Preparation method of enhanced CAR-T cell overexpressing LYAR, cell and application of enhanced CAR-T cell overexpressing LYAR

Abstract

The invention belongs to the technical fields of immunotherapy, bioengineering, gene therapy and cell therapy, and provides a preparation method of an enhanced CAR-T cell for overexpressing LYAR, and the enhanced CAR-T cell and application thereof in order to solve the problems that the killing function of the CAR-T cell in solid tumor treatment is limited, the durability is poor, and function depletion is likely to occur. CDNA of the LYAR gene is cloned to an overexpression plasmid vector and packaged into lentivirus, T cells are jointly infected by the lentivirus and the CAR expression virus, the LYAR gene is forced to express under the driving of an EF-1alpha promoter, and stable overexpression of the LYAR in the CAR-T cells is achieved; and the CAR is a CAR targeting EGFR (epidermal growth factor receptor) and CD19. According to the present invention, the gene coding LYAR and the CAR gene are simultaneously transferred into the T cell through the lentivirus infection technology, and are compulsively expressed under the action of the EF1 alpha promoter, such that the anti-tumor function of the CAR-T cell can be significantly enhanced, and the activation and killing functions of the immune cell can be improved;

Description

Technical Field

[0001] This invention belongs to the fields of immunotherapy, bioengineering and gene therapy, and cell therapy. Specifically, it relates to a method for preparing enhanced CAR-T cells overexpressing LYAR, the cells themselves, and their applications. The transcription factor LYAR serves as a functional factor that enhances the anti-tumor activity and anti-exhaustion ability of CAR-T cells. Background Technology

[0002] In recent years, T-cell therapy expressing chimeric antigen receptors (CARs) has made groundbreaking progress in the treatment of hematologic malignancies, becoming an important tool in cancer treatment. However, despite the significant efficacy of CAR-T therapy in hematologic malignancies, limitations remain in treating disease relapse and drug resistance. Meanwhile, the application of CAR-T cells in the treatment of solid tumors also faces multiple challenges, such as weak tumor invasiveness, high target heterogeneity, and T-cell exhaustion. Among these, T-cell exhaustion is widely considered a key factor limiting the efficacy of CAR-T cell therapy and has become an important research direction for optimizing its therapeutic effects.

[0003] In CAR-T cell functional regulation research, transcription factors have attracted much attention due to their core roles in T cell exhaustion, memory formation, and functional maintenance. Targeting and regulating the expression of key transcription factors can effectively delay T cell exhaustion and enhance its persistence and anti-tumor capabilities, thus becoming an important strategy for improving the efficacy of CAR-T cell therapy. However, current research on transcription factors is mostly based on traditional cell lines or animal models, which cannot fully reflect the dynamic evolution of T cell status in the real tumor microenvironment. Therefore, it is urgent to combine clinical samples, focus on the mechanisms of T cell fate evolution in the real tumor microenvironment, and deeply screen key factors regulating exhaustion status to provide a theoretical basis and target foundation for the precise optimization of CAR-T cells. Summary of the Invention

[0004] To address the current challenges faced by CAR-T cells in solid tumor treatment, such as limited killing function, poor persistence, and easy functional exhaustion, this invention provides a method for preparing enhanced CAR-T cells overexpressing LYAR, along with the cells themselves and their applications, targeting the key mechanisms by which T cell function is suppressed in the tumor immune microenvironment.

[0005] This invention is achieved by the following technical solution: a method for preparing enhanced CAR-T cells overexpressing LYAR, wherein the cDNA of the LYAR gene is cloned into an overexpression plasmid vector and packaged into a lentivirus, which is then co-infected with a chimeric antigen receptor CAR expression virus. The LYAR gene is forcibly expressed under the drive of the EF-1α promoter, thereby achieving stable overexpression of LYAR in CAR-T cells; the nucleic acid sequence of the LYAR gene is shown in SEQ ID NO. 1; the CAR is a CAR targeting EGFR and CD19, which contains an extracellular antigen-binding domain specifically targeting EGFR, the extracellular antigen-binding domain including a heavy chain variable region VH and a light chain variable region VL; VH contains the VH-CDR base sequence shown in SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 11, and VL contains the VL-CDR base sequence shown in SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 12.

[0006] Furthermore, the following steps are included: (1) Synthesize the nucleic acid sequence of the chimeric antigen receptor CAR that targets the epidermal growth factor receptor (EGFR) or the leukocyte differentiation antigen (CD19), and clone the nucleic acid sequence into the Fuw lentiviral vector to construct CAR expression plasmids, named CAR1-EGFR-GFP, CAR2-EGFR-GFP, and CAR-CD19-GFP; further package to obtain CAR lentivirus for transduction of T cells; (2) The human LYAR nucleic acid coding sequence was cloned into the Fuw lentiviral vector to construct the LYAR overexpression plasmid, named LYAR-mCherry; it was further used for packaging to obtain LYAR overexpression lentivirus and realize the transduction of T cells; (3) The CAR-infected lentivirus obtained in step (1) and the LYAR-overexpressing lentivirus obtained in step (2) are concentrated and then used to infect human primary CD3+ T cells to obtain enhanced CAR-T cells overexpressing LYAR.

[0007] The LYAR's CDS region nucleic acid sequence is shown in SEQ ID NO: 1; The CAR1-EGFR-GFP plasmid contains: an EF-1α promoter as shown in SEQ ID NO:13; an N-terminal CD8α signal peptide as shown in SEQ ID NO:4; a variable heavy chain region VH-CDR1 sequence as shown in SEQ ID NO:2; a variable light chain region VL-CDR1 sequence as shown in SEQ ID NO:3; a nucleic acid sequence of the CD8α hinge region as shown in SEQ ID NO:6; a nucleic acid sequence of the CD8α transmembrane region as shown in SEQ ID NO:5; a 4-1BB co-stimulatory domain as shown in SEQ ID NO:8; a CD3ζ signal transduction domain as shown in SEQ ID NO:7; a fusion peptide P2A; and a fluorescent protein GFP as shown in SEQ ID NO:14. The CAR2-EGFR-GFP plasmid contains: an EF-1α promoter as shown in SEQ ID NO:13; an N-terminal CD8α signal peptide as shown in SEQ ID NO:4; a variable heavy chain region VH-CDR2 sequence as shown in SEQ ID NO:9; a variable light chain region VL-CDR2 sequence as shown in SEQ ID NO:10; a nucleic acid sequence of the CD8α hinge region as shown in SEQ ID NO:6; a nucleic acid sequence of the CD8α transmembrane region as shown in SEQ ID NO:5; a 4-1BB co-stimulatory domain as shown in SEQ ID NO:8; a CD3ζ signal transduction domain as shown in SEQ ID NO:7; a fusion peptide P2A; and a fluorescent protein GFP as shown in SEQ ID NO:14. The CAR-CD19-GFP plasmid contains: an EF-1α promoter as shown in SEQ ID NO:13; an N-terminal CD8α signal peptide as shown in SEQ ID NO:4; a variable heavy chain region VH-CDR3 sequence as shown in SEQ ID NO:11; a variable light chain region VL-CDR3 sequence as shown in SEQ ID NO:12; a nucleic acid sequence of the CD8α hinge region as shown in SEQ ID NO:6; a nucleic acid sequence of the CD8α transmembrane region as shown in SEQ ID NO:5; a 4-1BB co-stimulatory domain as shown in SEQ ID NO:8; a CD3ζ signal transduction domain as shown in SEQ ID NO:7; a fusion peptide P2A; and a fluorescent protein GFP as shown in SEQ ID NO:14.

