Universal HER2 CAR-T cell for expressing HLA-G as well as preparation method and application of universal HER2 CAR-T cell

Abstract

The invention discloses a universal HER2 CAR-T cell for expressing HLA-G as well as a preparation method and application of the universal HER2 CAR-T cell, and belongs to the technical field of biological medicines. According to the present invention, a lentivirus transduction technology and a CRISPR / Cas9 technology are adopted to prepare the universal type CAR-T cell (the universal type HER2 CAR-T cell for expressing the HLA-G targeting HER2), wherein the universal type HER2 CAR-T cell is used for expressing the HLA-G targeting HER2; it is verified that the cell can kill HER2 positive non-small cell lung cancer cells, can resist immunological rejection of allogeneic PBMC cells and can also resist killing of NK92 cells, the survival number of the universal CAR-T is increased, premature exhaustion of the universal CAR-T is avoided, and in a word, a technical foundation is laid for killing HER2 positive tumor cells through the universal CAR-T.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a universal HER2 CAR-T cell expressing HLA-G, its preparation method, and its application. Background Technology

[0002] In recent years, the combination of tumor immunotherapy with surgery, radiotherapy, and chemotherapy has become a new trend in cancer treatment. Tumor immunotherapy mainly includes the use of immune checkpoint antibodies and chimeric antigen receptor T cells (CAR-T) cell therapy. Among them, CAR-T cell therapy is one of the most promising methods in the field of tumor immunotherapy. Its preparation process involves isolating the patient's own T lymphocytes, modifying them in vitro with the CAR gene to prepare CAR-T cells, and then reinfusing them into the patient to kill tumor cells. CAR-T cells are a cell therapy technology, and more than ten CAR-T products have been approved for marketing. These CAR-T cells are derived from the cancer patient's own body, i.e., autologous CAR-T cells. Autologous CAR-T cells are not conducive to large-scale production, belong to personalized treatment, have long waiting times for patients, are expensive, and cancer patients often have low immune function after chemotherapy and radiotherapy, and the number and quality of their own T cells are difficult to meet the requirements for in vitro expansion, which may result in the loss of the opportunity to prepare autologous CAR-T cell therapy. Based on the challenges of preparing autologous CAR-T cells, universal CAR-T cells are highly anticipated. Universal CAR-T cells can provide off-the-shelf cells for patients with the same target, while large-scale production also reduces the cost of CAR-T cell preparation.

[0003] Existing technologies include universal CAR-T cells targeting HER2, which can kill HER2-positive tumor cells, laying the foundation for the application of universal allogeneic CAR-T therapy in patients with HER2-positive tumors. These universal CAR-T cells knock out HLA-I molecules from T cells. HLA-I molecules are crucial molecules for T cells; normally, inhibitory receptors (such as KIRs / NKG2A) of natural killer (NK) cells bind to HLA-I molecules, transmitting inhibitory signals to NK cells. When HLA-I expression decreases or is absent, it triggers the "loss of self" mechanism of NK cells, leading to NK cell activation and expansion, recognizing and killing cells with low or no HLA-I expression. Since these HER2-targeting universal CAR-T cells lack HLA-I molecules, they need to be modified to avoid NK cell killing, prolong their survival time, prevent premature exhaustion, and improve their anti-tumor durability.

[0004] HLA-G belongs to the non-classical HLA-I class of molecules, characterized by low polymorphism and limited tissue distribution. The main receptors for HLA-G are immunoglobulin-like transcripts 2 and 4 (ILT2 and ILT4) and cytotoxic cell immunoglobulin-like receptor 2DL4 (KIR2DL4). ILT2 and ILT4 are inhibitory receptors; ILT2 is mainly expressed on NK cells, T cells, and antigen-presenting cells, while ILT4 is mainly expressed on myeloid cells. KIR2DL4 has both inhibitory and activating effects and is mainly expressed on NK cells and T cells. The function of NK cells is regulated by activating and inhibitory receptors on their surface. The balance between the activating signals transmitted by activating receptors and the inhibitory signals transmitted by inhibitory receptors determines the function of NK cells: killing target cells or achieving autoimmune tolerance. Summary of the Invention

