T cells, the number of surviving target cells is detected by flow cytometry to calculate the killing rate.

[0033] In some embodiments, the detection principle of the luciferase method is: target cells are labeled by the luciferase gene, and the fluorescence intensity of the luciferin produced by the luciferase gene is used to reflect the number of surviving target cells, thereby evaluating the killing ability of CAR-T cells.

[0034] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0035] The present invention discovered for the first time that the Luciferase tag constructed with the CMV promoter cannot indicate the true killing level of CAR-T cells. The unavailability of the CMV promoter indicates that the choice of promoter affects the accurate determination of the killing efficiency of CAR-T cells. In addition, the present invention evaluated the accuracy of the determination of CAR-T cell killing efficiency for five promoters commonly used to construct the target cell Luc tag - EF1α, SFFV, hPGK, CAG and SV40. For the first time, through comparative experiments, it was found that the EF1α promoter is more suitable for constructing the Luc reporter gene to indicate the killing function of CAR-T cells, and not all promoters are effective. This result is a technical effect that was unexpected by those skilled in the art based on the prior art. The present invention provides a favorable reference for the method of constructing a target cell line for determining the killing activity of CAR-T cells, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1: In vitro functional assays of three CAR-T cells, including: Figure a: Schematic diagram of the molecular construction of the three CARs; Figure b: FCM detection of CD19 expression levels on the surface of SKOV3-CD19 cells; Figure c: FCM detection of Her2 expression levels on the surface of B16F10-Her2 cells; Figure d: FCM detection of CAR expression levels on the surface of PDPN CAR-T cells, and RTCA detection of the killing effect of PDPN CAR-T on HGC-27 at a set effector-target ratio; Figure e: FCM detection of CAR expression levels on the surface of CD19 CAR-T cells, and RTCA detection of the killing effect of CD19 CAR-T on SKOV3-CD19 at a set effector-target ratio; Figure f: FCM detection of CAR expression levels on the surface of Her2 CAR-T cells, and RTCA detection of the killing effect of Her2 CAR-T on B16F10-Her2 at a set effector-target ratio;

[0037] Figure 2 : Luciferase-based chemiluminescence assay and flow cytometry were used to determine the killing efficiency of CAR-T cells. Panel a: Luciferase assay was used to detect the target cell Luc activity of the three CAR-T cells after 24 hours of co-incubation with their respective target cells at a set effector-target ratio. The Luc activity of the residual target cells was normalized to the NC group (n ≥ 3). Panel b: Flow cytometry was used to detect the relative number of residual target cells after 24 hours of co-incubation of the three CAR-T cells with their respective target cells under the same experimental conditions. The NC group was used as the reference (n = 3).

[0038] Figure 3 : Statistical analysis of CAR-T cell killing efficiency determined by luciferase-based chemiluminescence and flow cytometry. Panel a: Pearson correlation analysis was used to analyze the consistency of the killing trends of three CAR-T cells detected by luciferase-based chemiluminescence and flow cytometry; Panel b: Bland-Altman analysis was used to analyze the equivalence of the two detection methods. P>0.05 means that the two methods are equivalent.

[0039] Figure 4 : Luciferase assay and flow cytometry absolute count magnetic bead method were used to detect PDPN CAR-T cell cytotoxicity. Luciferase assay and flow cytometry absolute count magnetic bead method were used to detect PDPN CAR-T cell cytotoxicity. The target cells were HGC-27. Luc cells, normalized to the NC group;

[0040] Figure 5 Luciferase assay and flow cytometry absolute count magnetic bead method were used to detect the cytotoxicity of CD19 CAR-T cells. Luciferase assay and flow cytometry absolute count magnetic bead method were used to detect the cytotoxicity of CD19 CAR-T cells. The target cells were Nalm6.Luc cell;

[0041] Figure 6 : Luciferase assay and flow cytometry absolute count magnetic bead method were used to detect Her2 CAR-T cell cytotoxicity. Luciferase assay and flow cytometry absolute count magnetic bead method were used to detect Her2 CAR-T cell cytotoxicity. The target cells were B16F10-Her2 Luc cell;

[0042] Figure 7 : Statistical analysis of the CAR-T cell killing results determined by luciferase and flow cytometry. Figures ac: Pearson correlation analysis was used to analyze the consistency of the killing results trends of PDPN CAR-T, CD19 CAR-T, and Her2CAR-T detected by luciferase and flow cytometry. The Bland-Altman analysis was used to analyze the equivalence of the results of the two detection methods. P>0.05 means that the two methods are equivalent. DETAILED DESCRIPTION

[0043] The present invention will be further described below with reference to specific embodiments. The following specific embodiments are intended only to illustrate the present invention and are not to be construed as limiting the present invention. Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the claims and their equivalents.

[0044] The experimental consumables, reagents, and raw materials used in the present invention are readily available to those of ordinary skill in the art and, unless otherwise specified, can be obtained commercially. Experimental methods for which specific conditions are not specified in the present invention are generally performed under conventional conditions or as recommended by the manufacturer. In particular, the following examples are intended only to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are intended only to illustrate the present invention and should not, and will not, limit the present invention described in detail in the claims.