[0008] The difference between the CARs lies in the different single-chain Fv fragments (scFv) included in the extracellular antigen-binding domain. CAR1 and CAR2 are scFv fragments that target EGFR, while CD19CAR is the scFv fragment that targets CD19.

[0009] The CAR structure is forcibly expressed under the drive of the EF-1α promoter, and is linked to the fluorescent protein GFP via the fusion peptide P2A after the CAR structure, thereby achieving simultaneous CAR expression and GFP expression. The EF-1α nucleotide sequence is SEQ ID NO:13; the GFP nucleotide sequence is SEQ I NO:14.

[0010] The present invention also provides enhanced CAR-T cells overexpressing LYAR constructed according to the preparation method described above. These cells target tumor cells expressing EGFR or CD19.

[0011] The present invention also provides the use of the enhanced CAR-T cells overexpressing LYAR in the preparation of antitumor drugs.

[0012] Furthermore, the application of the enhanced CAR-T cells overexpressing LYAR in the preparation of drugs for treating esophageal squamous cell carcinoma.

[0013] The esophageal squamous cell carcinoma is an esophageal squamous cell carcinoma expressing EGFR. Enhanced CAR-T cells overexpressing LYAR specifically lyse EGFR-expressing esophageal squamous cell carcinoma cells in vitro at effector-to-target ratios of 0.01:1 and 0.005.

[0014] The present invention also provides the use of the enhanced CAR-T cells overexpressing LYAR in the preparation of drugs for treating pan-cancer.

[0015] The term pan-cancer includes solid tumors and hematologic malignancies.

[0016] The gene overexpression plasmid vector of the present invention includes: a promoter for driving gene expression and a GFP reporter gene; the vector can be further integrated with a chimeric antigen receptor (CAR) coding sequence targeting EGFR or CD19, or the CDS region of the LYAR gene, to achieve expression of different target genes.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Single-cell multi-omics sequencing technologies (including single-cell transcriptomics, chromatin accessibility analysis, and spatial transcriptomics) were used to systematically study the dynamic evolution of immune cells in the tumor microenvironment during the development and progression of esophageal squamous cell carcinoma. The study found that the transcription factor LYAR was persistently lowly expressed in exhausted T cell subsets, and its chromatin regions were in a closed state, suggesting that LYAR may play an important role in maintaining T cell effector function.

[0018] This invention reveals that the transcription factor LYAR is significantly downregulated in esophageal squamous cell carcinoma (ESCC) and other tumor types during T cell functional exhaustion. Therefore, linking LYAR to T cell functional exhaustion provides a scientific basis for its role as a potential functional regulator, offering a new target for enhancing CAR-T cell function.

[0019] This invention utilizes lentiviral infection technology to simultaneously introduce the gene encoding LYAR and the CAR gene into T cells. Forced expression of these genes under the action of the EF1α promoter significantly enhances the anti-tumor function of CAR-T cells and improves immune cell activation, killing, anti-exhaustion, and memory functions. This invention can also enhance CAR-T cells targeting any specific object by overexpressing LYAR.

[0020] Specifically, this invention achieves stable overexpression of LYAR in CAR-T cells by cloning the cDNA of the LYAR gene into an overexpression plasmid vector, packaging it into a lentivirus, and co-infecting T cells with a CAR expression virus. Forced expression of the LYAR gene under the drive of the EF-1α promoter significantly enhances the anti-tumor killing ability of CAR-T cells and strengthens their resistance to exhaustion and functional durability.

[0021] The technical solution provided by this invention is applicable to pan-cancer therapy and CAR structures targeting multiple targets, exhibiting broad adaptability. By genetically engineering T cells to achieve high expression of LYAR, it can be widely used to enhance the efficacy of CAR-T cells in the treatment of solid tumors, demonstrating promising application prospects and significant potential for wider application. The extracellular antigen-binding domain includes a single-chain Fv fragment (scFv). The overall CAR structure is as follows: Figure 3 As shown; as previously mentioned, the difference in the CAR structure lies in the different scFv segments.

[0022] Enhanced CAR-T cells can release more effector factors while killing tumor cells compared to ordinary CAR-T cells; they have longer persistence than ordinary CAR-T cells; they can differentiate into more effector memory T cells during repeated tumor stimulation experiments compared to ordinary CAR-T cells; they have fewer exhausted T cells during repeated tumor stimulation experiments compared to ordinary CAR-T cells; and different CAR structures can also play the same role in different tumors.

[0023] The remaining parts of the different CAR structures are consistent with those described previously. These functionally enhanced CAR-T cells exhibit better anti-tumor effects in tumor-bearing NCG mice. Attached Figure Description

[0024] Figure 1The results are from single-cell sequencing, showing genes with low expression and low chromatin openness in exhausted T cells. scRNA-seq and scATAC-seq revealed that LYAR (lower left-dashed circle marker) not only had significantly reduced transcriptional levels in exhausted CD8⁺ T cells, but also significantly reduced chromatin openness, indicating that LYAR may play a key role in maintaining T cell function, and its downregulation may be closely related to T cell exhaustion.

