Artificial antigen presenting cell for efficiently preparing chimeric antigen receptor T cell as well as construction and application of artificial antigen presenting cell

Abstract

The invention discloses an artificial antigen presenting cell for efficiently preparing a chimeric antigen receptor T cell (CAR-T) and a construction method of the artificial antigen presenting cell, and belongs to the field of cell therapy and immunotherapy, the artificial antigen presenting cell takes a K562 cell as a platform, an anti-CD3 single-chain antibody, an anti-CD28 single-chain antibody, a membrane binding type cell factor and a costimulatory ligand are stably expressed through genetic engineering, and the chimeric antigen receptor T cell (CAR-T) is obtained. And the local concentration of the anti-CD3 antibody is improved by adopting an HOTag3 tool. The multi-signal integration strategy obviously enhances the activation and proliferation capacity of the T cells, promotes the CAR-T cells to maintain a low differentiation state, and endows the CAR-T cells with excellent anti-tumor activity and durability. The artificial antigen presenting cell provides a novel efficient tool for the field of cellular immunotherapy, and is expected to promote development of more efficient and safer CAR-T therapy to cope with challenges of various malignant tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and cell immunotherapy, and specifically relates to an artificial antigen presenting cell for efficiently preparing chimeric antigen receptor T cells, and a construction method and application thereof. Background Art

[0002] CAR-T cell therapy, or chimeric antigen receptor T cell immunotherapy, is a precision targeted therapy for treating tumors. This therapy uses gene modification technology to introduce CAR molecules into T cells. CAR is a synthetic receptor that can make the recognition targets of T lymphocytes more extensive, guide T cells to specifically target tumor antigens and produce anti-tumor responses, thereby achieving the purpose of precision treatment of cancer. CAR-T cell therapy has achieved remarkable results in the treatment of hematological malignancies, but more and more clinical studies have found that the effect of CAR-T cell adoptive transfer therapy is not only related to the targeting specificity of T cells, but also closely related to their proliferation and persistence in the body. In the clinical treatment of hematological malignancies, the limited persistence of CAR-T cells in the body will inhibit their efficacy, which has become a major challenge that needs to be solved in CAR-T cell therapy.

[0003] During the in vitro manufacturing process of CAR-T cells, multiple steps and factors will affect the quality of the final product and thus the therapeutic effect. Among them, T cell activation, expansion and CAR gene transduction are key links in the in vitro manufacturing process. The efficiency and quality of these steps directly affect the therapeutic effect of the final CAR-T cell product; T cell activation is a key step because it can provide signals to T cells to guide T cell proliferation and differentiation. Therefore, the expansion rate of T cells, the persistence of the logarithmic growth phase, and the final CAR-T cell function are largely affected by T cell activation. In addition, effective T cell activation is crucial to improving the transduction efficiency of the CAR gene. Therefore, developing a fast, economical and efficient T cell activation technology to obtain high-quality CAR-T cell products is the core goal of current research.

[0004] The activation of T cells is a complex immune process that involves the precise coordination of multiple signals, including specific antigen recognition signals, nonspecific co-stimulatory signals, and cytokine signals. Moderate enhancement of antigen recognition signals can significantly enhance the activation and expansion of T cells while avoiding excessive differentiation and exhaustion of T cells. By enhancing the interaction between T cell receptors (TCRs) and antigens, the signal can be further amplified, thereby improving the proliferation capacity of T cells and the efficiency of gene transduction.

[0005] In order to enhance the antigen recognition signal of T cells, multivalent expression of anti-CD3 antibodies has become one of the ideal strategies. HOTags, or homo-oligomeric coiled coils, are formed by multiple identical α-helices winding around each other. They are highly stable and play an important role in many biological functions. The HOTags tool includes seven types of coiled coils, named HOTag1 to HOTag7, which can form tetramers, pentamers, hexamers, and heptamers. Studies have used them for the design of multivalent protein-protein interactions.

[0006] Many studies have shown that artificial antigen-presenting cells can effectively activate T cells. The strategy for designing artificial antigen-presenting cells is mainly to simulate the multiple activation signals of natural antigen presentation in a relatively simplified way. Since K562 cells (human chronic myeloid leukemia cell line) do not express MHC molecules and are easy to be genetically modified, they are commonly used materials for artificial antigen-presenting cells. Due to different activation levels and activation signals, T cells can have a series of differentiation states with heterogeneous functions, among which the initial T cells, memory stem cell T cells and central memory T cells with lower differentiation have stronger proliferation and self-renewal abilities. These poorly differentiated phenotypes show better implantation and anti-tumor activity. Therefore, simulating the complex functions of natural antigen-presenting cells while avoiding excessive differentiation and decreased persistence of CAR-T cells caused by excessive stimulation is a key issue for the use of artificial antigen-presenting cells in the preparation of CAR-T cells.