[0045] Example 1 The Luciferase tag constructed by the CMV promoter cannot indicate the true killing level of CAR-T cells

[0046] 1. Experimental Materials

[0047] Human embryonic kidney cell line HEK-293T, human B-lymphoid leukemia cell line Nalm6, mouse skin melanoma cell line B16F10, and human ovarian cancer cell line SKOV3 were purchased from the ATCC Biological Standard Resource Center in the United States; human gastric cancer cell line HGC-27 was purchased from the Cell Bank / Stem Cell Bank of the Chinese Academy of Sciences; human peripheral blood mononuclear cells (PBMCs) were donated by patients (with signed informed consent).

[0048] Luciferase substrate (30 mg / mL) was prepared by dissolving 1 g of D-Luciferin powder (purchased from Xuzhou Micro-Cormand, catalog number VIC1550) in 33.34 mL of ddH2O, sterilized by filtration using a 0.20 μm filter, and stored in a −80°C refrigerator after aliquoting.

[0049] SPF C57BL / 6J mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All mice were housed at the Experimental Animal Center of Xuzhou Medical University, maintained at a temperature of 23°C–25°C and a humidity of 40%–60%. They were kept on a 0-12 h day / night schedule and had free access to water and food. Experimental animal care complied with ethical animal welfare standards, with ethics number 202207S010.

[0050] 2. Experimental methods

[0051] 2.1 Construction of pCDH-CMV-Luciferase-HygroB plasmid and lentiviral packaging

[0052] (1) Obtain the luciferase fragment from the commercial pLVX-Luc (Addgene NO.#218630) plasmid and ligate it into the pCDH-CMV (Addgene NO.#72265) vector backbone. Design the following homologous recombination primer sequences;

[0053]

[0054] CMV-Luciferase-HygroB plasmid; (3) Prepare the enzyme digestion system according to the table below;

[0055]

[0056] (4) Prepare the PCR system according to the table below, using the Phanta Max Super-Fidelity DNA Polymerase Kit;

[0057]

[0058] (5) Prepare the homologous recombination system according to the table below, using the ClonExpress MultiS One Step Cloning Kit;

[0059]

[0060] After the configuration is completed, the reaction was carried out in a PCR instrument at 37℃ for 30 minutes. (6) Transformation of homologous recombination products: Thaw DH5α competent cells on ice, add 10μL homologous recombination products to 100μL competent cells, flick the bottom of the tube to mix, and let it stand on ice for 30 minutes; heat shock at 42℃ for 45 seconds, let it stand on ice for 2 minutes, add 900μL LB liquid medium, shake and culture at 37℃ at 200rpm for 1 hour, centrifuge at 5000rpm for 5 minutes, resuspend the bacteria to 100μL, evenly spread on AMP-resistant LB plates, and invert and culture at 37℃ overnight. (7) Pick a single clone and expand the culture: Prepare a 50mL centrifuge tube, add 20mL LB liquid medium and 20μL ampicillin (50mg / mL); pick a round, uniformly sized, and clearly edged single clone, inoculate it into the above centrifuge tube, and place it tilted in a constant temperature air shaker at 37℃ at 200rpm for 12 hours. (8) Extract the plasmid according to the instructions of the Novagen Endotoxin Removal Mini-Medium Extraction Kit. (9) CMV-Luciferase Lentivirus Packaging: On the first day, prepare HEK-293T cells in good condition and place them at 6×10 6 The cells were plated into 10 cm cell culture dishes. The next day, the pCDH-CMV-Luciferase recombinant plasmid was co-transfected with the auxiliary plasmids pMD2.G and psPAX2 into 293T cells. The cell culture supernatants were collected 48 h and 72 h after transfection, respectively. The cells were mixed evenly and filtered through a 0.45 μm filter. The lentivirus was concentrated by low-temperature ultracentrifugation and stored at -80°C after aliquoting.

[0061] 2.2 PDPN CAR and CD19 CAR Lentivirus Packaging and Titer Determination

[0062] (1) Lentivirus packaging: On the first day, 293T cells were plated into 10 cm dishes; on the second day, PDPN CAR or CD19CAR lentiviral shuttle plasmids: pMD2.G:psPAX2=7.5μg:2.5μg:5μg were co-transfected into 293T cells, and the cell culture supernatants were collected 48h and 72h after transfection, respectively. The cells were mixed and passed through a 0.45μm filter. The lentivirus was concentrated by ultracentrifugation and aliquoted and stored at -80℃. (2) Titer determination: On the first day, 293T cells were 1×10 5 Plate cells / dish into 24-well plates; count the number of cells in one well the next day, and infect 293T cells according to the system configuration in the table below; collect cells 48 hours after infection for CAR positive rate detection; the number of cells at the time of infection × (positive rate between 10% and 20%) / corresponding virus stock solution volume × 10 -3 (TU / mL) = virus titer.

