Novel TCR-CAR plasmid for expressing flagellin, CAR-T cell, preparation method of novel TCR-CAR plasmid and CAR-T cell, and application of novel TCR-CAR plasmid and CAR-T cell in tumor resistance

Abstract

The invention discloses a novel TCR-CAR plasmid for expressing flagellin, a CAR-T cell, a preparation method of the novel TCR-CAR plasmid and the CAR-T cell, and application of the novel TCR-CAR plasmid and the CAR-T cell in tumor resistance, and belongs to the technical field of CAR-T cells. By means of the innovative structural design, after CAR recognizes a target antigen, expression and secretion of flagellin can be started along with activation of T cells, the flagellin is released to a tumor site, after an activation signal disappears, release of the flagellin gradually fades away, and after the target antigen is recognized again, a new round of secretion and release is started. The technical problems that in the prior art, release of flagellin polypeptide cannot be accurately controlled, engineering immune cells release randomly, and then the anti-tumor effect cannot be achieved due to the fact that the engineering immune cells cannot effectively enter the tumor in the application of treating solid tumors can be effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of CAR-T cell technology, specifically relating to a novel TCR-CAR plasmid expressing flagellin, CAR-T cells and their preparation method, and their application in anti-tumor therapy. Background Technology

[0002] CAR-T therapy utilizes the specificity and persistence of immune cells in killing tumor cells, providing a more precise and long-lasting treatment modality for cancer patients, and exhibits significant anti-tumor activity in various hematopoietic malignancies. Several cell products targeting CD19 and B cell maturation antigen (BCMA) have been launched globally. However, while CAR-T cell therapy shows promise for hematologic malignancies, its efficacy against solid tumors, which constitute the vast majority of cancers, is much lower. Compared to hematologic malignancies, the high antigenic heterogeneity in solid tumors provides them with an effective mechanism to escape CAR-T cells. CAR-T cells typically encode specificity for a single antigen target, thus failing to recognize all cancer cells in the tumor. Secondly, the complex tumor microenvironment of solid tumors makes it difficult for CAR-T cells to infiltrate and achieve effective tumor killing. Overexpressed immunosuppressive ligands on tumor cells, such as PD-L1, can bind to PD-1 expressed on T cells, causing T cells to lose their killing ability. When CAR-T cell therapy is administered to solid tumors, concurrent administration of immune checkpoint pathway inhibitors can block the activation of the "brake" system, thereby enhancing the anti-tumor activity of CAR-T cells. Studies have shown that CAR-T cell therapy combined with or co-expressing cytokines such as IL-12, IL-18, and IL-15 enhances the cytotoxicity of CAR-T cells compared to CAR-T cell therapy alone, remodels the tumor microenvironment (TME), promotes T cell infiltration, increases the number of M1 macrophages and natural killer (NK) cells, and reduces the number of Tregs, thus enhancing the anti-tumor activity of CAR-T cells and other immune cells, and also increasing the persistence of CAR-T cells in vivo.

[0003] Toll-like receptors (TLRs) are type I transmembrane proteins and a novel type of pattern recognition receptor (PRR). They are considered a link between innate and adaptive immunity. TLRs participate in immune diseases by inducing signal transduction through the recognition of pathogen-associated molecular patterns (PAMPs) and some endogenous ligands. In mammals, some TLRs are located on the cell membrane, including TLR4 (recognizing bacterial LPS), TLR5 (recognizing bacterial flagellin), and TLRs 1, 2, and 6 (recognizing bacterial lipoproteins); while others are located intracellularly, such as TLR3 (recognizing double-stranded RNA), TLR7 and 8 (recognizing single-stranded RNA), TLR9 (recognizing unmethylated CpG containing ssDNA), and TLR13 (recognizing bacterial ribosomal RNA). Except for TLR3, the signaling pathways of other TLRs are primarily initiated by the MYD88 signaling pathway. TLR agonists can activate the immune system to kill tumor cells, thereby inducing strong anti-tumor activity. Therefore, the application of TLR-specific agonists has great potential in tumor treatment.