[0007] Although there have been reports on the use of engineered K562 cells to prepare CAR-T cells, the strategy of directly expanding CAR-T cells with K562 cells expressing a tumor antigen can only expand specifically targeted CAR-T cells and has a limited scope of application; although there have been reports on the use of anti-CD3 single-chain antibodies to activate T cells, there have been no reports on the use of multivalent expression of single-chain antibodies to enhance T cell activation signals; although there have been reports on multiple combinations of activation signals, there is a lack of further reports on the degree of differentiation and persistence of CAR-T cells. Summary of the invention

[0008] In view of the limitation of single antigen K562 amplification in existing CAR-T preparation, the lack of multivalent expression of single-chain antibodies to enhance T cell activation signals and the lack of reports on the effects of multiple activation signal combinations on CAR-T differentiation and persistence, the present invention aims to provide an artificial antigen presenting cell for efficiently preparing chimeric antigen receptor T cells and a method for constructing the same.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The present invention provides an artificial antigen presenting cell, which can activate T cells in PBMC in vitro; the artificial antigen presenting cell uses the HOTAgs tool to introduce multivalency, and stably expresses multivalent membrane-bound activating antibodies, membrane-bound activating antibodies, natural co-stimulatory ligands or molecules, and membrane-bound cytokines; the membrane-bound activating antibodies and membrane-bound cytokines are stably expressed on the T cell membrane using a transmembrane domain;

[0011] The membrane-bound activating antibody is selected from any one of an anti-CD3 single-chain antibody and an anti-CD28 single-chain antibody;

[0012] The HOTags tool is selected from HOTag3 or HOTag6, and is used to introduce multivalency to increase the local concentration of membrane-bound activating antibodies.

[0013] Further, the natural co-stimulatory ligand or molecule is selected from one or more of CD137L, CD30L, ICOSL, CD27L, OX40L, GITRL, HVEML, CD80, CD86 and CD40;

[0014] The membrane-bound cytokine is selected from one or more of IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18IL-21, IL-23, IFN-γ, TNF-α and GM-CSF;

[0015] The transmembrane domain is selected from any one of the CD8 transmembrane domain, CD28 transmembrane domain, CD3ζ chain transmembrane domain, CD4 transmembrane domain, Fas transmembrane domain, GP41 transmembrane domain, PD-1 transmembrane domain and EGFR transmembrane domain.

[0016] Furthermore, the artificial antigen presenting cell is a wild-type K562 cell that uses HOTAg3 hexamer and CD8 transmembrane domain to express multivalent membrane-bound anti-CD3 single-chain antibody, membrane-bound anti-CD28 single-chain antibody, natural co-stimulatory ligand HVEML and membrane-bound cytokine IL-21;

[0017] The amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO.1, containing a signal peptide located at positions 1 to 19 of its amino acid sequence, and the signal peptide sequence is shown in SEQ ID NO.2; the amino acid sequence of the anti-CD28 single-chain antibody is shown in SEQ ID NO.3, containing a signal peptide located at positions 1 to 21 of its amino acid sequence, and the signal peptide sequence is shown in SEQ ID NO.4; the amino acid sequence of the cytokine IL-21 is shown in SEQ ID NO.5; the amino acid sequence of the co-stimulatory ligand HVEML is shown in SEQ ID NO.6; the amino acid sequence of the HOTAg3 hexamer is shown in SEQ ID NO.7; and the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO.8.

[0018] The present invention provides an expression vector, which is used to prepare the above-mentioned artificial antigen-presenting cell;

[0019] The expression vector includes a gene encoding a multivalent membrane-bound activating antibody, a gene encoding a membrane-bound activating antibody, a gene encoding a membrane-bound cytokine, a gene encoding a transmembrane region, or a gene encoding a natural co-stimulatory ligand or molecule.

[0020] Furthermore, the expression vector comprises:

[0021] (1) A gene expression vector containing a coding sequence of a promoter SFFV for controlling the expression of an anti-CD3 single-chain antibody, a coding sequence of an activating antibody anti-CD3 single-chain antibody, a coding sequence of a CD8 transmembrane domain, a coding sequence of a HOTAg3 hexamer for controlling the multivalent expression of an anti-CD3 single-chain antibody, a coding sequence of BFP, and a post-transcriptional regulatory element WPRE for enhancing gene expression efficiency, which are sequentially connected;

[0022] (2) a gene expression vector containing the coding sequence of the promoter SFFV for controlling the expression of the anti-CD28 single-chain antibody, the coding sequence of the activating antibody anti-CD28 single-chain antibody, the coding sequence of the CD8 transmembrane domain, the coding sequence of GFP, and the post-transcriptional regulatory element WPRE for enhancing gene expression efficiency, which are sequentially connected;

[0023] (3) a gene expression vector containing the coding sequence of the promoter SFFV for controlling the expression of the cytokine IL-21, the coding sequence of the cytokine IL-21, the coding sequence of the CD8 transmembrane domain, the coding sequence of mCherry, and the post-transcriptional regulatory element WPRE for enhancing the gene expression efficiency, which are sequentially connected;

[0024] (4) a gene expression vector containing the coding sequence of the promoter SFFV for controlling the expression of the co-stimulatory ligand HVEML, the coding sequence of the co-stimulatory ligand HVEML, the coding sequence of the IRES connected to the puromycin resistance gene, the coding sequence of the puromycin resistance gene and the post-transcriptional regulatory element WPRE for enhancing the gene expression efficiency, which are sequentially connected;

[0025] The amino acid sequence of the BFP is shown in SEQ ID NO.9; the amino acid sequence of the GFP is shown in SEQ ID NO.10; the amino acid sequence of the mCherry is shown in SEQ ID NO.11; the amino acid sequence of the puromycin resistance gene is shown in SEQ ID NO.12; and the amino acid sequence of the IRES is shown in SEQ ID NO.13.