[0063]

[0064]

[0065] 2.3 Preparation of PDPN CAR-T and CD19 CAR-T cells

[0066] 2.3.1 Thawing and Purifying PBMCs and Activating T Cells

[0067] (1) Prepare the human T cell culture medium according to the solution preparation and preheat it in a 37°C water bath; (2) Take a 15 mL centrifuge tube, add 5 mL of RPMI 1640 culture medium, and preheat it to 37°C in a water bath; (3) Take out PBMC from the liquid nitrogen tank, quickly thaw it in a 37°C water bath, transfer it to the above 15 mL centrifuge tube, centrifuge it at 300g for 5 minutes, and discard the supernatant; (4) Resuspend the human T cell culture medium and mix it evenly by pipetting, filter the cell suspension through a 40 μm cell sieve and collect it in a new 15 mL centrifuge tube, and count the cells; (5) Use the human T cell purification kit to purify PBMC according to the instructions, obtain T cells and count them; (6) Wash the required number of human T cell CD3 / CD28 activation magnetic beads, mix them with purified T cells at a ratio of magnetic beads: cells = 1:1, and culture them in a cell culture incubator.

[0068] 2.3.2 PDPN CAR or CD19 CAR Lentivirus Infection of T Cells

[0069] (1) One day before infection, coat a 24-well plate with RetroNectin (7 μg / mL) at 300 μL / well at 4°C in the dark for 12 h; (2) Observe the activated T cells under a microscope for 72 h. After successful activation, collect and count the cells; (3) Remove the 24-well plate from step (1), remove the RetroNectin, add 300 μL of 2% BSA / PBS, and block at room temperature for 30 min. Then, wash twice with 1× PBS; (4) Configure the infection system according to the table below, transfer the cells along the side wall to the 24-well plate from step (3), and centrifuge at 1000 g for 2 h in a horizontal centrifuge. Set the temperature to 30°C and increase by 4 and decrease by 4.

[0070]

[0071] (5) After centrifugation, the cells were returned to the cell culture incubator for further culture. (6) 18 hours after centrifugation, fresh human T cell culture medium was replaced for further culture. (7) 48 hours after infection, the activated magnetic beads were removed, and the CAR-T cells were collected into a 15 mL centrifuge tube. The cell suspension volume was adjusted to 5 mL using RPMI 1640 culture medium. After mixing, the cells were immediately placed on a magnetic stand for 5 minutes. The supernatant was transferred to a new 15 mL centrifuge tube and centrifuged at 300 g for 5 minutes. The supernatant was discarded and the cells were resuspended in human T cell culture medium to adjust the cell density to 1×10 6 / mL, continue to culture and observe; (8) Detect the CAR positive rate by flow cytometry 3-5 days after infection.

[0072] 2.4 Detection of CAR-positive rates of PDPN CAR-T and CD19 CAR-T cells by flow cytometry

[0073] 2.4.1 Direct labeling method (using CD34-binding cell-expressed RQR8 sequence)

[0074] (1) 5×10 5 PDPN CAR-T cells were collected into 1.5 mL EP tubes and centrifuged at 300 g for 5 min; (2) washed twice with 1 mL of 1× PBS, centrifuged under the same conditions as above, and the supernatant was discarded; (3) cells were resuspended with CD34-FITC antibody diluted in 5% FBS / PBS and incubated on ice in the dark for 30 min; (4) after incubation, cells were washed twice with 1 mL of 1× PBS, centrifuged under the same conditions as above; (5) supernatant was discarded, cells were resuspended with 400 μL of 1× PBS, filtered with a 200-mesh nylon filter and transferred to a flow cytometer, and CD34-positive cells were detected on a flow cytometer.

[0075] 2.4.2 Indirect labeling method (using Protein L to bind to the CAR scFv sequence expressed on the cell surface)

[0076] (1) Take 5×10 5 (1) Place 100 CD19 CAR-T cells in a 1.5 mL EP tube and centrifuge at 300 g for 5 min; (2) Wash twice with 1 mL 1× PBS and centrifuge as above, discard the supernatant; (3) Resuspend the cells with Protein L-Biotin antibody diluted in 5% FBS / PBS and incubate on ice for 30 min; (4) Wash once with 1 mL 1× PBS and centrifuge as above, discard the supernatant; (5) Resuspend the cells with Biotin-BV421 secondary antibody diluted in 5% FBS / PBS and incubate on ice in the dark for 20 min; (6) After incubation, wash twice with 1 mL 1× PBS and centrifuge as above; (7) Discard the supernatant, resuspend the cells with 400 μL 1× PBS, filter with a 200-mesh nylon filter and transfer to a flow cytometer, and detect Protein L-positive cells on a flow cytometer.

[0077] 2.5 Her2 CAR retroviral packaging

[0078] (1) On the first day, 293T cells were cultured at 6×10 6(2) The Her2 CAR plasmid was co-transfected into 293T cells at 18 μg and 6 μg of pCL-Eco on the next day. The cell culture supernatants were collected 48 h and 72 h after transfection, mixed well, and filtered through a 0.45 μm filter to obtain the retroviral solution, which was aliquoted and stored at -80°C.