[0005] The purpose of this invention is to provide a universal HER2 CAR-T cell expressing HLA-G, its preparation method, and its application, in order to solve the problems existing in the prior art. The universal HER2 CAR-T cell expressing HLA-G prepared by this invention can kill HER2-positive non-small cell lung cancer cells, resist the immune rejection of allogeneic PBMC cells, and resist the killing by NK92 cells, thereby increasing the number of surviving universal CAR-T cells and preventing premature depletion.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for preparing HLA-G universal HER2 CAR-T cells, comprising the following steps:

[0008] 293-T cells were co-transfected with the lentiviral expression vector pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G, which simultaneously expresses HLA-G and the CAR gene targeting HER2, along with the lentiviral packaging plasmid psPAX2 and the lentiviral packaging plasmid pMD2.G, to obtain a lentivirus expressing the HLA-G-targeting HER2 CAR gene.

[0009] HLA-G HER2 CAR-T cells were obtained by infecting T cells with the lentivirus expressing the HLA-G-targeting HER2 CAR gene.

[0010] Knocking out the TCR and HLA-I molecules in the HLA-G HER2 CAR-T cells yields HLA-G universal HER2 CAR-T cells;

[0011] The pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G is constructed using pLVX as a backbone vector by inserting the Flag-P1h2-CAR-T2A-HLA-G gene into the pLVX vector.

[0012] The nucleotide sequence of the Flag-P1h2-CAR-T2A-HLA-G gene is shown in SEQ ID NO.1.

[0013] Optionally, the weight ratio of pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G, psPAX2, and pMD2.G is 2:1:1.

[0014] Optionally, the infection multiplicity of the infected T cells is 10.

[0015] Optionally, the T cells are CD3-transfected cells. + T cells are sorted and activated.

[0016] Optionally, knocking out the TCR and HLA-I molecules in the HLA-G HER2 CAR-T cells includes knocking out using sgRNA.

[0017] The present invention also provides HLA-G universal HER2 CAR-T cells prepared according to the preparation method described above.

[0018] The present invention also provides the application of the HLA-G universal HER2 CAR-T cells described above in the preparation of antitumor drugs.

[0019] Optionally, the tumor is a HER2-positive tumor.

[0020] Optionally, the HLA-G universal HER2 CAR-T cells expressed can resist NK cell killing.

[0021] The present invention also provides an antitumor drug comprising the aforementioned HLA-G universal HER2 CAR-T cells.

[0022] The present invention discloses the following technical effects:

[0023] To optimize a universal CAR-T cell line targeting HER2 and reduce NK cell attack on it, this invention utilizes lentiviral transduction technology to prepare CAR-T cells expressing HLA-G molecules. Furthermore, CRISPR / Cas9 technology is used to knock out the TCR and HLA-I molecules in these cells, thereby preparing a universal CAR-T cell line expressing HLA-G and targeting HER2. Verification showed that these cells can kill HER2-positive non-small cell lung cancer cells, resist immune rejection by allogeneic PBMC cells, and resist NK92 cell attack, increasing the number of surviving universal CAR-T cells and preventing premature depletion. In conclusion, this invention lays the technical foundation for using universal CAR-T cells to kill HER2-positive tumor cells. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 To detect CD3 in PBMC cells after sorting using flow cytometry + T cell positivity rate; A: Control group; B: CD3 in PBMCs + T cell percentage; C: sorted CD3 + T cell ratio;

[0026] Figure 2 Validation of HLA-G HER2 CAR-T cells; A: Schematic diagram of the lentiviral expression vector pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G that simultaneously expresses HLA-G and the CAR gene targeting HER2; B: Positive rate of control group cells; C: Positive rate of HLA-G HER2 CAR-T cells;

[0027] Figure 3 For the validation of GU HER2 CAR-T cells; A: Proportion of HLA-G HER2 CAR-T cells that are double-positive for both TCR and HLA-I molecules; B: Proportion of HLA-G HER2 CAR-T cells that are TCR-negative; C: Proportion of HLA-G HER2 CAR-T cells that are HLA-I-negative; D: Proportion of HLA-G HER2 CAR-T cells that are double-negative for both TCR and HLA-I molecules.