[0004] TLR5 can bind to the flagellin monomeric form of most Gram-negative bacteria, inducing MyD88-dependent signal transduction and activating the nuclear transcription factor NF-κB in epithelial cells, monocytes, and dendritic cells, thereby activating the body's innate immune response against flagellated bacteria. TLR5 is present on various cell types, including monocytes, macrophages, neutrophils, lymphocytes, NK cells, dendritic cells, epithelial cells, and lymph node stromal cells. Studies have shown that combining flagellin with CpG-ODN can induce the expression of IL-12 and IL-23 in dendritic cells (DCs), inducing Th1 cell immune responses and producing a synergistic anti-tumor response. Engineered Salmonella expressing Vibrio vulnificus FlaB flagellin can effectively target TLR5 in the tumor microenvironment to exert anti-tumor activity, and a significant increase in the M1 phenotype can be observed in tumor-infiltrating macrophages. The flagellin-Grp170 fusion protein drives DCs to convert to CD88. + T cells presented tumor antigens more effectively, subsequently inducing potent cytotoxic CD8 at the tumor site and throughout the body. +T cell response. T cells secreting TLR5 ligands can also improve the tumor microenvironment, thereby inducing anti-tumor responses, indicating that targeting intratumoral TLR5 can be a novel strategy for remodeling the tumor microenvironment. The recombinant Salmonella flagellin derivative CBLB502, when bound to TLR5, induces activation of the NF-κB signaling pathway. CBLB502 significantly enhances the role of CAR-NK in a mouse model of colon cancer and can activate endogenous immune cells to exert anti-tumor effects. For example, the patent publication number CN113164578B, entitled "Modified Immune Cells Expressing Flagellin Peptides," provides immune cells modified with flagellin peptides capable of binding to Toll-like receptors, specifically indicating that the Toll-like receptor is TLR5, using a partial flagellin sequence, and employing P2A linkage in the CAR-T process. However, this technology cannot precisely control the release of flagellin peptides; instead, it exhibits continuous release from cell secretion to the periphery, resulting in relatively random release of engineered immune cells and significant incompleteness.

[0005] In the application of CAR-T cells to treat solid tumors, their anti-tumor effects are hampered by their inability to effectively penetrate the tumor. Furthermore, CAR-T cells that infiltrate the tumor encounter complex tumor microenvironments, such as tumor heterogeneity and immunosuppressive microenvironments, leading to CAR-T cell depletion and loss of function. CAR-T therapy can be considered a typical example of personalized medicine, using genetic engineering to modify T cells to recognize tumors and exert anti-tumor effects. Currently, two CAR-T cell therapy products are on the market for treating B-cell lymphomas, representing a significant breakthrough in the treatment of hematological malignancies. However, regrettably, the efficacy of CAR-T cell therapy for solid tumors has remained unsatisfactory. Solid tumors and hematological malignancies possess different physiological and pathological characteristics, making them highly sensitive to CAR-T cell therapy. Tumor cells are usually present in the bloodstream, making them easily captured by CAR-T cells. The highly immunosuppressive tumor microenvironment severely hinders the infiltration, activation, and proliferation of T cells. Therefore, overcoming the suppressive tumor microenvironment remains a major obstacle for cell therapy in solid tumors. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a novel TCR-CAR plasmid expressing flagellin, CAR-T cells, their preparation method and their application in anti-tumor treatment. This invention can effectively solve the technical problem that the existing technology cannot precisely control the release of flagellin peptides, and the release of engineered immune cells is relatively random. As a result, in the application of solid tumor treatment, the immune cells cannot effectively enter the tumor and thus cannot exert their anti-tumor effect.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a novel TCR-CAR plasmid for expressing flagellin, which adds the nucleic acid sequence of flagellin, a flagellin protein, to a second-generation CAR-T cell by NFAT-induced expression of promoter elements and codon optimization.

[0008] Preferably, the 5' end of the plasmid is connected to the NFAT-induced expression promoter element and the Flagellin gene, and the Flagellin gene is independently induced and driven to express by the NFAT-induced expression promoter element.

[0009] More preferably, an anti-CD19 CAR without the NFAT-Flagellin sequence is used as a control, and the amino acid sequence of the anti-CD19 CAR without the NFAT-Flagellin sequence is shown in SEQ ID NO: 1.

[0010] Furthermore, the amino acid sequence of the codon-optimized flagellin is shown in SEQ ID NO: 2.