[0026] The present invention provides a recombinant virus, wherein the recombinant lentivirus is prepared by co-transfecting HEK293T cells with the above-mentioned expression vector and a packaging helper plasmid.

[0027] The present invention provides a method for constructing the above-mentioned artificial antigen-presenting cells, comprising introducing the above-mentioned recombinant virus into a host cell, and obtaining positive clones by screening using antibiotics or fluorescent markers; the host cell is selected from any one of K562 cells, HEK293T cells, Jurkat cells, CHO cells, Raji cells or other cell lines that are easy to be genetically modified.

[0028] Further, the host cell is a wild-type K562 cell line;

[0029] Furthermore, the packaging plasmid and envelope plasmid of the lentiviral transduction method are psPAX2 and pMD2.G; the lentiviral packaging method of the lentiviral transduction method is the calcium phosphate method;

[0030] Furthermore, the natural co-stimulatory ligand or molecule used for antibiotic screening is connected with a resistance gene through a bicistronic vector element to obtain a stable cell line, and the resistance gene is selected from any one of the puromycin resistance gene, neomycin resistance gene, hygromycin resistance gene, pyrimidinemycin resistance gene, and Zeocin resistance gene.

[0031] Furthermore, the resistance gene is a puromycin resistance gene, whose amino acid sequence is shown in SEQ ID NO.12, and the bicistronic vector element is selected from 2A peptide (P2A, T2A, E2A, F2A) and internal ribosome entry site IRES, whose nucleotide sequence is shown in SEQ ID NO.13.

[0032] Furthermore, the membrane-bound activating antibody and membrane-bound cytokine fusion fluorescent protein for fluorescent labeling screening are used to visualize transduction efficiency and flow cytometry sorting, and the fluorescent protein used is selected from any one of GFP, YFP, CFP, BFP, RFP, mCherry, mOrange, mVenus, mKate2, mPlum, and smURFP;

[0033] Furthermore, the anti-CD3 single-chain antibody is fused with a blue fluorescent protein BFP, whose amino acid sequence is shown in SEQ ID NO.9, the anti-CD28 single-chain antibody is fused with a green fluorescent protein GFP, whose amino acid sequence is shown in SEQ ID NO.10, and the cytokine IL-21 is fused with a red fluorescent protein mCherry, whose amino acid sequence is shown in SEQ ID NO.11.

[0034] The present invention provides a culture medium, which comprises the artificial antigen presenting cells; the culture medium also comprises exogenous cytokine IL-2 with a final concentration of 200 IU / mL and fetal bovine serum with a volume percentage of 10%, and the culture fluid of the culture medium is RPMI-1640 culture medium.

[0035] The present invention provides use of the artificial antigen presenting cell, the expression vector, the recombinant virus and the culture medium in preparing a T cell-based tumor immunotherapy product.

[0036] The present invention provides a method for preparing a T cell tumor immunity product, wherein the tumor immunity product is a CAR-T cell. The specific preparation method comprises activating peripheral T lymphocytes and culturing CAR-T cells with a high positive rate and maintained in a low differentiation state.

[0037] The conditions for activating peripheral T lymphocytes are: peripheral blood T lymphocytes and the above-mentioned artificial antigen presenting cells that have been lethally irradiated are co-cultured in a ratio of 2:1.

[0038] The tumor cells are hematological malignant tumor cells or solid tumor cells, wherein the hematological malignant tumor cells are selected from any one of human chronic myeloid leukemia cell line K562 cells, human B cell lymphoma cell line Raji cells, human Burkitt lymphoma cell line Daudi cells, and precursor B cell acute lymphoblastic leukemia cell line NALM-6 cells;

[0039] The solid tumor cells are selected from any one of human epidermal cancer cell line A431 cells, human ovarian cancer cell line SKOV3 cells, human colorectal cancer cell line LoVo cells, non-small cell lung cancer cell line A549 cells, glioblastoma cell line U87 cells, triple-negative breast cancer cell line MDA-MB-231 cells, hepatocellular carcinoma cell line HepG2 cells, colorectal cancer cell line HT-29 cells and prostate cancer cell line PC-3 cells;

[0040] The CAR-T cells are selected according to the targeted antigen from: any one of anti-CD19 CAR-T cells, anti-CD20 CAR-T cells, anti-BCMACAR-T cells, anti-HER2 CAR-T cells, anti-EGFR CAR-T cells, anti-Mesothelin CAR-T cells, anti-CD22 CAR-T cells, anti-CEA CAR-T cells, and anti-PSMA CAR-T cells;