[0028] Figure 4Schematic diagram of HER2 CAR-T, U HER2 CAR-T, and GU HER2 CAR-T cells;

[0029] Figure 5 To detect the in vitro killing effect of different CAR-T cells on HER2-positive tumor cells using the CCK-8 assay;

[0030] Figure 6 The results of detecting the expression of cytokines GM-CSF (A), IFN-γ (B), IL-2 (C), TNF-α (D) and IL-6 (E) in GU HER2 CAR-T cells killing tumor cells;

[0031] Figure 7 Results of GU HER2 CAR-T cell proliferation assay;

[0032] Figure 8 To detect the immune rejection response of GU HER2 CAR-T cells to allogeneic PBMCs by co-culturing with PBMCs;

[0033] Figure 9 To quantify the cytotoxicity of NK92 cells against CAR-T cells using luciferase. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1

[0040] 1. Materials and Methods

[0041] 1.1 Materials

[0042] The lentiviral expression vector pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G, which simultaneously expresses HLA-G and the CAR gene targeting HER2 (structure as shown in Figure 1), is used. Figure 2 The following cells (as shown in A) were provided by the Tumor Immunology Laboratory of the School of Medical Technology, Henan University of Medical Sciences: lentiviral packaging plasmids psPAX2 and pMD2.G, human embryonic kidney 293-T cells, HER2-positive non-small cell lung cancer PC9 cells and NK92 cells.

[0043] The lentiviral expression vector pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G was constructed using pLVX (purchased from Takara) as a backbone by inserting the codon-optimized "Flag-P1h2-CAR-T2A-HLA-G" gene (nucleotide sequence shown in SEQ ID NO.1) between the XhoI and EcoRI restriction sites in the pLVX vector. In this embodiment, the lentiviral expression vector pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G was constructed by Sangon Biotech (Shanghai) Co., Ltd.

[0044] SEQ ID NO.1:

[0045]

[0046] HER2-targeted TRAC and B2M dual-gene knockout universal CAR-T cells (U HER2 CAR-T) and HER2-targeted CAR-T cells (HER2 CAR-T) were prepared according to the method described in the literature Song Yuan, Li Yang, Yan Bo, et al. Preparation of HER2-targeted TRAC and B2M dual-gene knockout universal CAR-T cells and their killing effect on non-small cell lung cancer cells [J]. Journal of Cellular and Molecular Immunology, 2022, 38(05):432-438. DOI:10.13423 / j.cnki.cjcmi.009435.

[0047] Human peripheral blood lymphocyte separation medium was purchased from Dakowei Biotechnology Co., Ltd.; X-VIVO15 cell culture medium was purchased from LONZA; Dynabeads TM Human T-Activator CD3 / CD28, fetal bovine serum, Lipofectamine 2000 cell transfection reagent, and biotin-labeled anti-Flag tag antibody were purchased from Thermo Fisher Scientific. + T lymphocyte isolation kit, fluorescein isothiocyanate (FITC) labeled anti-CD3 antibody (CD3-FITC), phycoerythrin (PE) labeled anti-HLA-G antibody (HLA-G-PE), FITC-labeled anti-TCRα / β antibody (TCRα / β-FITC), allophycocyanin-cyanine 7. Anti-HLA-A,B,C antibodies labeled with APC / Cy7 (HLA-A,B,C-APC / Cy7), anti-Flag-tagged antibodies labeled with allophycocyanin (APC) (Flag-APC), anti-TCRα / β antibodies labeled with biotin (TCRα / β-Biotin), anti-HLA-A,B,C antibodies labeled with biotin (HLA-A,B,C-Biotin), anti-human CD62L antibodies labeled with PE, anti-human CD45RO antibodies labeled with APC, streptomycin-avidin magnetic beads, and LEGENDplex. TM The multifactor detection kit was purchased from Biolegend; peripheral venous blood was collected from the elbow vein of healthy individuals, and the use of blood products for this project was reviewed and approved by the Ethics Committee of Xinxiang Medical University.