[0011] Preferably, in second-generation CAR-T, the CAR structure consists of a scFV targeting human CD19, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling activation domain. At the same time, a Flag tag is introduced at the N-terminus, and its expression is driven by the EF1α promoter.

[0012] The present invention also discloses a recombinant lentivirus obtained by transfecting host cells with the novel TCR-CAR plasmid expressing flagellin.

[0013] Preferably, the recombinant lentivirus provided by the present invention includes human primary T cells as the host cell.

[0014] This invention also discloses a flagellin-expressing CAR-T cell, obtained by infecting T cells with the aforementioned recombinant lentivirus; wherein: The T cells include: peripheral blood T cells, umbilical cord blood T cells, or stem cell-induced T cells.

[0015] This invention also discloses the application of the above-mentioned novel TCR-CAR plasmid expressing flagellin, recombinant lentivirus, or flagellin-expressing CAR-T cells in the preparation of anti-solid tumor drugs, wherein the drugs are those capable of controlling flagellin expression and secretion.

[0016] The present invention also discloses an antitumor drug composition comprising an effective amount of the aforementioned flagellated CAR-T cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for expressing flagellin in engineered CAR-T cells. The innovative structural design allows flagellin expression and secretion to be initiated upon T cell activation after CAR recognition of the target antigen, and released into the tumor site. When the activation signal disappears, flagellin release gradually subsides, and a new round of secretion and release is initiated upon re-recognition of the target antigen. This invention utilizes the immunostimulatory activity of flagellin to activate immune cells, especially innate immune cells within the tumor, mobilizing their anti-tumor activity and transforming the tumor from a "cold" tumor to a "hot" tumor. While existing technologies present difficulties in engineered T cells entering solid tumors, they are not impossible. According to our design, first-line cells, upon entry, recognize tumor cells and secrete flagellin. This protein effectively activates internal immune cells, altering the tumor microenvironment (the release of higher levels of inflammatory factors and chemokines encourages CAR-T cell entry). This alteration further facilitates CAR-T cell infiltration into the tumor. Second-line cells, upon recognizing the tumor, also continue to secrete flagellin, further enhancing this process. In this invention, CAR-T cells are induced to express and secrete flagellin, thereby achieving precise regulation of flagellin. This means that flagellin is precisely released to the tumor site, while avoiding side effects.

[0018] This invention discloses a method for activating endogenous immune cells by inducing flagellin expression in engineered CAR-T cells to promote the application of CAR-T cells in solid tumors. The invention mainly relates to a scheme for preparing flagellin-expressing CAR-T cells from engineered T cells (including T cells induced from human peripheral blood, umbilical cord blood, and stem cells), and the application of novel CAR-T cells prepared by this method in the field of tumor therapy. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the lentiviral plasmid design of the present invention; Figure 2 This is a graph showing the results of flow cytometry analysis of gene transduction efficiency. Figure 3 The image shows the results of flow cytometry detection of flagellin expression. Figure 4 The result of cell killing; Figure 5 The images show the detection results for IL-2 and IFN-γ; the left image represents IL-2, and the right image represents IFN-γ. Detailed Implementation

[0020] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0021] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0022] This invention discloses a novel TCR-CAR plasmid for expressing flagellin, which adds the nucleic acid sequence of flagellin, a flagellin protein, to a second-generation CAR-T cell by NFAT-induced expression of promoter elements and codon optimization.

[0023] The "second-generation CAR-T" described in this invention refers to second-generation chimeric antigen receptor (CAR) T-cell therapy. CAR-T cell therapy is a treatment method that uses genetic engineering technology to modify T cells isolated from a patient or allogeneic donor to express chimeric antigen receptors (CARs), thereby specifically recognizing and killing tumor cells. Second-generation CAR-T adds one or more co-stimulatory domains, such as CD28 or 4-1BB, compared to first-generation CAR-T. The structure of second-generation CAR-T cells includes an extracellular antigen recognition and binding domain, an intracellular signaling domain, and hinge and transmembrane domains connecting the two. These co-stimulatory domains are a significant improvement of second-generation CAR-T cells compared to first-generation CAR-T cells, significantly enhancing T cell survival, proliferation, and persistence.