[0025] Figure 2 The figures show the expression characteristics of LYAR in pan-cancer CD8+ T cells; A: Overall subtype distribution of pan-cancer T cells; B: Heatmap showing the expression of LYAR and typical exhausted maker cells in subpopulations; C: Expression distribution of LYAR in T cell subpopulations; D: Violin plot showing the expression of LYAR in different T cell subpopulations of pan-cancer cells; E: Expression of LYAR in T cells of classical Hodgkin lymphoma; F: Expression of LYAR in T cells of multiple myeloma; G: Expression of LYAR in T cells of basal cell carcinoma; H: Expression of LYAR in T cells of squamous cell carcinoma. The results indicate that the transcription factor LYAR shows low expression in exhausted CD8+ T cell subpopulations across multiple tumor types, suggesting that low LYAR expression has a universal characteristic across cancer types during T cell exhaustion. Figure 3 The diagram shows the overexpression of CAR and LYAR plasmids; in the diagram: A: Schematic diagram of the CAR1 plasmid structure targeting EGFR; B: Schematic diagram of the CAR2 plasmid structure targeting EGFR; C: Schematic diagram of the CAR plasmid structure targeting CD19; D: Detailed schematic diagram of different CAR structures; E: Schematic diagram of the LYAR plasmid; the sequence details are as described above; Figure 4 To validate the overexpression of LYAR on CAR-T cells; In the figure: A: Flow cytometry (FACS) shows the efficiency of CAR expression in T cells; B: Flow cytometry (FACS) shows that CAR-T cells simultaneously express CAR and LYAR, with CAR on the vertical axis and LYAR on the horizontal axis; C: qPCR shows that LYAR mRNA expression is upregulated in CAR-T cells; D: Western blot shows the protein expression of LYAR; In summary, both CAR and LYAR can be successfully overexpressed on T cells; Figure 5To enhance the cytotoxic activity, cytokine secretion, and proliferation of CAR-T cells through LYAR overexpression; Figure: (A): Under low target-efficacy ratios (E:T = 0.01:1 and 0.005:1), the specific lysis ability of LYAR-overexpressed (OE) and control (NC) CAR-T cells against esophageal squamous cell carcinoma cells (KYSE150L) was evaluated. The results showed that the OE group had stronger cytotoxic activity; (B–C): The secretion of effector factors after CAR-T cell stimulation was detected by ELISA, namely Granzyme B in Figure B and IFN-γ in Figure C. The results were compared under different ESCC cell lines (KYSE150Luc, KYSE450Luc, TE1Luc). The OE group showed significantly enhanced cytokine secretion; (D–E): After labeling with CellTrace Violet dye, the proliferation of T cells on day 4 was analyzed by flow cytometry. D represents the NC group, and E represents the OE group. The percentage of positive cells is shown in parentheses. The results showed that OE The OE group had a higher proliferation rate; (F): Statistical analysis of the proportion of positive cells in the flow cytometry showed that the proliferation of T cells in the OE group was significantly enhanced; (G): T cell counting was performed during continuous culture to assess the expansion capacity of cells in the NC group and the OE group. The OE group showed a significant proliferation advantage on days 4 and 7. Overall, the results showed that LYAR overexpression can significantly enhance the tumor-killing effect, cytokine secretion capacity and proliferation potential of CAR-T cells, suggesting that LYAR plays an important role in maintaining CAR-T cell function and anti-tumor activity. Note: Statistical significance analysis: P<0.01 ( P<0.0001 Error bars represent ± standard deviation (SD). Figure 6The LYAR-overexpressing CAR-T cells exhibit stronger anti-exhaustion ability under repeated tumor stimulation. Figure A: CAR-T cell killing ability under multiple rounds of killing experiments, showing that the LYAR-overexpressing group (OE) maintained strong tumor cell clearance efficiency even after repeated tumor stimulation; B: Proliferation of Donor1-derived CAR-T cells in multiple rounds of killing experiments, indicating that the OE group has superior proliferation capacity under long-term stimulation conditions; C and D: Flow cytometry (FACS) analysis shows that after multiple rounds of tumor cell stimulation, the OE group CAR-T cells had more memory T cells. Subpopulations (including central memory and effector memory T cells) suggest that their differentiation tends to form a more persistent memory phenotype; EF: FACS results showed that after multiple rounds of tumor stimulation, the proportion of PD1 positive cells in CAR-T cells in the OE group was significantly lower than that in the control group (NC), indicating that LYAR overexpression can effectively slow down the functional exhaustion of CAR-T cells; the above results indicate that LYAR overexpression not only enhances the killing activity and proliferation capacity of CAR-T cells under long-term stimulation, but also promotes the formation of memory T cells and reduces the proportion of PD1 positive cells, thereby enhancing their anti-exhaustion characteristics. Note: Statistical analysis was performed using a two-tailed t-test. P<0.05, P<0.01, P<0.001; Figure 7To verify the enhancement of CAR-T cell killing activity, cytokine secretion, and proliferation by LYAR overexpression in different donors; Figure: (A): Under low-efficiency target ratios (E:T = 0.01:1 and 0.005:1), the specific lysis ability of LYAR-overexpressing (OE) and control (NC) CAR-T cells against esophageal squamous cell carcinoma (KYSE150L) was evaluated. The results showed that the killing efficiency of the OE group was significantly enhanced; (B–C): The secretion of effector factors after CAR-T cell stimulation was detected by ELISA, namely Granzyme B in Figure B and IFN-γ in Figure C. The results were compared under different ESCC cell lines (KYSE150Luc, KYSE450Luc, TE1Luc). The results showed that under different ESCC cell lines such as KYSE150L, ​​KYSE450L, and TE1, the secretion levels of Granzyme B (B) and IFN-γ (C) of CAR-T cells in the OE group were significantly higher than those in the control group; (D–E) CellTrace After labeling with Violet dye, T cell proliferation on day 4 was analyzed by flow cytometry. D represents the NC group, and E represents the OE group. The percentage of positive cells in parentheses is the percentage of positive cells. The results showed that the proportion of positive cells in the OE group (E) was significantly higher than that in the control group (D). (F): Statistical analysis of the proportion of positive cells in the flow cytometry plots showed that T cell proliferation in the OE group was significantly enhanced, indicating that the proliferative capacity of T cells in the OE group was significantly enhanced. (G): T cell counting was performed during continuous culture to assess the cell proliferation capacity of the NC and OE groups. The OE group showed a significant proliferative advantage on days 4 and 7. The results showed that the OE group showed a significant proliferative advantage on both days 4 and 7. The above results indicate that LYAR overexpression can significantly enhance