[0048] 1.2 Methods

[0049] 1.2.1 Isolation and activation of T cells

[0050] 10 mL of peripheral venous blood was collected from healthy volunteers and diluted with an equal volume of 1640 culture medium. Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium and analyzed using CD3+. + T lymphocyte isolation kits were used to isolate T cells from PBMCs. The isolated T cells were stained with CD3-FITC, and the purity of the T cells was detected by flow cytometry. T cells were cultured in X-VIVO medium (containing 10% fetal bovine serum and 100 U / mL IL-2) in a 5% CO2 cell culture incubator, and Dynabeads were added. TM Human T-Activator CD3 / CD28 stimulates T cell activation.

[0051] 1.2.2 Preparation of lentiviruses expressing the HLA-G-targeting HER2 CAR gene

[0052] 293-T cells were cultured in 10 cm diameter cell culture dishes with DMED containing 10% serum. When the cell density reached approximately 80%, 10 μg of pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G, 5 μg of psPAX2, and 5 μg of pMD2.G plasmids were co-transfected into 293-T cells using 60 μL of Lipofectamine 2000. After 72 h of transfection, the cell supernatant was collected and filtered through a 0.45 μm filter. 50% PEG-6000 and 3 mol / L NaCl solution were added to the filtrate. The cells were centrifuged at 7000 g and 4 °C for 30 min. The precipitate was dissolved in serum-free X-VIVO15 medium and stored at -80 °C.

[0053] 1.2.3 Preparation and identification of HLA-G-expressing HER2-targeting CAR-T cells

[0054] Take the T cells isolated and cultured in 1.2.1, and infect them with the prepared lentivirus at a multiplicity of infection (MOI) of 10. Remove the virus 24 hours after infection, and continue culturing the cells in X-VIVO15 medium. Take 1×10 6 T cells infected with the virus were stained with Flag-APC and HLA-G-PE antibodies at 4°C in the dark for 30 min, and the expression of HLA-G and CAR genes was detected by flow cytometry. 1×10⁶ cells were collected. 7Cells transduced with lentivirus were washed with sorting buffer, centrifuged at 300g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL of sorting buffer. 5 μL of biotin-labeled anti-Flag antibody was added, and the cells were incubated on ice for 15 min. Subsequently, 10 μL of streptavidin magnetic beads were added to the cells, mixed thoroughly, and incubated on ice for 15 min. After washing with sorting buffer and resuspending the cells, they were magnetically attached to a grid for 5 min, and the target cells were collected and cultured in X-VIVO15 complete medium. Successfully transduced T cells were named HLA-G HER2 CAR-T cells.

[0055] 1.2.4 Knockout and Identification of HLA-G HER2 CAR-T Cell TCR and HLA-I Molecules