[0041] The CAR-T cells are selected according to the CAR structure: any one of the first generation CAR containing an antigen domain and an activation signal domain, the second generation CAR containing an antigen domain and a co-stimulatory domain, the third generation CAR containing two co-stimulatory domains, the fourth generation CAR containing an inducibly expressed cytokine gene, and the fifth generation CAR fused with multiple signal transduction domains.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The artificial antigen presenting cells constructed by the present invention can effectively activate T cells in PBMC in vitro, and use the HOTAgs tool to stably express multivalent T cell membrane-bound activating antibodies. The local concentration of membrane-bound activating antibodies is increased by introducing multivalency, thereby effectively activating T cells and significantly improving their proliferation ability. Using a multi-signal integration strategy, the integration of co-stimulatory signals and cytokine signals not only further promotes T cell activation, proliferation and gene transduction, but also significantly improves the functions of various CAR-T cells, and obtains CAR-T cells with a high positive rate and a low differentiation state, thereby giving them stronger and more lasting anti-tumor activity, providing a new approach for tumor treatment, and providing a solid foundation for the future development of more efficient and safer immune cell therapies.

[0044] Furthermore, artificial antigen-presenting cells can stably express multivalent membrane-bound anti-CD3 single-chain antibodies, anti-CD28 single-chain antibodies, cytokine IL-21 and co-stimulatory ligand HVEML. The combination of these molecules can accurately construct the required artificial antigen-presenting cells, further enhance the activation and expansion of T cells, and improve the efficacy of immunotherapy.

[0045] The expression vector provided by the present invention for constructing the above-mentioned artificial antigen presenting cell comprises a gene sequence of a multivalent membrane-bound activating antibody encoding gene, a membrane-bound activating antibody encoding gene, a membrane-bound cytokine encoding gene, a transmembrane region encoding gene, a natural co-stimulatory ligand or a molecule encoding gene, which can be flexibly adjusted according to demand, providing a basis for batch preparation of artificial antigen presenting cells, and is conducive to promoting the clinical application of T cell immunotherapy. Further, through the combination of specific promoters, transmembrane domains, HOTAg polymers, fluorescent proteins and post-transcriptional regulatory elements, precise regulation and enhancement of gene expression are achieved; the introduction of fluorescent proteins allows transduction efficiency and expression to be easily monitored by fluorescence microscopy; the use of fluorescent protein fusion expression realizes visual monitoring of transduction efficiency; at the same time, by connecting the puromycin resistance gene to IRES, efficient screening of stable cell lines is achieved.

[0046] The recombinant virus provided by the present invention uses HEK 293T cells as virus packaging cells, and through calcium phosphate co-transfection technology, psPAX2, pMD2.G and vector plasmids are efficiently transferred into cells, and lentiviral particles are successfully packaged. The transfection efficiency is high and a large number of virus particles can be produced, which provides a sufficient virus source for the subsequent infection of K562 cells, ensures the high quality and stability of the artificial antigen-presenting cells finally obtained, and provides important technical support and experimental basis for tumor immunotherapy and other cell-based therapies.

[0047] The method for constructing artificial antigen-presenting cells provided by the present invention is simple to operate and highly efficient. By introducing the recombinant virus into K562 cells and using antibiotics and flow cytometry to screen positive clones, artificial antigen-presenting cells with high purity and activity are successfully constructed, providing a reliable cell source for subsequent T cell activation.

[0048] The culture medium provided by the present invention contains artificial antigen-presenting cells and necessary growth factors and nutrients, such as exogenous cytokine IL-2 and fetal bovine serum. The culture medium provides a suitable environment for the growth and expansion of artificial antigen-presenting cells, ensures the quality and quantity of the cells, and lays a foundation for the subsequent preparation of cell therapy products.

[0049] The method for preparing T cell tumor immunity products provided by the present invention increases the positive rate and low differentiation state of CAR-T cells by activating peripheral blood T lymphocytes, thereby enhancing their anti-tumor effect and persistence; provides a variety of CAR-T cell classifications and targeted antigen selections, and can perform personalized treatments according to different tumor types and patient needs; different types of CAR-T cells have different anti-tumor effects, and different types of tumor cells can be efficiently treated; precise treatment of different types of tumor cells improves the treatment effect and patient survival rate, and the prepared CAR-T cells have good killing ability and persistence, which can significantly improve the treatment effect and survival rate of tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 A flow chart for constructing the artificial antigen presenting cell K3H28-21H of the present invention;

[0051] Figure 2 Figure 1 is a diagram of the gene expression vectors of four compositions in the artificial antigen presenting cell K3H28-21H cell line of the present invention;

[0052] Figure 3 The present invention is a flow cytometric detection of surface molecules of the artificial antigen presenting cell K3H28-21H cell line; wherein A is AlexaFluor488-A dye, B is Pacific Blue-A dye, C is PE-A dye, and the control is a wild-type K562 cell line without gene transduction;