[0079] 2.6 Her2 CAR-T cell preparation, optimal retroviral infection volume, and CAR positivity rate monitoring

[0080] (1) Sterilize scissors and forceps with high-pressure steam in advance, isolate the mesenteric and inguinal lymph nodes of C57 mice (6-8 weeks old) in a clean bench, grind through a 40 μm filter membrane into a mouse T cell-specific culture medium, and centrifuge at 300 g for 5 minutes to obtain a cell pellet; (2) During the centrifugation, prepare the mouse T cell activation culture medium, aspirate 5 mL, resuspend by pipetting and mix thoroughly, and then count the cells to adjust the cell density to 1×10 6 / mL, 1mL per well was plated in a 48-well plate and cultured in a 37℃ cell culture incubator for 24h; (3) After 24h of activation, one well was divided into three wells and the volume of each well was supplemented with mouse T cell culture medium to 500μL; (4) 0, 50, 100, 150, 200, and 400μL of Her2 CAR-BFP retroviral solution were added respectively, centrifuged at 26℃ 1500g for 2h, 4 rises and 4 drops, and continued to culture in a 37℃ cell culture incubator for 48h; (5) CAR-T cells were collected 48h after infection with retrovirus and cell counts were performed; (6) CAR positive rate was detected by flow cytometry: the percentage of BFP positive cells co-expressed with Her2 CAR sequence was detected, which was the CAR positive rate. Take 5×10 5 Cells were centrifuged at 300 g for 5 min and the supernatant was discarded. The cells were washed with 1× PBS and centrifuged as before. The supernatant was discarded. The cells were resuspended in 400 μL of 1× PBS and filtered through a 200-mesh nylon filter. The percentage of BFP-positive cells was determined by flow cytometry.

[0081] 2.7 CAR-T cell killing function detection

[0082] 2.7.1RTCA detection of CAR-T killing efficiency

[0083] (1) Observe the adherent cells under a microscope and see if they are in good condition. Digest the cells into a single cell suspension, mix thoroughly by pipetting, and then count the cells. (2) Adjust the density of HGC-27 cells to 1×10 5 The SKOV3-CD19 cell density was adjusted to 8×10 4 The B16F10-Her2 cell density was adjusted to 6×10 4 / mL; (3) Add 50μL DMEM complete medium to the detection plate to determine the baseline; (4) Inoculate the cells at 100μL / well in the RTCA cell culture plate and culture in a 37℃ cell culture incubator for 24 hours to monitor the cell growth signal; (5) Prepare PDPN CAR-T, CD19 CAR-T, and Her2 CAR-T cells in good condition, and follow the same steps as before; (6) After the tumor cells adhere to the wall and the growth curve reaches about 1, add PDPN CAR-T (target ratio of 0.1:1, 0.2:1), CD19 CAR-T (target ratio of 0.25:1, 0.5:1), and Her2 CAR-T (target ratio of 0.25:1, 0.5:1, 1:1) cells to the culture wells of the corresponding target cells, and continue to observe the killing curve of CAR-T cells against tumor cells.

[0084] 2.7.2 Detection of CAR-T killing efficiency by absolute counting magnetic beads method using flow cytometry

[0085] (1) As before, tumor cells were converted into single-cell suspensions, target cells were labeled with CFSE (5 μM), and the HGC-27 cell density was adjusted to 1.2 × 10 5 The B16F10-Her2 cell density was adjusted to 1.2×10 5 / mL, 500 μL per well was plated into a 24-well plate, with 3 replicates per group; (2) The density of suspended Nalm6 cells was adjusted to 1×10 6 500 μL per well was plated into a 24-well plate, and CD19 CAR-T cells were directly added at an effector-target ratio of 0.25:1 or 0.5:1 for co-incubation; (3) After the adherent cells adhered, PDPN CAR-T cells were added to HGC-27 cells at an effector-target ratio of 0.1:1 or 0.2:1, and Her2 CAR-T cells were added to B16F10-Her2 cells at an effector-target ratio of 0.25:1 or 0.5:1. After co-incubation for 24 h, the cells were removed from the 24-well plate and incubated using CountBright according to the instructions. TM Absolute counting beads were used to detect the number of CFSE-positive cells. The number of CFSE-positive cells represented the number of tumor cells. The relative cell viability of each group was calculated with the negative control group at the corresponding time point as a reference.

[0086] 2.7.3 Luciferase-based Chemiluminescence Method (Luciferase Method) for Detecting CAR-T Killing Efficiency

[0087] (1) As before, the three tumor cells were converted into single cell suspensions, and the density of HGC-27 cells was adjusted to 1.2×10 5The B16F10-Her2 cell density was adjusted to 1.2×10 cells / mL 5 The cell density of Nalm6 cells was adjusted to 1×10 6 / mL; (2) Target cells were plated into a white transparent bottom 96-well cell culture plate; (3) Suspended Nalm6 cells were directly added to CD19 CAR-T cells at the set effector-target ratio for co-incubation; (4) After the adherent cells attached, PDPN CAR-T cells were added to HGC-27 cells at the set effector-target ratio, and Her2 CAR-T cells were added to B16F10-Her2 cells at the set effector-target ratio; (5) After co-incubation for 24 hours, 10 μL of D-Luciferin with a final concentration of 3 mg / mL was added to each well, and the mixture was shaken at 1000 rpm for 30 seconds. The Luc chemiluminescence intensity was detected by a multifunctional microplate reader. The relative cell Luc activity of each group was calculated using the negative control group at the corresponding time point as a reference.