[0056] TRAC sgRNA1 and B2M sgRNA1 targeting the TRAC and B2M genes were synthesized (the nucleotide sequences of sgRNA and sgRNA transcription primers were referenced in the literature Song Yuan, Li Yang, Yan Bo, et al. Preparation of HER2-targeted TRAC and B2M dual-gene knockout universal CAR-T cells and their killing effect on non-small cell lung cancer cells [J]. Journal of Cellular and Molecular Immunology, 2022, 38(05):432-438.DOI:10.13423 / j.cnki.cjcmi.009435.). The transcription product sgRNA was prepared using an in vitro one-step transcription kit. The synthesis system was: 2 × sgRNA Reaction Buffer 10 μL, sgRNA transcription primer 2 μL, Enzyme Mix 2 μL, and RNase Free Water to 20 μL. Reaction conditions: Incubate at 37℃ for 2 hours, then add 1 μL of DNase I and react at 37℃ for 30 minutes to remove the DNA template. Incubate the reaction product at 75℃ for 5 minutes to obtain the transcription product sgRNA. (Prepared at 1 × 10⁻⁶) 6Collect the required number of cells per 2 mm electroporation cuvette, remove the activating magnetic beads, and wash the cells twice with sterile Duchenne phosphate-buffered saline (D-PBS). Mix 1 μg TRAC sgRNA1 and 1 μg B2M sgRNA1 with 5 μg Cas9 protein, and incubate at room temperature for 10 min to form a ribonucleoprotein (RNP) complex. Resuspend the cells and RNP complex in Opti-MEM medium to a final volume of 25 μL, and add this to the bottom of the electroporation cuvette. Set the electroporation apparatus to 180 V and the drive voltage to 45 V. When the resistance of the electroporation cuvette reaches approximately 200 Ω, electroporate HLA-G HER2 CAR-T cells. After electroporation, resuspend the cells in X-VIVO15 medium for culture. Five days after electroporation, collect cells from the control group (un-electroplated HLA-G HER2 CAR-T cells) and the experimental group. Take 1 × 10⁶ cells... 6 Add 1 cell to a 1.5 mL EP tube, add 5 μL of TCRα / β-FITC antibody and 5 μL of human HLA-A,B,C-APC / Cy7 antibody, and detect the knockout efficiency of TCR and HLA-I molecules.

[0057] 1.2.5 Screening of TCR and HLA-I class double-negative cells

[0058] Take 1×10 7 After electroporation, cells were washed with sorting buffer, centrifuged at 300g for 5 min, and the supernatant was discarded. Cells were resuspended in 100 μL of sorting buffer. 5 μL of biotin-labeled anti-TCRα / β antibody and 5 μL of biotin-labeled anti-HLA-A,B,C antibody were added, and the cells were incubated on ice for 15 min. After resuspending the cells in magnetic bead vortex, 10 μL of streptavidin magnetic beads were added, and the mixture was thoroughly mixed and incubated on ice for 15 min. Cells were washed with sorting buffer, resuspended, and magnetically attached for 5 min. The target cells were then collected and cultured in X-VIVO15 complete medium. The expression of TCR and HLA-I class molecules in the sorted cells was detected by staining with TCRα / β-Biotin antibody and HLA-A,B,C-Biotin antibody. The HLA-G HER2 CAR-T cells with dual knockout of TCR and HLA-I class molecules were named GU HER2 CAR-T cells.

[0059] 1.2.6 Killing effect of GU HER2 CAR-T cells on HER2-positive PC9 tumor cells

[0060] Take 1 × 10 4 PC9 (HER2) +Cells were cultured in 96-well plates. After tumor cells adhered, HER2 CAR-T, U HER2 CAR-T, GU HER2 CAR-T, and T cells were added to target cells at effector-to-target ratios of 4:1, 2:1, 1:1, and 0.5:1, respectively, and resuspended in 200 μL of 1640 complete culture medium. Each group had four replicates, with separate wells for tumor cells and blank cells. The 96-well plates were co-incubated in an incubator. After 12 h, the supernatant was discarded, and the cells were washed twice by centrifugation at 800 rpm for 5 min with 1 × PBS. CCK8 reagent was added to detect target cell viability. 100 μL of diluted CCK8 reagent was added to each well to resuspend the cell pellet, and the plates were incubated for another 2 h. The absorbance was read at 450 nm.

[0061] 1.2.7 Detection of GU HER2 CAR-T cell cytokine secretion levels in killing tumor cells

[0062] PC9 tumor cells were seeded into 96-well plates, and HER2 CAR-T cells, GUHER2 CAR-T cells, and T cells were added at a 1:1 effector-to-target ratio for co-incubation. 200 μL of cytokine-free X-VIVO15 medium was added to each well to resuspend the cells. After 24 h of culture, 50 μL of culture medium was collected and used in LEGENDplex. TM The multifactor kit was used to detect the levels of GM-CSF, IFN-γ, IL-2, TNF-α and IL-6.