[0053] Figure 4 This is the EVOS live cell fluorescence imaging of K562-aCD3(H)-aCD28 cell line; the control is K562-aCD3-aCD28 cell line;

[0054] Figure 5 The flow cytometric detection of anti-CD3 single-chain antibody on the surface of K562-aCD3-aCD28 cell line and K562-aCD3(H)-aCD28 cell line; the control is K562-aCD3-aCD28 cell line;

[0055] Figure 6 The proliferation and phenotypic changes of T cells after T cell activation were statistically analyzed for the K562-aCD3-aCD28 cell line, where A is the expression of proliferation and activation markers on T cells, B is the T cell expansion multiple, and C is the differentiation phenotypic changes. The control is the K562-aCD3-aCD28 cell line;

[0056] Figure 7Flow cytometry analysis of CFSE-labeled T cells stimulated by K562-aCD3(H)-aCD28 cell line, and the controls were Dynabeads and K562-aCD3-aCD28 cell line;

[0057] Figure 8 The figure shows the flow cytometry detection of transduction efficiency and clustering phenotype of K562-aCD3(H)-aCD28 cell line in the experiment of generating anti-CD19 CAR-T cells, wherein A is the CAR positivity rate, B is the clustering phenotype of CAR-T cells, and the controls are Dynabeads and K562-aCD3-aCD28 cell line;

[0058] Fig. 9 The in vitro short-term killing and specific killing detection of anti-CD19 CAR-T cells generated by K562-aCD3(H)-aCD28 cell line activation, wherein A is the in vitro short-term killing rate, B is the specific killing rate, and the controls are Dynabeads and K562-aCD3-aCD28 cell line;

[0059] Fig.10 Flow cytometry detection of activation, proliferation, transduction efficiency and clustering phenotype in the experiment of generating anti-DR5 CAR-T cells from K562-aCD3(H)-aCD28-mbIL21 cell line, where A is the expression of activation markers on T cells, B is the CAR positive rate, C is the differentiation phenotype, D is the expansion fold, and the control is K562-aCD3(H)-aCD28 cell line;

[0060] Fig.11 The flow cytometry analysis shows that K3H28-21H cell line of the present invention generates anti-CD19 CAR-T cells, wherein A is the expression of activation markers on T cells, B is the CAR positivity rate, and the controls are Dynabeads and K562-aCD3(H)-aCD28-mbIL21 cell lines. DETAILED DESCRIPTION

[0061] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0062] Any simple modification or equivalent change made to the embodiments according to the technical points of the present invention without departing from the content of the present invention, such as replacing K562 cells with other cells that do not express human MHC molecules and are easy to be genetically modified, using other multimeric proteins to achieve multivalent expression of single-chain antibodies, using other methods to achieve stable expression of multivalent anti-CD3 single-chain antibodies, anti-CD28 single-chain antibodies, membrane-bound cytokines IL-21 and co-stimulatory ligands HVEML on the cell membrane surface, replacing or increasing or decreasing cytokines and co-stimulatory ligands expressed on artificial antigen presenting cells, improving the activation conditions of T cells and the culture conditions of CAR-T cells, etc., these changes or improvements should also be regarded as the scope of protection of the present invention. Based on the embodiments of the present invention, any technician familiar with the art may change the above technical content into other equivalent embodiments, which should belong to the scope of protection of the present invention.

[0063] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0064] Example 1: Method for constructing artificial antigen presenting cell K3H28-21H cell line

[0065] (1) Design of gene expression vector

[0066] The flowchart of artificial antigen presenting cell construction is attached Figure 1 .

[0067] The gene expression vectors of the four compositions in artificial antigen presenting cells are shown in the attached Figure 2 .

[0068] An anti-CD3 single-chain antibody (SEQ ID NO.1), comprising a signal peptide (SEQ ID NO.2) located at positions 1 to 19 of the amino acid sequence of the anti-CD3 single-chain antibody;

[0069] An anti-CD28 single-chain antibody (SEQ ID NO.3), comprising a signal peptide (SEQ ID NO.4) located at positions 1 to 21 of the amino acid sequence of the anti-CD28 single-chain antibody (SEQ ID NO.3);

[0070] Cytokine IL-21 (SEQ ID NO. 5);

[0071] co-stimulatory ligand HVEML (SEQ ID NO. 6);

[0072] HO-Tag3 hexamerization tool (SEQ ID NO. 7);

[0073] CD8 transmembrane domain (SEQ ID NO. 8);

[0074] Fluorescent proteins: BFP (SEQ ID NO.9), GFP (SEQ ID NO.10), and mCherry (SEQ ID NO.11) are used to label anti-CD3 single-chain antibody, anti-CD28 single-chain antibody, and cytokine IL-21, respectively;

[0075] Puromycin resistance gene (SEQ ID NO.12): marking the co-stimulatory ligand HVEML;

[0076] Internal ribosome entry site IRES (SEQ ID NO. 13);

[0077] See the table below for the specific sequence.