the killing function, cytokine secretion capacity, and proliferative activity of CAR-T cells from different donor sources, suggesting that this effect is stable and universal. Note: Statistical significance analysis: P<0.01 ( P<0.0001 Error bars represent ± standard deviation (SD). Figure 8This study demonstrates the anti-exhaustion ability of LYAR-overexpressing CAR-T cells from different donors in multiple rounds of tumor stimulation experiments. Figure A shows the killing ability of Donor2-CAR-T cells in multiple rounds of tumor cell stimulation, with the LYAR-overexpressing group (OE) maintaining stronger killing activity even under continuous stimulation. Figure B shows the cell proliferation of Donor2-CAR-T cells in multiple rounds of tumor cell stimulation, with the OE group exhibiting a significant expansion advantage. Figures C and D show the LYAR-overexpressing CAR-T cells after multiple rounds of tumor cell stimulation. The differentiation of memory T cell subsets in CAR-T cells was investigated. The results showed that under repeated tumor stimulation, the proportions of memory T cell subsets in OE group CAR-T cells were significantly different, suggesting a memory differentiation advantage. Flow cytometry (FACS) analysis showed that the proportion of PD1⁺ positive cells in OE group CAR-T cells was lower, indicating that LYAR overexpression can effectively alleviate CAR-T cell functional exhaustion. These results indicate that LYAR overexpression can enhance the durability and anti-exhaustion ability of CAR-T cells under multiple rounds of tumor stimulation in CAR-T cells from different donor sources. Note: Statistical analysis was performed using a two-tailed t-test. P<0.05, P<0.01, P<0.001; Figure 9CAR-T cells overexpressing LYAR also exhibit stronger function in pan-cancer studies; Figure: A: In a lung cancer cell model, the killing ability of CAR-T cells in the OE group and NC group was tested under low target ratio conditions (E:T = 0.01:1 or less), with the OE group showing stronger lytic activity; B: In a multi-round killing experiment in lung cancer, the proliferation ability of CAR-T cells in the OE group was significantly better than that in the NC group; C: In a gallbladder cancer cell model, the killing ability of CAR-T cells in the OE group and NC group was tested under low target ratio conditions (E:T = 0.05 or less), with the OE group showing stronger lytic activity; D: In a multi-round killing experiment in gallbladder cancer, the proliferation ability of CAR-T cells in the OE group was significantly better than that in the NC group; E: In a colorectal cancer cell model, the killing ability of CAR-T cells in the OE group was tested under low target ratio conditions (E:T = 0.01:1 or less), with the OE group showing stronger lytic activity; F: In a multi-round killing experiment in colorectal cancer, the proliferation capacity of CAR-T cells in the OE group was significantly better than that in the NC group. G: In a skin cancer cell model, under low target-to-cell ratio (E:T below 0.01), the killing capacity of CAR-T cells in the OE group and the NC group was tested, and the OE group showed stronger lytic activity. H: In a multi-round killing experiment in skin cancer, the proliferation capacity of CAR-T cells in the OE group was significantly better than that in the NC group. The above results show that LYAR overexpression can generally enhance the killing activity and proliferation capacity of CAR-T cells in various tumor backgrounds (including lung cancer, gallbladder cancer, colon cancer, and epidermoid carcinoma), indicating that this strategy has broad applicability. Note: Statistical analysis was performed using a two-tailed t-test. P<0.05, P<0.01, P<0.001; Figure 10 To enhance the function of CAR-T cells overexpressing LYAR under different CAR structures; Figure: A: Under CAR2 construction, the lytic activity of OE and NC CAR-T cells against tumor cells was detected under low target-to-cell ratio (E:T ≤ 0.04:1), showing that the OE group had a stronger killing effect; B: Under the CAR2 background, the proliferation capacity of OE group CAR-T cells was significantly better than that of NC group; C: In CD19-CAR construction and Raji-Luc cell model, the killing activity of OE and NC CAR-T cells was compared under low target-to-cell ratio (E:T ≤ 0.1:1), with the OE group showing stronger lytic activity; D: Under the CD19-CAR structure, the proliferation level of OE group CAR-T cells was also significantly enhanced; These results reveal that LYAR overexpression can generally enhance the killing activity and proliferation capacity of CAR-T cells under different CAR construction backgrounds, suggesting its potential value in broad-spectrum anti-tumor therapy. Note: Statistical analysis was performed using a two-tailed t-test. P<0.05, P<0.01, P<0.001; Figure 11 To demonstrate the effect of LYAR on the in vivo anti-esophageal squamous cell carcinoma ability of CAR-T cells; Figure: A: In the NCG tumor-bearing mouse model, after injection of LYAR-overexpressing CAR-T cells, tumor volume progression was significantly slowed, showing its enhanced in vivo anti-esophageal squamous cell carcinoma effect; B: Conversely, LYAR knockout CAR-T cells showed poorer anti-tumor ability in vivo, with tumor burden increasing faster compared to the CTRL group; The above results show that YAR has a significant positive regulatory effect on the anti-tumor function of CAR-T cells in vivo. Note: Statistical analysis was performed using a two-tailed t-test. P<0.05, P<0.01, P<0.001; Figure 12 The study verified that LYAR-overexpressing CAR-T cells enhanced in vivo anti-tumor function in different mouse models of tumors. In the figure: A: In a mouse model of lung cancer, CAR-T cells in the OE group significantly inhibited tumor growth; B: In a mouse model of cholangiocarcinoma, CAR-T cells in the OE group also showed a stronger anti-tumor effect. The above results show that LYAR overexpression endows CAR-T cells with a broad-spectrum in vivo anti-tumor advantage, and its universality has been verified in multiple tumor models. Note: Statistical analysis was performed using a two-tailed t-test. P<0.05, P<0.01, P<0.001. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0028] Equivalent technologies of the specific embodiments described herein that are apparent to those skilled in the art through routine experimentation are included in this application.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0030] Example 1: Plasmid structure design, construction, and virus packaging: (1) The sequence required for this invention was found from existing patent WO 2022068870 A1 and NCBI, and after gene synthesis, the sequence was respectively arranged according to Figure 3 The corresponding nucleic acid sequences of the structures shown were cloned into the Fuw lentiviral vector to construct CAR expression plasmids and LYAR overexpression plasmids, named CAR1-EGFR-GFP, CAR2-EGFR-GFP, CAR-CD19-GFP, and LYAR-mCherry. The nucleic acid sequence of the CDS region of LYAR is shown in SEQ ID NO: 1; the CAR structural sequences differ in that the scFv segment is different.