[0063] 1.2.8 Detection of GU HER2 CAR-T cell proliferation

[0064] Isolated T cells and prepared GU HER2 CAR-T cells were cultured in 96-well plates, with 1 × 10⁶ cells per well. 4 Each cell type was cultured in 15 wells using X-VIVO medium (containing 10% fetal bovine serum and 100 U / mL IL-2). Cells from three wells were randomly selected each time from day 1 to 5 after culture and counted under a microscope. Cell proliferation curves were plotted based on the counting results.

[0065] 1.2.9 Detection of GU HER2 CAR-T cell differentiation subsets

[0066] Take 1×10 6GU HER2 CAR-T cells were resuspended in 500 μL of flow cytometry washing buffer and centrifuged at 500g for 5 min. The cell pellet was then resuspended again in flow cytometry washing buffer and centrifuged at 500g for 5 min. The cells were then resuspended in 100 μL of PBS. 2 μL of PE-labeled anti-human CD62L antibody and 2 μL of APC-labeled anti-human CD45RO antibody were added to the cells, and the cells were incubated at 4°C for 30 min, vortexing three times during incubation. After washing the cells twice with cell washing buffer, the cells were analyzed by flow cytometry.

[0067] 1.2.10 Detection of rejection response of GU HER2 CAR-T cells to allogeneic PBMCs

[0068] Allogeneic PBMCs were co-cultured with HER2 CAR-T, U HER2 CAR-T, and GU HER2 CAR-T cells at a ratio of 5:1. CAR-T cells were labeled with carboxyfluorescein diacetate succinimide (CFSE). 5 × 10⁶ cells were cultured in 200 μL of X-VIVO15 complete culture medium. 4 One PBMC and 1 × 10 4 HER2 CAR-T, UHER2 CAR-T, and GUHER2 CAR-T cells were cultured in 96-well plates, with four replicate wells for each culture. The number of CFSE-labeled CAR-T cells was detected by flow cytometry on days 2, 5, and 7 after culture.

[0069] 1.2.11 The resistance of GU CAR-T to NK kills

[0070] CAR-T cell viability was assessed using a quantitative luciferase assay to evaluate the cytotoxic effect of NK92 on CAR-T cells. All CAR-T cells used in this study expressed the René luciferase reporter gene. NK92 cells were co-cultured with HER2 CAR-T, U HER2 CAR-T, and GU HER2 CAR-T cells, respectively, at ratios of NK92 cells to the three CAR-T cells of 10:1, 3:1, and 1:1. After 20 h of co-culture, the luciferase substrate coelenterin h was added. The cell lysis ratio was calculated by measuring fluorescence values ​​to reflect CAR-T cell viability and assess the cytotoxic effect of NK92 cells on CAR-T cells.

[0071] 1.2.12 Data Analysis

[0072] The data in this study were statistically analyzed using GraphPad Prism 5. The experiment was repeated three times, and the data were presented as follows: This indicates that a t-test was used to compare the two groups, and p < 0.05 was considered statistically significant.

[0073] 2. Results

[0074] 2.1 Sorting of T cells

[0075] PBMCs were isolated from venous blood of healthy individuals using density gradient centrifugation with lymphocyte separation medium, and T cells were sorted from the PBMCs using a T lymphocyte separation kit. Results showed ( Figure 1 CD3 in PBMC + The proportion of T cells was 90.20%, and the sorted CD3... + The positive rate of T cells was 98.33%.