[0088] 2.8 Correlation Analysis

[0089] (1) GraphPad Prism 9.0 software was used for plotting and statistical analysis. Pearson correlation analysis was performed on the correlation between the killing levels of CAR-T cells detected by flow cytometry and luciferase assay. r>0 indicates a positive correlation, r<0 indicates a negative correlation, and |r|>0.7 indicates a high correlation. The higher the correlation, the better the consistency between the two methods. (2) MedCalc software was used for Bland-Altman analysis to determine the equivalence of the results of the two detection methods. It is a combination of quantitative and qualitative analysis. The difference between y and x is the Yb variable, and the mean of y and x is the Xb variable. A Bland-Altman scatter plot was drawn, and the two straight lines of the Yb mean ±1.96×SD were used as reference lines. If the scatter points are evenly distributed above and below the Yb=0 horizontal line, most of the scatter points are within the limits of agreement (95% reference value of the mean difference D), and the P value is greater than 0.05, it indicates that the two methods have a high degree of equivalence, that is, one method is equivalent to the other method.

[0090] 2.9 Data Analysis and Processing

[0091] Flow cytometry data were analyzed and processed using Flowjo V10 software, Bland-Altman analysis was performed using MedCalc software, and other data were statistically analyzed using GraphPad Prism 9.0 software. The t-test was used to compare the means between the two groups. A p value of < 0.05 was considered statistically significant.

[0092] 3. Experimental results

[0093] 3.1 Construction and Cytotoxicity Testing of Human PDPN CAR-T, CD19 CAR-T, and Murine Her2 CAR-T Cells

[0094] Based on the CAR-T cell therapy product targeting PDPN constructed by the inventor's research group in the early stage [WANG X, WANG P, LIAO Y, et al. Expand available targets for CAR-T therapy to overcome tumor drug resistance based on the "Evolutionary Traps" [J]. Pharmacological research, 2024, 204: 107-221.] ( Figure 1 a) To evaluate the killing function of PDPN CAR-T cells, we constructed the HGC-27 gastric cancer cell line based on the HGC-27 gastric cancer cell line that highly expresses PDPN and overexpressed the Luc tag using the CMV promoter. CMV-Luc Cell line. But surprisingly, the experimental results showed that after PDPN CAR-T attacked the target cells, the measured value of Luc expression in the target cells increased ( Figure 2 a). This is contrary to the killing results determined by RTCA ( Figure 1 d). Given that PDPN CAR-T cells target solid tumor cells, in order to verify the true universality of the above results, we constructed two other CAR-T cell killing target cell systems ( Figure 1 a) This includes the currently mature assay system for CD19 CAR-T cells targeting human leukemia cells to kill Nalm6 cells, and the assay system for mouse CAR-T cells targeting Her2 to kill B16F10-Her2 cells, a mouse melanoma cell line that overexpresses Her2. RTCA was first used to verify that both CAR-Ts could significantly cause target cell death ( Figure 1 ef).

[0095] 3.2 The Luc tag constructed by the CMV promoter cannot indicate the true killing level of CAR-T cells

[0096] In order to verify whether the above phenomenon is accidental, we used the same technical method to construct Nalm6 CMV-Luc 、B16F10-Her2 CMV-Luc Cell lines. Subsequently, the luciferase assay was used to detect the Luc values ​​of CD19 CAR-T and Her2 CAR-T cells after they killed their respective target cells. The results showed that the phenomenon of increased Luc values ​​after the above-mentioned CAR-T cells killed target cells also existed ( Figure 2a). To confirm that the increase in Luc values ​​was abnormal, we used CFSE to label target cells while maintaining consistent experimental conditions. We then used absolute counting magnetic beads to measure the number of target cells remaining after CAR-T cell killing by flow cytometry. The experimental results also showed that the above-mentioned CAR-T cells were able to significantly cause target cell death ( Figure 2 b) Therefore, we speculate that in these CAR-T cell-killing target cell systems, the elevated Luc values ​​of target cells are abnormal.

[0097] Given that different detection methods have differences in measurement accuracy and equivalence, the absolute counting magnetic bead method of flow cytometry can accurately count the absolute number of target cells after CAR-T cell attack. Based on this, we believe that the killing data calculated by flow cytometry results can truly reflect the killing efficiency of CAR-T cells. In response to the problems of accuracy and equivalence of different detection methods, we used Pearson correlation analysis to analyze the trend similarity of the killing results obtained by the Luc-based chemiluminescence method and the flow cytometry absolute counting magnetic bead method. At the same time, Bland-Altman analysis was used to calculate the equivalence of the killing results obtained by the Luc-based chemiluminescence method and the flow cytometry absolute counting magnetic bead method. The results showed that in the above three CAR-T cell killing target cell systems, the P values ​​of the Bland-Altman analysis results were all less than 0.05, and the Pearson coefficient r was less than 0, suggesting that the data trends of the Luc-based chemiluminescence method and the flow cytometry determination were opposite and not equivalent ( Figure 1-3 ab). Therefore, in the aforementioned CAR-T cell killing target cell system, the elevated Luc value of target cells is indeed an abnormal phenomenon. Therefore, we conclude that the Luc tag constructed using the CMV promoter cannot accurately indicate the true killing level of CAR-T cells.

[0098] Example 2: The EF1α promoter is more suitable for constructing a Luc reporter gene to indicate the killing function of CAR-T cells

[0099] 1. Experimental Materials

[0100] Same as described in Example 1.