[0031] The nucleic acid sequence of the variable heavy chain (VH) region of the CAR1-anti-EGFR antibody is shown in SEQ ID NO: 2; the nucleic acid sequence of the variable light chain (VL) region of the CAR1-anti-EGFR antibody is shown in SEQ ID NO: 3; the nucleic acid sequence of the CD8α signal peptide is shown in SEQ ID NO: 4; the nucleic acid sequence of the CD8α transmembrane region is shown in SEQ ID NO: 5; the nucleic acid sequence of the CD8α hinge region is shown in SEQ ID NO: 6; the nucleic acid sequence of the CD3ζ signal transduction domain is shown in SEQ ID NO: 7; and the nucleic acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO: 8.

[0032] The nucleic acid sequence of the variable heavy chain (VH) region of the CAR2-anti-EGFR antibody is shown in SEQ ID NO: 9; the nucleic acid sequence of the variable light chain (VL) region of the CAR2-anti-EGFR antibody is shown in SEQ ID NO: 10.

[0033] The nucleic acid sequence of the antibody variable heavy chain (VH) region in the CD19 CAR structure is shown in SEQ ID NO: 11; the nucleic acid sequence of the antibody variable light chain (VL) region in the CD19 CAR structure is shown in SEQ ID NO: 12.

[0034] Using a Lipo3000 (Thermo Fisher, L3000015), 293T cells were co-transfected with the three-plasmid system vector, pMD2.G, and psPAX2. Lentiviral supernatant was collected at 48 and 72 hours, and filtered through a 0.45 μm filter (Merck Millipore, SLHVR33RB) to obtain lentiviral supernatant, which was then concentrated using a concentration column (Merck Millipore, UFC910096).

[0035] (2) Isolation, stimulation, and expansion of CD3+ T cells: Fresh PBMCs from commercially available healthy donors were purchased. T cells were isolated using the EasySep human T cell enrichment kit (STEMCELL, 17751), and anti-CD3 / CD28 Dynabeads (Gibco, 11131D) were added according to the instructions to activate the sorted T cells at a 1:1 ratio. The T cell culture medium was X-VIVO15 (Lonza, 04-418Q) medium supplemented with 5% (v%v) heat-inactivated fetal bovine serum (Gibco) and 400 IU / ml recombinant human IL-2 (R&D Systems, 202-IL-010).

[0036] (3) Preparation of CAR-T cells: Human primary CD3+ T cells were stimulated with magnetic beads for 24 hours and then infected with lentiviruses. The cells were infected with CAR and LYAR overexpressing lentiviruses, and the cell density was adjusted to 2x10⁻¹². 6 / ml, with the addition of polybrene (APExBIO, K2701) as a transfection aid, to a final polybrene concentration of 10ug / ml. CAR-T cells were detected by flow cytometry 48h after infection, and then double-positive CAR-T cells were sorted using a flow cytometry sorter (BD, FACSAriaⅢ). Figure 4 AB). For example Figure 4 As shown in AB, T cells were successfully infected with CAR and LYAR-expressing lentiviruses; both CAR and LYAR were successfully overexpressed on T cells.

[0037] Example 2: Detection of LYAR overexpression in CAR-T cells (1) qPCR detection of LYAR overexpression in CAR-T cells: CAR-T cells from different groups were collected, centrifuged, and the supernatant was removed. Total RNA was extracted according to the instructions of the RNA extraction kit (QIAGEN, 74104).

[0038] 2 μg of RNA was used for reverse transcription to obtain cDNA; the expression level of the LYAR gene was detected by real-time quantitative PCR using SYBR Green Master Mix (Vazyme, Q711-02). GAPDH was used as an internal control gene. Figure 4 As shown in C, the LYAR mRNA level was higher than that in the NC group.

[0039] (2) Western blot detection of LYAR protein expression in CAR-T cells: CAR-T cells from different groups were collected, centrifuged, and after removing the supernatant, 1 mL of pre-cooled PBS was added for resuspending. The cells were then washed and centrifuged once more. The cell pellet was lysed with strong RIPA lysis buffer, sonicated at 40% power for 30 seconds, and then centrifuged at 14000g for 15 minutes. The supernatant was collected. Depending on the system, 6× Loading Buffer (Sangon Biotech) was added to the sample, and the mixture was boiled in a water bath at 95℃ for 10 minutes before SDS-PAGE electrophoresis. LYAR protein expression was detected using Western blot, with GAPDH as an internal control. Figure 4 As shown in Figure D, the LYAR protein level was significantly higher than that in the NC group.

[0040] (3) FACS detection of mCherry fluorescent protein expression (indirectly reflecting LYAR plasmid infection efficiency): CAR-T cells from different groups were collected, and mCherry fluorescence signals were detected using flow cytometry (Beckman Coulter, CytoFLEX LX). The proportion of positive cells was counted. Figure 4 B).

[0041] like Figure 4 As shown, CAR-T cells simultaneously express CAR and LYAR, and LYAR mRNA expression is upregulated in CAR-T cells; the results show that both CAR and LYAR can be successfully overexpressed on T cells.

[0042] Example 3: In vitro functional detection of CAR-T cells (1) CAR-T cell in vitro killing experiment (Luciferase detection method): KYSE-150-Luc, H226-Luc, HUCCT1-Luc, HT29-Luc and Raji-Luc cells were constructed: KYSE150, H226, HUCCT1, HT29 and Raji cell lines were infected with lentivirus (Hanheng Biotechnology) expressing luciferase and Puromycin resistance selection genes, and then selected with Puromycin to obtain the above cell lines that stably express luciferase.

[0043] Killing Assay: The target cells were resuspended in 1640 complete medium. The target cell suspension was seeded into opaque 96-well plates (CORNING, 3610), 100 μl per well. Different numbers of effector CAR-T cells were added at different effector-to-target ratios (0.1-0.005), with 3-4 replicates for each ratio. The final volume per well was 200 μl. The plates were incubated and removed at different time points to assess the killing efficiency. For detection, 10 μl of Steady-glo luciferin substrate (Promega, E2520) was added to each well, and the fluorescence value was immediately detected using a microplate reader. The killing efficiency of effector cells against target cells was calculated based on the fluorescence value of each well: Killing efficiency (%) = (1 - RLU effector cells plus target cells / RLU target cells) × 100 (RLU: relative optical units). We found that in CAR-T cells from multiple donors, LYAR overexpression exhibited stronger killing ability against esophageal squamous cell carcinoma (ESCC) and other tumor cells. This enhancing effect was consistent across different CAR structural designs. For detailed experimental data, please refer to [link to relevant data]. Figure 5 A, Figure 7 A, Figure 9 A, Figure 9 C Figure 9 E, Figure 10 A and Figure 10 C.