[0076] 2.2 Lentiviral Packaging and Preparation of HLA-G HER2 CAR-T Cells

[0077] The structure of the lentiviral plasmid pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G expressing HLA-G and targeting HER2 is as follows: Figure 2 As shown in Figure A, this plasmid was co-transfected with lentiviral packaging plasmids psPAX2 and pMD2.G into 293-T cells to package lentivirus. Lentiviral cells were harvested and used to infect T cells. Five days after infection, HLA-G expression was detected by flow cytometry. The results showed that the positive rate of HLA-GHER2 CAR-T cells was 42.11%, compared to 0.78% in the control group. Figure 2 China B- Figure 2 (C)

[0078] 2.3 Preparation of GU HER2 CAR-T cells

[0079] The RNP complex targeting the TRAC and B2M genes was co-transduced into HLA-G HER2 CAR-T cells via electrotransduction. After electrotransduction and culturing for 5 days, cells with simultaneous TCR and HLA-I class I knockout were screened using magnetic beads. Results showed that 99.67% of HLA-G HER2 CAR-T cells were double-positive for both TCR and HLA-I class I molecules. Figure 3 In HLA-G HER2 CAR-T cells, after the RNP complex targeting the TRAC gene was transfected into TCR-T cells, the proportion of TCR-negative cells was 56.76%. Figure 3 In HLA-G HER2 CAR-T cells, the proportion of HLA-I negative cells was 19.32% after the RNP complex targeting the B2M gene was transfected into HLA-G HER2 CAR-T cells. Figure 3 (C). After negative screening with magnetic beads, the proportion of TCR and HLA-I double-negative cells was 83.92%. Figure 3In the study, cells that simultaneously lack both TCR and HLA-I molecules were named GU HER2 CAR-T cells.

[0080] 2.4 GU HER2 CAR-T cells on HER2 + PC9 tumor cell killing

[0081] HER2 CAR-T, U HER2 CAR-T, and GU HER2 CAR-T (structural diagrams shown in the figure) Figure 4 (As shown) and primary T cells, according to effector-target ratios of 4:1, 2:1, 1:1, and 0.5:1 with HER2 + After co-culturing PC9 tumor cells for 12 h, CCK8 reagent was added to detect target cell viability. After 2 h of incubation, absorbance was measured at 450 nm. The results showed that, compared with primary T cells, HER2 CAR-T, UHER2 CAR-T, and GU HER2 CAR-T cells could specifically kill HER2-positive tumor cells at different effector-to-target ratios. The three CAR-T cell groups showed the best killing effect on tumor cells at an effector-to-target ratio of 4:1. There was no significant difference in killing effect among the different CAR-T groups. Figure 5 ).

[0082] 2.5 Cytokine Detection Results

[0083] HER2 CAR-T cells, GU HER2 CAR-T cells, and T cells were co-incubated with PC9 cells at a 1:1 effector-target ratio for 24 h. The supernatant from the culture medium was collected to detect cytokine secretion. The results are as follows: Figure 6 As shown, GU HER2 CAR-T cells released lower levels of GM-CSF, IFN-γ, IL-2, TNF-α, and IL-6 cytokines than HER2 CAR-T cells, but both were higher than those released by T cells. Despite the difference in cytokine release between HER2 CAR-T and GU HER2 CAR-T cells, there was no significant difference in their tumor-killing ability in vitro, suggesting that GU HER2 CAR-T cells may have a lower risk of generating a cytokine storm.

[0084] 2.6 Results of GU HER2 CAR-T cell proliferation assay

[0085] Both GU HER2 CAR-T cells and T cells showed significant proliferation upon IL-2 stimulation, as shown in the proliferation curves of the two cell types. Figure 7As shown, T cells began to proliferate significantly on day 3 of culture, while GU HER2 CAR-T cells began to proliferate on day 4 of culture. The number of proliferating GU HER2 CAR-T cells was slightly lower than that of T cells, but the difference was not significant.

[0086] 2.7 Immune rejection of allogeneic PBMC cells by GU HER2 CAR-T cells

[0087] Allogeneic PBMCs were co-cultured with HER2 CAR-T, U HER2 CAR-T, and GU HER2 CAR-T cells at a ratio of 5:1. The three types of CAR-T cells were labeled with CFSE. The mixed cells were cultured in 96-well plates with four replicates. The number of CFSE-labeled CAR-T cells was detected by flow cytometry on days 2, 5, and 7. Results are as follows: Figure 8 As shown, over time, compared to HER2 CAR-T cells, U HER2 CAR-T and GU HER2 CAR-T cells had a higher number of surviving cells, with GU HER2 CAR-T cells having the highest number of surviving cells. This indicates that both types of cells can resist immune rejection of allogeneic PBMCs.