[0101] 2. Experimental methods

[0102] 2.1 Construction of pCDH-EF1α / SFFV / hPGK / CAG / SV40-Luciferase Plasmid and Lentivirus Packaging

[0103] (1) Using the constructed pCDH-CMV-Luciferase plasmid as the vector backbone, the SV40 promoter sequence was obtained by PCR from the existing lentiviral vector in the laboratory, the CAG promoter sequence was obtained from the Addgene NO.#218630 ​​plasmid from the Addgene website, the SFFV promoter sequence was obtained from the Addgene NO.#45578 plasmid, and the hPGK promoter sequence was obtained from the ddgene NO.#101852 plasmid. The CAG, SFFV, and hPGK promoter sequences were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and ligated into the pCDH-promotor-Luciferase vector backbone; using the existing pCDH-EF1α plasmid in the laboratory as the backbone, the Luc fragment was fished out from the pLVX-Luc (Addgene NO.#218630) plasmid and ligated into the above backbone, and the following homologous recombination primer sequences were designed;

[0104]

[0105]

[0106] (2) The primers were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and the above fragments were fished out and synthesized into a new pCDH-promotor-Luciferase plasmid by homologous recombination; (3) The enzyme digestion system was prepared as shown in the table below;

[0107]

[0108] (4) Prepare the PCR system according to the instructions of the Novozymes Phanta Max Super-Fidelity DNA Polymerase Kit as in Example 1 to fish out the desired fragments; (5) Prepare the homologous recombination system according to the instructions of the Novozymes ClonExpress MultiS OneStep Cloning Kit as in Example 1, and react in a PCR instrument at 37°C for 30 minutes after the preparation is completed; (6) Transform the homologous recombination product as in Example 1; (7) Pick and expand the single clone culture as in Example 1; (8) Extract the plasmid according to the instructions of the Novozymes Endotoxin-Free Mini-Medium Kit; (9) EF1α / SFFV / hPGK / CAG / SV40-Luciferase Lentivirus Packaging: Perform lentivirus packaging and ultra-speed freezing and centrifugation concentration as in Example 1 to obtain lentivirus, and divide into 50 μL / tube and store in a -80°C refrigerator.

[0109] 2.2 Construction of HGC-27, Nalm6, and B16F10-Her2 cells overexpressing EF1α / SFFV / hPGK / CAG / SV40-Luciferase (1) As in Example 1, HGC-27 and B16F10-Her2 cells were converted into single-cell suspensions, and the cell density of HGC-27 cells was adjusted to 2.5×10 5 The B16F10-Her2 cell density was adjusted to 2.5×10 5 / mL, 2mL per well was plated into a 6-well plate; after the cells were placed in a cell culture incubator and attached to the wall, 50μL of EF1α / SFFV / hPGK / CAG / SV40-Luciferase lentivirus was used to infect the target cells per well. After 8 hours, fresh cell complete culture medium was replaced and culture was continued; (2) Nalm6 cells were converted into a single cell suspension as in Example 1, and the Nalm6 cell density was adjusted to 2×10 6 1 mL was plated into each well of a 12-well plate, and 25 μL of EF1α / SFFV / hPGK / CAG / SV40-Luciferase lentivirus was used to infect target cells. The cells were centrifuged at 1000 g for 2 h, with the speed of both the upper and lower speeds being 4. Fresh complete cell culture medium was replaced after 8 h, and the culture was continued. (3) 72 h after lentiviral infection, 1×10 cells of each type were taken. 6 Cells were cultured and the Luc values ​​of the cells were detected according to the steps in Example 1 to determine whether the cells were successfully constructed; (4) To obtain cells that were successfully infected, G418 was used as a screening agent for cells that needed to construct EF1α-Luciferase, and Hygro B was used as a screening agent for cells that needed to construct SFFV / hPGK / CAG / SV40-Luciferase. After the cell growth state stabilized, G418 and Hygro B were added to HGC-27 and B16F10-Her2 cells at a final concentration of 500 μg / mL, and Hygro B was added to Nalm6 cells at a final concentration of 600 μg / mL. The cells were then cultured routinely. During the culture period, cells that were not successfully infected and did not have G418 or Hygro B resistance gradually died, while cells that were successfully infected and acquired G418 or Hygro B resistance genes were able to survive and proliferate in the culture medium containing G418 or Hygro B.

[0110] 2.3 CAR-T cell killing function detection

[0111] 2.3.1 Flow cytometry absolute counting magnetic bead method

[0112] (1) As in Example 1, the tumor cells were converted into a single cell suspension, and the target cells were labeled with CFSE (5 μM).Luc The cell density was adjusted to 1.2 × 10 5 / mL, B16F10-Her2 Luc The cell density was adjusted to 1.2 × 10 5 cells / mL, 500 μL per well was plated into 24-well plates, with 3 replicates per group; (2) Suspended cells Nalm6 Luc The cell density was adjusted to 1 × 10 6 500 μL per well was plated into a 24-well plate, and CD19 CAR-T cells were directly added at an effector-target ratio of 0.25:1 or 0.5:1 for co-incubation; (3) After the adherent cells were attached to the wall, HGC-27 Luc PDPN CAR-T cells and B16F10-Her2 were added at effector-target ratios of 0.1:1 and 0.2:1. Luc Her2 CAR-T cells were added at effector-target ratios of 0.25:1 and 0.5:1, and the detection method and data analysis were the same as described in Example 1.