[0044] Figure 5 A shows that under low target-to-cell ratio (E:T = 0.01:1 and 0.005:1), the LYAR overexpression (OE) group showed stronger killing activity against esophageal squamous cell carcinoma cells (KYSE150L) than the control group (NC). Figure 7 A shows that under low target-to-cell ratio (E:T = 0.01:1 and 0.005:1), the LYAR overexpression (OE) group and the control group (NC) CAR-T cells showed a significant increase in the specific lysis ability of esophageal squamous cell carcinoma cells (KYSE150L). Figure 9 A shows that in a lung cancer cell model, the killing ability of CAR-T cells in the OE group and NC group was tested under low target-to-cell ratio conditions (E:T = 0.01:1 or less), and the OE group showed stronger lysis activity. Figure 9 C shows that in a gallbladder cancer cell model, the killing ability of CAR-T cells in the OE group and NC group was tested under low target-to-cell ratio conditions (E:T = below 0.05), and the OE group showed stronger lysis activity. Figure 9 E shows that in a colorectal cancer cell model, the killing ability of CAR-T cells in the OE group and NC group was detected under low target-to-cell ratio conditions (E:T = 0.05:1 or less), and the OE group showed stronger lysis activity. Figure 10A shows that, under CAR2 construction and low target-to-cell ratio (E:T ≤ 0.04:1) conditions, the ability of CAR-T cells in the OE group and NC group to lyse tumor cells was detected, and the results showed that the OE group had a stronger killing effect. Figure 10 C shows that in CD19-CAR construction and Raji-Luc cell model, under low target-to-cell ratio (E:T≤0.1:1) conditions, the killing activity of CAR-T cells in the OE group and NC group was compared. The OE group showed stronger lysis function. These results indicate that LYAR overexpression can effectively enhance the broad-spectrum anti-tumor activity of CAR-T cells and has good application potential.

[0045] (2) Repeated tumor stimulation experiment (multiple rounds of antigen stimulation experiment): CAR-T cells and tumor cells were co-cultured at a 1:1 effector-to-target ratio. After complete lysis of tumor cells, the CAR-T cells were counted, and new tumor cells were added again to maintain the 1:1 effector-to-target ratio for the next round of co-culture. This process was repeated every other day until a significant difference in the killing efficiency of CAR-T cells between different treatment groups was observed. The above experimental results are shown in […]. Figure 6 AB Figure 8 AB Figure 9 B. Figure 9 D、 Figure 9 F, Figure 10 B and Figure 10 D.

[0046] Figure 6 The results of the AB study showed that the LYAR overexpression group (OE) maintained a strong tumor cell clearance efficiency after repeated tumor stimulation; the OE group had better CAR-T cell proliferation capacity under long-term stimulation conditions. Figure 8 The results of the AB study showed that the LYAR overexpression group (OE) maintained stronger killing activity under continuous stimulation, and the OE group showed a significant amplification advantage. Figure 9 Results B, D, and F showed that in the multi-round killing experiment for lung cancer, the proliferation capacity of CAR-T cells in the OE group was significantly better than that in the NC group; in the multi-round killing experiment for gallbladder cancer, the proliferation capacity of CAR-T cells in the OE group was significantly better than that in the NC group; and in the multi-round killing experiment for colorectal cancer, the proliferation capacity of CAR-T cells in the OE group was significantly better than that in the NC group. Figure 10 Results B and D showed that, under the CAR2 background, the proliferation capacity of CAR-T cells in the OE group was significantly better than that in the NC group; under the CD19-CAR structure, the proliferation level of CAR-T cells in the OE group was also significantly enhanced; this experiment further verified the enhancing effect of LYAR on the sustained killing ability of CAR-T cells and that it can maintain a higher proliferation capacity than NC under sustained killing effect.

[0047] (3) Cell proliferation detection: The proliferation capacity of CAR-T cells was detected using the CellTrace™ Violet Cell Proliferation Detection Kit (ThermoFisher, C34557). The specific operation was as follows: Before use, prepare the stock solution immediately by adding 20 μL of DMSO to one bottle of CellTrace™ Violet reagent and mixing thoroughly to prepare a 5 mM stock solution. Then, add 1 μL of this stock solution to 1 mL of cell suspension in PBS to bring the final dye concentration to 5 μM. Incubate the cells at room temperature or 37°C in the dark for 20 minutes. After incubation, add 5 times the volume of chromosomes in complete culture medium to the stained cells and continue incubation for 5 minutes to remove residual free dye. Centrifuge to collect the cells and resuspend them in preheated complete culture medium, continuing incubation for at least 10 minutes to ensure that the CellTrace™ reagent completes acetate hydrolysis. After cell staining, culture for 4 and 6 days respectively, count the CAR-T cells, and detect their proliferation by flow cytometry on day 4. Figure 5 DG, Figure 7 As shown in DG, Figure 5 DG results showed that flow cytometry analysis of T cell proliferation on day 4 revealed a higher proliferation rate in the OE group, indicating significantly enhanced T cell proliferation. During continuous culture, T cell counting in the OE group showed a significant proliferative advantage on days 4 and 7. Overall, these results indicate that LYAR overexpression significantly enhances the tumor-killing effect, cytokine secretion capacity, and proliferative potential of CAR-T cells, suggesting that LYAR plays an important role in maintaining CAR-T cell function and anti-tumor activity. Figure 7 The DG results showed that after labeling with CellTrace Violet dye, flow cytometry analysis of T cell proliferation on day 4 revealed a significantly higher proportion of positive cells in the OE group compared to the control group. T cell proliferation in the OE group was significantly enhanced, indicating a significantly stronger proliferative capacity. T cell counting during continuous culture was performed to assess the proliferation capacity of cells in the NC and OE groups. The OE group showed a significant proliferative advantage on both days 4 and 7. These results indicate that LYAR overexpression significantly enhances the cytotoxic function, cytokine secretion capacity, and proliferative activity of CAR-T cells from different donor sources, suggesting that this effect is stable and universal. Overall, the proliferation capacity of CAR-T cells in the LYAR-overexpression group was significantly higher than that in the NC group.