[0088] 2.8 The resistance of GU HER2 CAR-T to NK92 cell killing

[0089] NK92 cells were co-cultured with HER2 CAR-T, U HER2 CAR-T, and GU HER2 CAR-T cells at effector-target ratios of 10:1, 3:1, and 1:1, respectively. After 20 hours of co-culture, the luciferase substrate coelenterin h was added, and the survival rate of the three CAR-T cells was compared by detecting fluorescence values. This assessed the cytotoxicity of NK92 cells against the three CAR-T cell types, i.e., their killing ability. Results are as follows: Figure 9 As shown, when the effector-target ratio is 1:1, compared with the cytotoxicity of NK92 cells to HER2 CAR-T and U HER2 CAR-T, NK92 cells showed the least cytotoxicity to GU HER2 CAR-T cells, indicating that GU HER2 CAR-T can resist the killing effect of NK92 cells.

[0090] In summary, to optimize a universal CAR-T cell targeting HER2 and reduce NK cell attack on universal CAR-T cells, this invention uses lentiviral transduction technology to prepare CAR-T cells expressing HLA-G molecules, and then uses CRISPR / Cas9 technology to knock out the TCR and HLA-I molecules in these cells, thereby preparing a universal CAR-T cell expressing HLA-G and targeting HER2. Verification showed that these cells can kill HER2-positive non-small cell lung cancer cells, resist immune rejection by allogeneic PBMC cells, and resist NK92 cell attack, increasing the number of surviving universal CAR-T cells and preventing premature depletion. In conclusion, this invention lays the technical foundation for using universal CAR-T cells to kill HER2-positive tumor cells.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing HLA-G universal HER2 CAR-T cells, characterized in that, Includes the following steps: 293-T cells were co-transfected with the lentiviral expression vector pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G, which simultaneously expresses HLA-G and the CAR gene targeting HER2, along with the lentiviral packaging plasmid psPAX2 and the lentiviral packaging plasmid pMD2.G, to obtain a lentivirus expressing the HLA-G-targeting HER2 CAR gene. T cells were infected with the lentivirus expressing the HLA-G-targeting HER2 CAR gene to obtain HLA-G HER2 CAR-T cells; Knocking out the TCR and HLA-I molecules in the HLA-G HER2 CAR-T cells yields HLA-G universal HER2 CAR-T cells; The pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G is constructed using pLVX as a backbone vector by inserting the Flag-P1h2-CAR-T2A-HLA-G gene into the pLVX vector. The nucleotide sequence of the Flag-P1h2-CAR-T2A-HLA-G gene is shown in SEQ ID NO.

1.

2. The preparation method according to claim 1, characterized in that, The weight ratio of pLVX-EF1pro-Flag-P1h2-CAR-T2A-HLA-G, psPAX2, and pMD2.G is 2:1:

1.

3. The preparation method according to claim 1, characterized in that, The infection multiplicity of the infected T cells is 10.

4. The preparation method according to claim 1, characterized in that, The T cells are CD3-mediated. + T cells are sorted and activated.

5. The preparation method according to claim 1, characterized in that, The knockout of TCR and HLA-I molecules in the HLA-G HER2 CAR-T cells includes knockout using sgRNA.

6. HLA-G universal HER2 CAR-T cells prepared by the preparation method according to any one of claims 1-5.

7. The application of the HLA-G universal HER2 CAR-T cells as described in claim 6 in the preparation of antitumor drugs.

8. The application as described in claim 7, characterized in that, The tumor is a HER2-positive tumor.

9. The application as described in claim 7, characterized in that, The HLA-G universal HER2 CAR-T cells expressed in this study are able to resist NK cell killing.

10. An antitumor drug, characterized in that, The drug comprises the HLA-G universal HER2 CAR-T cells as described in claim 6.

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