[0113] 2.3.2 Luciferase-based chemiluminescence method (luciferase method)

[0114] (1) As in Example 1, three tumor cells were converted into single cell suspensions, and HGC-27 Luc The cell density was adjusted to 1.2 × 10 5 / mL, B16F10-Her2 Luc The cell density was adjusted to 1.2 × 10 5 / mL, Nalm6 Luc The cell density was adjusted to 1 × 10 6 cells / mL, and plated into a 96-well cell culture plate with a white transparent bottom; (2) Suspended cells Nalm6 Luc Directly add CD19 CAR-T cells according to the set effector-target ratio for co-incubation; (3) After the adherent cells adhere to the wall, HGC-270 Luc PDPN CAR-T cells, B16F10-Her2 Luc Her2CAR-T cells were added according to the set effector-target ratio; (4) After 24 hours of co-incubation, detection and data analysis were performed as described in Example 1.

[0115] 2.4 Correlation Analysis

[0116] The data statistical analysis was performed in the same manner as in Example 1.

[0117] 2.5 Data Analysis and Processing

[0118] Flow cytometry data were analyzed and processed using Flowjo V10 software, Bland-Altman analysis was performed using MedCalc software, and luciferase-based chemiluminescence (luciferase) data were processed using Microsoft Excel 2021. Other data were statistically analyzed using GraphPad Prism 9.0 software. Student's t-test was used to compare the means between the two groups. A p value of < 0.05 was considered statistically significant.

[0119] 3. Experimental results

[0120] 3.1EF1α promoter is more suitable for constructing Luc reporter gene to indicate CAR-T cell killing function

[0121] To identify suitable promoters for constructing Luc-tagged target cells in a CAR-T cell cytotoxicity assay, we selected five commonly used promoters that drive gene expression in mammalian cell lines: EF1α, SFFV, hPGK, CAG, and SV40. We constructed lentiviral expression vectors encoding Luc tags for each promoter. We then prepared lentiviruses using these five promoter-driven Luc-tagged lentiviral expression vectors and transduced them into HGC-27, Nalm6, and B16F10-Her2 target cells, respectively.

[0122] First, the luciferase assay was used to detect the Luc values ​​of target cells after the three CAR-T cells attacked target cells. The results showed that in the PDPN CAR-T cell-killing HGC-27 cell and CD19 CAR-T cell-killing Nalm6 cell systems, the target cell Luc values ​​decreased gradually compared with the normal control group with the increase of the effector-target ratio ( Figure 4 、 Figure 5 In the Her2 CAR-T cell-killing B16F10-Her2 cell system, the Luc tag driven by different promoters behaved differently in target cells. For the EF1α and SFFV promoters, as the effector-target ratio increased, the target cell Luc value decreased gradually compared with the normal control group. For the hPGK promoter, although the target cell Luc value decreased after CAR-T attack, it did not decrease with the increase of the effector-target ratio. For the CAG and SV40 promoters, the target cell Luc value increased after CAR-T attack. Figure 6 Under the same experimental conditions, the efficiency of CAR-T cells in killing target cells in the above three CAR-T killing systems was detected by flow cytometry absolute counting magnetic beads method, and the results of luciferase assay were compared. The results showed that the above CAR-T cells were able to significantly cause the death of target cells ( Figure 4 、 Figure 5、 Figure 6 ).

[0123] Existing detection methods have limitations in terms of accuracy and comparability between methods. Flow cytometry using absolute counting magnetic beads can quantify the absolute number of target cells after CAR-T cell activation. Therefore, we believe that the killing data calculated by flow cytometry can more accurately reflect the killing efficiency of CAR-T cells. Therefore, to identify which promoters can be used to indicate CAR-T cell killing efficiency, we evaluated the equivalence and dose-response consistency of the Luc-based assay and the absolute counting magnetic bead method for flow cytometry in assessing CAR-T cell cytotoxicity.

[0124] The experimental results showed that the Luc tags driven by five promoters behaved differently in three CAR-T cell toxicity detection systems. In the mouse Her2 CAR-T cell cytotoxicity system targeting B16F10-Her2 cells, the Luc assay data driven by the CAG and SV40 promoters showed a negative correlation with the dose-response trend measured by flow cytometry, indicating that these promoters are not suitable for this assay system and have poor universality. In addition, although the data generated using the hPGK promoter showed a positive correlation, the correlation was weak and could be used as an alternative ( Figure 6 However, in the other two human CAR-T cell cytotoxicity systems, Pearson correlation analysis statistical analysis showed a positive correlation ( Figure 7 ).

[0125] In addition, we observed that the killing efficiency of CAR-T cells measured by luciferase was generally lower than the relative value detected by flow cytometry at the same effector-target ratio. Therefore, even in a system with a positive dose-response trend, the results of Bland-Altman analysis indicate that the two cytotoxicity measurement methods are not always statistically equivalent. The Luc values ​​of the EF1α and SFFV promoters showed good consistency with the flow cytometry results in all three CAR-T cell toxicity assays. Considering that the trend consistency of the SFFV promoter in Her2 CAR-T cell-mediated B16F10-Her2 cell killing was significantly less than that of the EF1α promoter, in summary, the EF1α promoter is the most suitable choice for constructing target cells carrying Luc tags for CAR-T cell toxicity testing (see the table below). That is, this application discovered for the first time through comparative experiments that the EF1α promoter is more suitable for constructing Luc reporter genes to indicate CAR-T cell killing function, and not all promoters are effective. This result is a technical effect that was unexpected by those skilled in the art based on the prior art.