[0078]

[0079]

[0080] (2) Construction of vector plasmid

[0081] Obtaining target genes: obtaining DNA fragments of anti-CD3 single-chain antibody, anti-CD28 single-chain antibody, HOTAg3 hexamer tool, CD8 transmembrane domain, BFP, GFP, mCherry, puromycin resistance gene, and IRES from plasmid cloning templates containing anti-CD3 single-chain antibody, anti-CD28 single-chain antibody, HOTAg3 hexamer tool, CD8 transmembrane domain, BFP, GFP, mCherry, puromycin resistance gene, and IRES by polymerase chain reaction (PCR), agarose gel electrophoresis, and gel recovery; obtaining DNA fragments of cytokine IL-21 and costimulatory ligand HVEML by whole gene synthesis;

[0082] Obtaining the vector backbone: Obtaining the vector through double enzyme digestion reaction and agarose gel electrophoresis and gel recovery;

[0083] Connect gene fragments: Use Gibson Mix to connect DNA fragments and vectors to form recombinant plasmids;

[0084] Transformation of recombinant plasmid: transform the ligation product into competent cells, spread the transformed bacterial solution on LB agar plates with ampicillin resistance, and culture at 37°C for 12 to 14 hours;

[0085] Screening sequencing: Perform colony PCR identification on the colonies grown on the resistant plate, and send the colonies that can produce the target band of the correct size to the sequencing company for sequencing. After the sequencing results are compared, notify the sequencing company to return the sample bacterial solution with the correct sequencing results;

[0086] Amplification and purification: After the returned sample bacterial solution is expanded and frozen, a high-purity endotoxin-free plasmid extraction kit is used to obtain a vector plasmid that can be used for lentiviral packaging.

[0087] (3) Collecting lentiviral particles by transfection of HEK 293T cells

[0088] Plasmid preparation: Use a high-purity endotoxin-free extraction kit to obtain the three plasmids psPAX2, pMD2.G, and vector plasmid;

[0089] Cell preparation: Take HEK 293T cells in the logarithmic growth phase, plate 0.9×10^6 cells in each well of a 6-well cell culture plate, use 2 mL of DMEM complete medium, and allow the cells to adhere overnight.

[0090] Cell transfection: After 16-20 hours, when the cell density reaches 80%-90% and the cells are in good condition, transfection can be performed. The transfection uses a three-plasmid transfection system, i.e., psPAX2: pMD2.G: vector plasmid at a ratio of 10:3:12 for lentivirus packaging. Transfection uses calcium phosphate co-transfection. When using a 6-well plate to package lentivirus, the total transfection reagent system is 200μL, which is divided into tube A and tube B. Only 100μL of phosphate buffer solution 2×HBS is added to tube A, and tube B is a mixed solution with 10μL of CaCl2 solution. The dosage of the three plasmids is 2.25μg, 0.75μg and 3μg respectively. The remaining volume is supplemented to 100μL with deionized water. The solution in tube B is added dropwise to the solution in tube A and gently mixed. After standing for 15-20 minutes, calcium phosphate-DNA precipitation is formed. The transfection reagent is slowly added to the HEK 293T cells and placed in the incubator;

[0091] Cell medium replacement: 6-8 hours after transfection, when the cells are in good condition, aspirate the original culture medium, replace the supernatant containing the transfection reagent with 2 mL of DMEM complete culture medium, and continue culturing in the incubator;

[0092] Virus supernatant collection: 48 hours after transfection, collect the cell culture supernatant with a centrifuge tube, add 2 mL of DMEM complete medium, and store the collected virus supernatant in a 4°C refrigerator; 72 hours after transfection, collect the cell culture supernatant and mix it with the 48-hour virus supernatant;

[0093] Virus concentration: The collected 48-hour and 72-hour virus supernatants were centrifuged to remove cell debris at 500×g, 5min, 4°C. After centrifugation, the supernatant was transferred to a new centrifuge tube, and the virus concentrate was added and mixed. The tube was placed in a 4°C refrigerator. The next day, the tube was centrifuged at 4°C, 3500-4000×g for 20-30 minutes. The supernatant was aspirated, and the tube was resuspended in 1×PBS solution or culture medium and stored in a -80°C refrigerator. If used in the short term, the tube was stored in a 4°C refrigerator.

[0094] (4) Lentivirus infection of K562 cells

[0095] Cell preparation: Take K562 cells in good cell condition, inoculate the cells at a density of 1×10^6 cells / well in a 24-well culture plate, and add 0.5 mL of RPMI-1640 complete medium;

[0096] Stable cell transfection: Add an appropriate amount of lentivirus solution to K562 cells according to the virus titer and place them in an incubator for continued culture;

[0097] Cell medium replacement: 12-16 hours after lentiviral infection, centrifuge the cell suspension containing lentiviral particles at 300×g for 3 minutes, remove the supernatant containing lentiviral particles, resuspend the cell pellet in RPMI-1640 complete medium, plate it, and culture it in an incubator;

[0098] Stable transfection efficiency detection: The expression of fluorescent protein in K562 cells after stable transfection was detected by EVOS live cell fluorescence imaging system and flow cytometry to obtain the efficiency of stable transfection of K562 cells.