[0024] The "flagellin" described in this invention is a granular protein that constitutes bacterial flagellum fibers. Flagellin is a subunit protein that polymerizes to form bacterial flagellum filaments. The flagellum filament is the longest part of the flagellum and is composed of repeating flagellin subunits, forming a helical filament. Flagella are the organ of movement for bacteria, and flagellin, as a component of the flagellum, plays an important role in bacterial motility, immune activation, anti-tumor activity, and radiation protection.

[0025] The "scFv targeting human CD19" described in this invention refers to scFv, a small protein molecule composed of the heavy chain variable region (VH) and light chain variable region (VL) of an antibody linked by a linker peptide, which can specifically recognize and bind to the target antigen. In CAR-T cell therapy targeting human CD19, scFv, as the antigen-binding domain of a chimeric antigen receptor (CAR), can recognize and bind to CD19-positive tumor cells.

[0026] The "CD8α hinge and transmembrane domain" described in this invention are key components in CAR-T cell design, playing a crucial role in the structure and function of the CAR. The CD8α hinge region, also known as the spacer / hinge, connects the scFv (single-chain variable fragment) and the transmembrane domain. The CD8α hinge is designed based on derivatives of the CD8α extracellular domain. The CD8α transmembrane domain is responsible for connecting the extracellular portion of the CAR to the intracellular signaling domain and anchoring the receptor to the T cell membrane. The CD8α transmembrane domain is one of the commonly used transmembrane domains. Its main function is to immobilize the CAR on the T cell membrane and ensure that the CAR can properly interact with the target antigen. It can also influence the surface expression and stability of the CAR.

[0027] The "4-1BB co-stimulatory domain" described in this invention is an important target in tumor immunotherapy. 4-1BB is a type I transmembrane glycoprotein belonging to the tumor necrosis factor receptor (TNFR) superfamily. Its structure includes four cysteine-rich domains (CRDs), a short transmembrane domain, and a C-terminal cytoplasmic domain. This structure allows 4-1BB to bind to other molecules, thereby transducing signals. 4-1BB has been successfully applied in the design of CAR-T cells for CAR-T therapy. By introducing the 4-1BB co-stimulatory domain, the activity, persistence, and anti-tumor efficacy of CAR-T cells can be enhanced. The "CD3ζ signaling activation domain" described in this invention is a key component of the T cell receptor (TCR) complex, crucial for T cell activation and signal transduction. CD3ζ is a subunit of the CD3 molecule, which consists of four subunits: CD3ε, CD3δ, CD3γ, and CD3ζ, and forms the core of the TCR-CD3 complex. During T cell activation, when the TCR / CD3 complex is stimulated by an antigen, the ITAM domain of the CD3ζ intracellular domain is phosphorylated, thereby recruiting and activating downstream signaling molecules, such as ζ-chain-associated protein kinase (ZAP70), which in turn mediates the activation of downstream signaling pathways, such as calmodulin, MAPK, and NF-κB signaling pathways.

[0028] The present invention also discloses a recombinant lentivirus obtained by transfecting host cells with the novel TCR-CAR plasmid expressing flagellin.

[0029] Preferably, the host cell includes human primary T cells.

[0030] The "human primary T cells" described in this invention refer to T lymphocytes isolated from human blood that have not been cultured or treated in vitro and have normal physiological functions and immune activity; human primary T cells are a crucial component of the immune system and have a variety of biological functions and immune activities.

[0031] The present invention also discloses a flagellin-expressing CAR-T cell, which is obtained by infecting T cells with the above-mentioned recombinant lentivirus; wherein: the T cells include: peripheral blood T cells, umbilical cord blood T cells or stem cell-induced T cells.

[0032] The "T cells" described in this invention are short for T lymphocytes. T cells possess various characteristics, including specific recognition capabilities, immune memory function, cytotoxic activity, and regulatory functions. Common T cells are classified according to function, including: naive T cells, helper T cells (Th cells), cytotoxic T cells (CTL cells), regulatory T cells (Tregs cells), and memory T cells. Furthermore, all T cells originate from hematopoietic stem cells in the bone marrow and complete their maturation process in the thymus.