[0126]

Claims

1. Application of the EF1α promoter in constructing a reporter gene for indicating the killing function of CAR-T cells, characterized in that: The sequence of the EF1α promoter is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The reporter gene is Luciferase, GFP, eGFP, mGFP, RFP, mCherry, tdTomato or Luciferase-GFP; Optionally, the reporter gene is Luciferase.

3. The use according to claim 1, characterized in that The application is achieved by constructing a lentiviral expression vector in which the EF1α promoter drives the expression of the Luciferase gene, transducing the vector into target cells, and detecting the function of CAR-T cells in killing target cells.

4. The use according to claim 3, characterized in that The lentiviral expression vector in which the EF1α promoter drives the expression of the Luciferase gene is a lentiviral vector expressing the pCDH-EF1α-Luciferase plasmid.

5. The use according to claim 1, characterized in that The CAR-T cells are PDPN CAR-T cells, CD19 CAR-T cells or Her2 CAR-T cells; Optionally, the PDPN CAR in the PDPN CAR-T cell is composed of EF1α, RQR8, T2A, PDPN antibody, CD8 transmembrane domain, 4-1BB costimulatory signaling domain, and CD3ε intracellular signaling domain connected in series; Optionally, the CD19 CAR in the CD19 CAR-T cell is obtained by sequentially connecting EF1α, RQR8, T2A, CD19 antibody, CD8 transmembrane domain, 4-1BB costimulatory signaling domain, and CD3ε intracellular signaling domain; Optionally, the Her2 CAR in the Her2 CAR-T cell is obtained by sequentially connecting a CD8α signal peptide, a Her2 antibody, a CD8 hinge region, a CD8 transmembrane domain, a CD28 costimulatory signaling domain, and a CD3ε intracellular signaling domain; Optionally, the amino acid sequence of the heavy chain variable region of the PDPN antibody is as shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 8; Optionally, the amino acid sequence of the CD19 antibody heavy chain variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 12; Optionally, the amino acid sequence of the heavy chain variable region of the Her2 antibody is shown in SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16; Optionally, the sequence of EF1α in the PDPN CAR or CD19 CAR is shown in SEQ ID NO: 18, the amino acid sequence of RQR8 is shown in SEQ ID NO: 19, the amino acid sequence of T2A is shown in SEQ ID NO: 21, the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO: 23, the amino acid sequence of the 4-1BB costimulatory signaling domain is shown in SEQ ID NO: 25, and the amino acid sequence of the CD3ε intracellular signaling domain is shown in SEQ ID NO: 27; Optionally, the amino acid sequence of the CD8α signal peptide in the Her2 CAR is shown as SEQ ID NO: 31, the amino acid sequence of the CD8 hinge region is shown as SEQ ID NO: 33, the amino acid sequence of the CD8 transmembrane domain is shown as SEQ ID NO: 35, the amino acid sequence of the CD28 co-stimulatory signal domain is shown as SEQ ID NO: 37, and the amino acid sequence of the CD3ε intracellular signaling domain is shown as SEQ ID NO:

39.

6. The use according to claim 1, characterized in that The killing function of the CAR-T cells was detected by flow cytometry absolute counting magnetic bead method or luciferase method.

7. A plasmid for EF1α promoter-driven Luciferase gene expression, characterized in that: The plasmid is pCDH-EF1α-Luciferase plasmid; The sequence of the EF1α promoter is shown in SEQ ID NO:

1.

8. A lentiviral vector driven by EF1α promoter to express Luciferase gene, characterized in that: The lentiviral vector is a lentiviral vector expressing the pCDH-EF1α-Luciferase plasmid; The sequence of the EF1α promoter is shown in SEQ ID NO:

1.

9. The method for constructing a lentiviral vector according to claim 8, wherein: The method comprises the following steps: (1) Using the pCDH-EF1α plasmid as a backbone, the Luciferase gene fragment was ligated into the backbone to obtain the plasmid pCDH-EF1α-Luciferase plasmid in which the EF1α promoter drives the expression of the Luciferase gene, which is the target plasmid; (2) co-transfecting the target plasmid with psPAX2 and pMD2.G into host cells; (3) 48-72 hours after transfection, the virus-containing supernatant is collected, wherein the virus is the lentiviral vector according to claim 6; Optionally, the target plasmid: psPAX2: pMD2.G = 7.5 μg: 5 μg: 2.5 μg; Optionally, the host cell is a 293T cell, a HEK293 cell, a 293FT cell, a HT1080 cell or a NIH / 3T3 cell; Optionally, the host cell is a 293T cell.

10. A method for detecting the killing function of CAR-T cells, characterized in that: The method comprises the following steps: (1) using the lentiviral vector of claim 8 to construct tumor cells overexpressing EF1α; (2) The tumor cells are co-cultured with the CAR-T cells, and the killing function of the CAR-T cells is detected by flow cytometry absolute counting magnetic bead method or luciferase method.

Images