[0099] (5) Obtaining a stable K562 cell line by flow cytometry sorting

[0100] Cells carrying fluorescent protein marker genes are screened by flow cytometry.

[0101] Sample preparation: According to the efficiency of K562 cell stable transfection, K562 cells were cultured to a sufficient number for sorting, and single-cell suspension was prepared using sterile PBS buffer containing 2% FBS. The cell density was controlled between 1×10^6 and 1×10^7 cells / mL, and the cells were labeled and stored on ice.

[0102] Instrument calibration: Start the flow cytometer and use microspheres for calibration to ensure that the droplets are stable, the droplet delay time is accurate, and the fluorescence detection channel is normal;

[0103] Cell sorting: In the analysis mode, obtain the data of the cell sample, determine the non-target cell population and the target cell population according to the signal characteristics of the cell's side scattered light and fluorescence intensity, accurately circle the cell population to be sorted, add an appropriate amount of culture medium to the collection tube to maintain cell activity, set the sorting outlet, and place the collection bin before starting the sorting mode;

[0104] Cell processing: After sorting, the collected cells are properly processed, centrifuged, resuspended and plated according to the number of collected cells, and placed in an incubator for further culture;

[0105] Sorting efficiency detection: Flow cytometry was used to detect the expression of fluorescent protein in K562 cells after stable transfection and sorting to evaluate the efficiency and purity of sorting.

[0106] When the positive rate of fluorescent protein reaches more than 95%, the K562-aCD3(H)-aCD28-mbIL21 cell line stably expressing the target gene is obtained.

[0107] (6) Drug screening to obtain a stable K562 cell line:

[0108] Cells carrying the puromycin resistance gene can be selected by adding puromycin antibiotic to the culture medium.

[0109] Preparation of antibiotics: Use a 0.22 μm filter to filter and sterilize the puromycin antibiotic solution, prepare 1000× puromycin antibiotic with a final concentration of 2 μg / mL, and store in a -20°C refrigerator away from light after aliquoting;

[0110] Drug addition screening: 48-72 hours after lentiviral infection, K562 cells were expanded to a certain number, with a concentration of 0.6-0.8×10 6 The cells were plated into a 6-well culture plate at a density of 100 cells / mL, the volume of RPMI-1640 complete medium was 2 mL, 2 μL of 1000× concentration of puromycin antibiotic was added, and the plate was returned to the incubator for continued culture;

[0111] Repeated screening: 48 hours after drug addition, the cell status was observed under a microscope, the cells were collected and centrifuged, the supernatant containing a large number of dead cells was removed, the remaining cells were passaged 1-2 times, and then the puromycin antibiotic was added again for repeated screening to obtain the artificial antigen presenting cell K3H28-21H cell line of the present invention.

[0112] Flow cytometry was used to confirm the expression of the target gene on the surface of the K3H28-21H cell line. The results are shown in the attached Figure 3 . Figure 3 The data shows that Figure 3In the figure, A is AlexaFluor488-A dye, B is Pacific Blue-A dye, C is PE-A dye, and the control is a wild-type K562 cell line without gene transduction. The expression difference between the K3H28-21H cell line of the present invention and the original K562 cells under specific staining agents reflects the successful expression of the target gene on the surface of K3H28-21H cells.

[0113] Example 2

[0114] The artificial antigen presenting cell K3H28-21H cell line obtained in Example 1 was used to activate peripheral blood lymphocyte T cells.

[0115] Isolation and cryopreservation of peripheral blood mononuclear cells (PBMC): Collect human blood, remove multinuclear cells and red blood cells using density gradient centrifugation and other methods, and resuspend the obtained PBMC in a cryopreservation solution containing 40% FBS for cryopreservation;

[0116] Pretreatment of artificial antigen-presenting cells: Take an appropriate number of K3H28-21H cell lines in a 6-well plate and place them under ultraviolet light for irradiation for 30-60 minutes to obtain artificial antigen-presenting cells that have lost their proliferation ability and stably and continuously express the molecules of target genes that activate T cells. After irradiation, collect the cells into a centrifuge tube, centrifuge, resuspend, and count.

[0117] Ex vivo activation of peripheral blood T cells: thaw frozen PBMCs one day in advance, resuspend them in RPMI-1640 complete medium and place them in an incubator. Count them on the second day of thaw, co-culture PBMCs with lethally irradiated artificial antigen-presenting cells at a cell number ratio of 2:1, add 200 IU / mL of IL-2 to the culture medium, and continue culturing in the incubator.

[0118] Example 3

[0119] CAR-T cells were prepared using the T cells activated in Example 2.

[0120] Lentivirus preparation: HEK 293T cells were plated two days before PBMC recovery, and lentivirus packaging was performed one day before PBMC recovery. The concentrated lentivirus solution was obtained the day after T cell activation.