[0048] (4) Flow cytometry: Cultured cells were collected and stained with cell surface markers according to the product instructions of each antibody. The stained cells were then analyzed using a flow cytometer and a spectroscopic flow cytometer. The antibodies used in this experiment included: CD3-BV510 (BioLegend, 300448), CD4-APC-Cy7 (BioLegend, 317418), CD8-AF700 (BioLegend, 344724), PD1-BV421 (BioLegend, 329920), CCR7-BV785 (BioLegend, 353230), and CD45RO-PE / Cy7 (BioLegend, 304230).

[0049] like Figure 6 CF, CD: Flow cytometry (FACS) analysis showed that after multiple rounds of tumor cell stimulation, the OE group CAR-T cells had more memory T cell subsets (including central memory and effector memory T cells), suggesting that their differentiation tended to form a more persistent memory phenotype; EF: FACS results showed that after multiple rounds of tumor stimulation, the proportion of PD1 positive cells in the OE group CAR-T cells was significantly lower than that in the control group (NC), indicating that LYAR overexpression can effectively slow down the functional exhaustion of CAR-T cells.

[0050] Figure 8 As shown in CF, CD: Flow cytometry (FACS) showed the differentiation of memory T cell subsets in CAR-T cells overexpressing LYAR after multiple rounds of tumor cell stimulation. The results showed that under repeated tumor stimulation, the proportions of memory T cell subsets in CAR-T cells of the OE group were significantly different, suggesting its memory differentiation advantage. EF: Flow cytometry (FACS) showed that the proportion of PD1⁺ positive cells in CAR-T cells of the OE group was lower, indicating that LYAR overexpression can effectively reduce the functional exhaustion of CAR-T cells. These results indicate that LYAR overexpression can enhance the durability and anti-exhaustion ability of CAR-T cells under multiple rounds of tumor stimulation in CAR-T cells from different donor sources.

[0051] Overall, under experimental conditions simulating repeated tumor cell killing, LYAR-overexpressing CAR-T cells exhibited a higher proportion of central memory T cells (CD45RO) compared to the control group (NC group). + CCR7 + The results also showed a lower proportion of PD1-positive CAR-T cells. This suggests that LYAR may play a key role in maintaining the memory phenotype of CAR-T cells and inhibiting their depletion.

[0052] (5) ELISA detection: To assess the immune activity of CAR-T cells under different target cell stimulation conditions, enzyme-linked immunosorbent assay (ELISA) was used to detect the level of cytokine release. The specific steps are as follows: CAR-T cells from each group were co-cultured with different types of target cells at a predetermined effector-target ratio. After co-culture, the supernatant sample was collected, placed in a centrifuge tube, and centrifuged at 300×g for 10 minutes to remove cells and debris. The supernatant was then used for further processing. Subsequently, the concentrations of cytokines such as interferon-γ (IFN-γ, DaYou, 1110002) and granzyme B (GZMB, DaYou, 1118502) were detected according to the ELISA kit instructions. The absorbance values ​​were read at 450 nm using a microplate reader, and the factor concentrations in the samples were calculated using a standard curve for statistical analysis.

[0053] The results are as follows Figure 5 BC: Under different ESCC cell lines (KYSE150Luc, KYSE450Luc, TE1Luc), the OE group showed significantly enhanced cytokine secretion capacity. Figure 7 As shown in Figure BC, comparisons were made under different ESCC cell lines (KYSE150Luc, KYSE450Luc, TE1Luc). The results showed that under different ESCC cell lines such as KYSE150L, ​​KYSE450L, and TE1, the secretion levels of Granzyme B (B) and IFN-γ (C) in the OE group CAR-T cells were significantly higher than those in the control group. The results also showed that the LYAR overexpression group CAR-T cells exhibited stronger IFN-γ and GZMB secretion abilities in different esophageal squamous cell carcinoma cell lines, suggesting enhanced killing activity and improved immune effector function.

[0054] Example 4: Detection of in vivo antitumor function of CAR-T cells in a tumor-bearing mouse model The experimental animals were 6-week-old female NCG mice. Human KYSE150Luc tumor cells were resuspended in PBS, and 75 μL of the cell suspension was mixed with 75 μL of Matrigel (CORNING, 354234) at a 1:1 ratio. The mixture was then subcutaneously injected into the right back of each mouse, with approximately 2 × 10⁻⁶ cells injected per mouse. 6 One tumor cell. Wait until the tumor volume reaches approximately 300 mm. 3 Then, the mice were randomly divided into groups of 5 mice each, based on the size of the tumor.

[0055] CAR-T cells were injected once via the tail vein according to the prescribed dose. Tumor volume was measured and recorded every three days from the date of CAR-T cell injection. The results showed that, compared with the control group, LYAR-overexpressing CAR-T cells exhibited stronger anti-tumor activity in vivo, manifested in a significantly slower tumor growth rate in mice (see...). Figure 11 A); In the LYAR knockout CAR-T cell treatment group, the tumor control ability was significantly reduced, suggesting that LYAR plays a positive regulatory role in the tumor killing function of CAR-T cells in vivo.

[0056] Example 5: Detection of in vivo anti-tumor function of CAR-T cells in different tumor-bearing mouse models The experimental animals were 6-week-old female NCG mice. Human H226Luc and HUCCT1Luc tumor cells were resuspended in PBS, and 75 μL of the cell suspension was mixed with 75 μL of Matrigel (CORNING, 354234) at a 1:1 ratio. The mixture was then subcutaneously injected into the right back of each mouse, with approximately 2 × 10⁻⁶ cells injected per mouse. 6 One tumor cell. Wait until the tumor volume grows to approximately 150 mm. 3 Then, the mice were randomly divided into groups of 5 mice each, based on the size of the tumor.

[0057] CAR-T cells were administered twice via tail vein injection according to the prescribed dosage. Tumor volume was measured and recorded every three days from the date of CAR-T cell injection. Experimental results are as follows: Figure 12 As shown: In a mouse model of lung cancer, CAR-T cells in the OE group significantly inhibited tumor growth; in a mouse model of cholangiocarcinoma, CAR-T cells in the OE group also showed a stronger anti-tumor effect; these results indicate that LYAR overexpression endows CAR-T cells with a broad-spectrum in vivo anti-tumor advantage, and its universality has been verified in multiple tumor models. In summary, these results show that compared with the control group, LYAR-overexpressing CAR-T cells have a stronger anti-tumor ability in vivo, manifested in a significantly slower growth rate of mouse tumors (see...). Figure 12 ).