[0033] The present invention will now be described in further detail with reference to the accompanying drawings: The NFAT (Nuclear factor of activated T-cells) element disclosed in this invention is a family of transcription factors. It was first reported as a transcription factor in activated T cells capable of binding to the promoter of human interleukin-2 (IL-2) and rapidly inducing its expression. Proteins in the NFAT family, except for their non-conserved C-terminus, possess two adjacent, highly conserved regions: the NFAT homology region (NHR) and the Rel-homology domain (RHD). As transcription factors, NFATs play a crucial role in inducing gene transcription in immune responses. They can recognize and bind to specific DNA sequences, which are referred to as NFAT binding motifs or response elements.

[0034] 1. Design and plasmid construction of novel TCR-CAR structures This invention constructs a flagellin nucleic acid sequence based on a conventional second-generation CAR-T cell by adding an NFAT-inducible promoter element and codon optimization. The CAR structure consists of a scFV targeting human CD19, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling activation domain. A flag tag is introduced at the N-terminus, and its expression is driven by the EF1α promoter. The flagellin gene is expressed by a 4×NFAT element (Fla-anti-CD19 CAR), with a control (anti-CD19 CAR) lacking the NFAT-Flagellin sequence. See [link to relevant documentation]. Figure 1 . Figure 1 In the figure, IL2p is a minimized interleukin-2 promoter region, and p stands for promoter; this inducible promoter is composed of four NFAT promoters cascaded together plus the IL2 promoter. Also, hCD19 CAR and CD19 CAR in the figure are the same element.

[0035] The amino acid sequence of anti-CD19 CAR is shown in SEQ ID NO: 1, and the amino acid sequence of Flagellin is shown in SEQ ID NO: 2.

[0036] 2. Packaging and Concentration of Lentivirals 1) Seed HEK293T cells into 10 cm cell culture dishes one day in advance, and wait until the cell density reaches 70%; 2) Add 10 μg of Fla-anti-CD19 CAR or anti-CD19CAR plasmid, 7.5 μg of psPAX (lentiviral packaging plasmid psPAX2), and 2.5 μg of pMD2.G to 500 μL of Opti-MEM medium. After mixing well, add 60 μL of transfection reagent PEI, vortex to mix well, and let stand at room temperature for 15 min to obtain the transfection complex. 3) Replace the cells with fresh, preheated culture medium, add the transfection complex from the previous step, gently shake to mix, and then place in an incubator to continue culturing. 4) After adding the transfection complex for 6-8 hours, replace the cells with fresh culture medium and continue culturing in the incubator; 5) 48 h after transfection, collect cell supernatant and filter it through a 0.45 μm filter. Add an appropriate amount of 5×PEG8000 virus concentration reagent according to the ratio, mix by inverting, and place at 4 ℃. Invert 3 times every 30 min, repeat twice, and then let stand overnight. 6) Centrifuge the virus supernatant at 4000 rpm and 4 °C for 1 h, remove the supernatant, resuspend the virus pellet in appropriate RPMI-1640 complete medium, and use it directly to infect cells or store it at -80 °C.

[0037] 3. Lentiviral infection of T cells 1) Take 1×10 7 Add 3 × 10 PBMC cells 7 Add one anti-CD3 / CD28 activated magnetic bead and add interleukin-2 to a final concentration of 100 U / mL, then incubate in a cell culture incubator for 48 h. 2) Take an appropriate amount of cells, add concentrated lentivirus and polybrene to a final concentration of 8 μg / mL, and continue culturing; 3) After 12-24 hours, replace the cell culture medium with a new one and continue culturing.

[0038] 4. Flow cytometry to detect gene transduction efficiency 1) Three days after viral infection, collect an appropriate amount of T cells, add PE anti-Flag antibody, and incubate on ice for 25 min; 2) Wash cells three times with PBS buffer containing 1% FBS; 3) Finally, resuspend the cells in 200 μL of PBS buffer with 1% FBS and analyze the samples using flow cytometry.

[0039] Flow cytometry results of gene transduction efficiency are as follows Figure 2 As shown, Figure 2The CAR-T group consisted of anti-CD19 CARs, a control design based on a traditional CAR-T structure; the Mock group served as a blank control, similar to UTD (untransduction) from... Figure 2 As can be seen, lentiviruses carrying the flagellin (Fla) gene can efficiently integrate the CAR gene and transduce it into T cells, and there is no difference from conventional second-generation anti-hCD19 CAR-T.