[0058] In summary, LYAR-overexpressing CAR-T cells exhibited good anti-tumor activity in different donors, different CAR structures, and various solid tumor models, effectively delaying tumor progression and enhancing sustained killing effects. This mechanism of action may be closely related to its promotion of memory phenotype differentiation and inhibition of T cell exhaustion, suggesting that LYAR, as a key factor regulating CAR-T function, has broad clinical translational potential.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing enhanced CAR-T cells overexpressing LYAR, characterized in that: The cDNA of the LYAR gene was cloned into an overexpression plasmid vector and packaged into a lentivirus. This lentivirus, along with an expression virus expressing a chimeric antigen receptor (CAR), was used to co-infect T cells. The LYAR gene was forcibly expressed under the drive of the EF-1α promoter, achieving stable overexpression of LYAR in CAR-T cells. The nucleic acid sequence of the LYAR gene is shown in SEQ ID NO.

1. The CAR is a CAR targeting EGFR or CD19, containing an extracellular antigen-binding domain specifically targeting EGFR or CD19. The extracellular antigen-binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL). VH contains the VH-CDR base sequence shown in SEQ ID NO: 2, SEQ ID NO: 9, or SEQ ID NO: 11, and VL contains the VL-CDR base sequence shown in SEQ ID NO: 3, SEQ ID NO: 10, or SEQ ID NO:

12.

2. The method for preparing enhanced CAR-T cells overexpressing LYAR according to claim 1, characterized in that: Includes the following steps: (1) Synthesize the nucleic acid sequence encoding the chimeric antigen receptor CAR that targets the epidermal growth factor receptor, i.e., EGFR or CD19, and clone the nucleic acid sequence into the Fuw lentiviral vector to construct CAR expression plasmids, named CAR1-EGFR-GFP, CAR2-EGFR-GFP, and CAR-CD19-GFP; further package to obtain CAR-infected lentiviruses to achieve transduction of T cells; (2) The human LYAR nucleic acid coding sequence was cloned into the Fuw lentiviral vector to construct the LYAR overexpression plasmid, named LYAR-mCherry; it was further used for packaging to obtain LYAR overexpression lentivirus and realize the transduction of T cells; (3) The CAR-infected lentivirus obtained in step (1) and the LYAR-overexpressing lentivirus obtained in step (2) are concentrated and then used to infect human primary CD3+ T cells to obtain enhanced CAR-T cells overexpressing LYAR.

3. The method for preparing enhanced CAR-T cells overexpressing LYAR according to claim 2, characterized in that: The LYAR's CDS region nucleic acid sequence is shown in SEQ ID NO: 1; The CAR1-EGFR-GFP plasmid contains: an EF-1α promoter as shown in SEQ ID NO:13; an N-terminal CD8α signal peptide as shown in SEQ ID NO:4; a variable heavy chain region VH-CDR1 sequence as shown in SEQ ID NO:2; a variable light chain region VL-CDR1 sequence as shown in SEQ ID NO:3; a nucleic acid sequence of the CD8α hinge region as shown in SEQ ID NO:6; a nucleic acid sequence of the CD8α transmembrane region as shown in SEQ ID NO:5; a 4-1BB co-stimulatory domain as shown in SEQ ID NO:8; a CD3ζ signal transduction domain as shown in SEQ ID NO:7; a fusion peptide P2A; and the fluorescent protein GFP as shown in SEQ ID NO:

14. The CAR2-EGFR-GFP plasmid contains: an EF-1α promoter as shown in SEQ ID NO:13; an N-terminal CD8α signal peptide as shown in SEQ ID NO:4; a variable heavy chain region VH-CDR2 sequence as shown in SEQ ID NO:9; a variable light chain region VL-CDR2 sequence as shown in SEQ ID NO:10; a nucleic acid sequence of the CD8α hinge region as shown in SEQ ID NO:6; a nucleic acid sequence of the CD8α transmembrane region as shown in SEQ ID NO:5; a 4-1BB co-stimulatory domain as shown in SEQ ID NO:8; a CD3ζ signal transduction domain as shown in SEQ ID NO:7; a fusion peptide P2A; and a fluorescent protein GFP as shown in SEQ ID NO:

14. The CAR-CD19-GFP plasmid contains: an EF-1α promoter as shown in SEQ ID NO:13; an N-terminal CD8α signal peptide as shown in SEQ ID NO:4; a variable heavy chain region VH-CDR3 sequence as shown in SEQ ID NO:11; a variable light chain region VL-CDR3 sequence as shown in SEQ ID NO:12; a nucleic acid sequence of the CD8α hinge region as shown in SEQ ID NO:6; a nucleic acid sequence of the CD8α transmembrane region as shown in SEQ ID NO:5; a 4-1BB co-stimulatory domain as shown in SEQ ID NO:8; a CD3ζ signal transduction domain as shown in SEQ ID NO:7; a fusion peptide P2A; and a fluorescent protein GFP as shown in SEQ ID NO:

14.

4. The gene overexpression plasmid vector constructed according to the preparation method described in claim 1 or 2, characterized in that: The vector comprises: an EF-1α promoter that drives gene expression, an integration with a chimeric antigen receptor CAR coding sequence targeting EGFR or CD19, or the CDS region of the LYAR gene, and a GFP reporter gene.

5. Enhanced CAR-T cells overexpressing LYAR constructed according to the preparation method described in claim 1 or 2.

6. The enhanced CAR-T cells overexpressing LYAR according to claim 5, characterized in that: The enhanced CAR-T cells overexpressing LYAR are tumor cells that target EGFR or CD19, or CAR-T cells that target any target and are enhanced by overexpressing LYAR.

7. The use of the enhanced CAR-T cells overexpressing LYAR as described in claim 5 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that: The application of the enhanced CAR-T cells overexpressing LYAR in the preparation of drugs for the treatment of esophageal squamous cell carcinoma.

9. The application according to claim 8, characterized in that: The esophageal squamous cell carcinoma is an esophageal squamous cell carcinoma expressing EGFR. Enhanced CAR-T cells overexpressing LYAR specifically lyse EGFR-expressing esophageal squamous cell carcinoma cells in vitro at effector-to-target ratios of 0.01:1 and 0.

005.

10. The use of the enhanced CAR-T cells overexpressing LYAR as described in claim 5 in the preparation of a medicament for treating pan-cancer.

11. The application according to claim 10, characterized in that: The term pan-cancer includes solid tumors and hematologic malignancies.

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