[0040] Flow cytometry results of flagellin expression are as follows Figure 3 As shown, from Figure 3 As can be seen, Fla-anti-CD19 CAR-T cells only trigger the expression and secretion of flagellin when cultured with CD19-positive tumor cells, thus achieving precise control over its expression.

[0041] 5. Detection of CAR-T cell tumor killing effect 1) One day in advance, HCT116 (CD19) was seeded into black opaque flat-bottomed 96-well plates. + )c cells, 20,000 per well; 2) Effector cell preparation: Fla-anti-CD19 CAR-T cells, anti-CD19 CAR-T cells and Mock-T cells were washed and resuspended in fresh RPMI-1640 medium for later use; 3) Add effector cells to each well according to the effector-to-target ratio, and set up separate control wells for effector cells and target cells; 4) Gently mix and then return to the incubator for 6 hours; 5) After removing the culture supernatant, add 100 μL of culture medium containing luciferase substrate to each well, incubate at 37 °C for 5 min, and then read the luminescence signal.

[0042] Cell killing rate (%) = [(-target cell luminescence value - effector cell autoluminescence value - experimental luminescence value) / (target cell autoluminescence value - target cell maximum luminescence value)] × 100% See the experimental results. Figure 4 This indicates that Fla-anti-CD19 CAR-T cells can enhance the anti-tumor activity of CAR-T cells.

[0043] 6. Cytokine secretion detection 1) Transfer the supernatant from the previous cell-killing step into a clean centrifuge tube, centrifuge at 12000 g for 20 min, and collect the supernatant for detection; 2) According to the ELISA kit instructions, perform the detection of IL2 and IFN-γ in the cell supernatant.

[0044] The results are shown in Figure 5. Figure 5As can be seen, Fla-anti-CD19 CAR-T cells can secrete more IL2 and IFN-γ compared to traditional anti-CD19 CAR-T cells.

[0045] In summary, the experimental results of this invention demonstrate that the engineered CAR-T cells prepared in this invention express flagellin, which enhances the anti-tumor activity of CAR-T cells, thus providing a new potential approach for cell immunotherapy.

[0046] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A novel TCR-CAR plasmid expressing flagellin, characterized in that, The second-generation CAR-T cell contains the nucleic acid sequence of flagellin, a flagellin protein induced by NFAT and optimized with promoter elements and codons.

2. The novel TCR-CAR plasmid expressing flagellin as described in claim 1, characterized in that, The plasmid connects the NFAT-induced expression promoter element and the Flagellin gene at its 5' end. The Flagellin gene is independently induced and driven to express by the NFAT-induced expression promoter element.

3. The novel TCR-CAR plasmid expressing flagellin as described in claim 2, characterized in that, The amino acid sequence of the anti-CD19 CAR without the NFAT-Flagellin sequence is shown in SEQ ID NO:

1.

4. The novel TCR-CAR plasmid expressing flagellin as described in any one of claims 1 to 3, characterized in that, The amino acid sequence of the codon-optimized flagellin is shown in SEQ ID NO:

2.

5. The novel TCR-CAR plasmid expressing flagellin as described in any one of claims 1 to 3, characterized in that, In second-generation CAR-T, the CAR structure consists of a human CD19-targeting scFV, a CD8α hinge and transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling activation domain. At the same time, a Flag tag is introduced at the N-terminus, and its expression is driven by the EF1α promoter.

6. A recombinant lentivirus, characterized in that, It is obtained by transfecting host cells with the novel TCR-CAR plasmid expressing flagellin as described in any one of claims 1 to 5.

7. The recombinant lentivirus as described in claim 6, characterized in that, Host cells include human primary T cells.

8. A CAR-T cell expressing flagellin, characterized in that, Obtained by T cell expression after infection with the recombinant lentivirus as described in claim 6 or 7; wherein: The T cells include: peripheral blood T cells, umbilical cord blood T cells, or stem cell-induced T cells.

9. The use of the novel TCR-CAR plasmid expressing flagellin according to any one of claims 1 to 5, the recombinant lentivirus according to claim 6 or 7, or the flagellin-expressing CAR-T cells according to claim 8 in the preparation of anti-solid tumor drugs, characterized in that, The drug described is one that can control the expression and secretion of flagellin.

10. An antitumor drug composition, characterized in that, The antitumor drug composition comprises an effective amount of the flagellin-expressing CAR-T cells as described in claim 8.

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