[0121] Lentiviral transduction: 48 hours after T cell activation, add the lentiviral solution to PBMCs and culture them in an incubator. After 12-16 hours, centrifuge and resuspend the PBMCs, remove the supernatant containing lentiviral particles, and replace with culture medium containing 200 IU / mL IL-2 to continue culturing.

[0122] Expansion culture: CAR-T cells begin to proliferate rapidly on the 5th to 6th day after activation. It is necessary to replenish the culture medium for CAR-T cells every day and adjust the cell density to 1-3×10^6 cells / mL to prevent cell exhaustion and maintain strong proliferation ability until the CAR-T cells enter a resting state and no longer actively proliferate;

[0123] CAR-T cell product quality testing: During the culture process of CAR-T cells, fluorescently labeled antibodies and flow cytometry are used to detect and record the CAR expression level, differentiation phenotype, and proliferation ability of CAR-T cells. After the CAR-T cells enter the resting state, short-term killing experiments and long-term proliferation experiments are used to further detect the tumor killing ability and persistence of CAR-T cells.

[0124] CAR-T cell freezing and resuscitation: High-quality CAR-T cell products can be frozen using cryopreservation fluid after entering resting state, and can still maintain good activity and function when revived for testing or treatment.

[0125] Example 4

[0126] The multivalent expression of anti-CD3 single-chain antibody was verified by fluorescence imaging and flow cytometry.

[0127] During the construction of artificial antigen presenting cells, the K562-aCD3-aCD28 cell line and the K562-aCD3(H)-aCD28 cell line were observed under a 10× microscope using the EVOS live cell fluorescence imaging system. The differences in fluorescence imaging of the two cell lines are shown in the attached figure. Figure 4 The expression of anti-CD3 single-chain antibody on the surface of these two cells was detected by Pacific Blue channel in flow cytometry. The results are shown in the attached Figure 5 .

[0128] By the attached Figure 4The data show that the K562-aCD3(H)-aCD28 cell line was modified to express a multivalent anti-CD3 single-chain antibody structure, and a stronger blue fluorescence signal was seen because the antibody was fused with the BFP fluorescent protein, and the multivalent expression on the cell surface led to the accumulation and enhancement of the fluorescence signal. The K562-aCD3-aCD28 cell line expressed a conventional single-chain antibody on the cell surface, and the fluorescence intensity was relatively moderate. For GFP-aCD28, the GFP channel shows the expression of anti-CD28 single-chain antibody on the surface of K562 cells, and the fluorescence signals of the two cells are relatively uniform, indicating that the expression of anti-CD28 single-chain antibody is stable; for BFP-aCD3, the K562-aCD3(H)-aCD28 cell line shows more dense fluorescent spots or fluorescent clusters, and these areas are rich in anti-CD3 single-chain antibodies. In contrast, the fluorescence distribution of the K562-aCD3-aCD28 cell line may be more uniform or sparse; in the Merge image, both cell lines show overlap of blue and green fluorescence, indicating co-localization of aCD3 and aCD28 antibodies on the cell surface. The overall fluorescence intensity of the K562-aCD3(H)-aCD28 cell line is significantly higher than that of the K562-aCD3-aCD28 cell line, further supporting that multivalent modification improves the expression or detection signal of anti-CD3 single-chain antibody on the cell surface.

[0129] By the attached Figure 5 The data show that by comparing the flow cytometry detection of anti-CD3 single-chain antibodies on the surface of different cell lines, under the PacificBlue channel, the fluorescence peak of the K562-aCD3-aCD28 cell line shifted to the right compared with the negative control NC, indicating that the monovalent anti-CD3 single-chain antibody was successfully expressed on the cell surface; the fluorescence peak of the K562-aCD3(H)-aCD28 cell line shifted further to the right and the signal was stronger, indicating that the multivalent modification enhanced the expression of the anti-CD3 single-chain antibody on the cell surface, thereby enhancing the fluorescence detection signal. This change directly reflects the multivalent expression of the anti-CD3 single-chain antibody.

[0130] Example 5

[0131] Verify the function of multivalently expressed anti-CD3 single-chain antibody in activating T cells.

[0132] Use activator Gibco TM Dynabeads CD3 / CD28 CTS magnetic beads, hereinafter referred to as Dynabeads, were used as a control with the K562-aCD3-aCD28 cell line obtained in the process of the present invention to detect the function of the K562-aCD3(H)-aCD28 cell line in activating T cells. From day 1 to day 14 after T cell activation, flow cytometry and fluorescently labeled antibodies were used to detect and record the activation level, proliferation, and differentiation phenotype changes of T cells. The results are shown in the attached Figure 6 .

[0133] By the attached Figure 6 The data shows that Figure 6 In the figure, A is the expression of proliferation and activation markers on T cells, B is the T cell expansion multiple, C is the differentiation phenotype change, and the control is the K562-aCD3-aCD28 cell line. In the K562-aCD3(H)-aCD28 cell line, multivalent expression of anti-CD3 single-chain antibodies can effectively activate T cells and promote T cell proliferation and function. The transformation to a higher proportion of naive T cells and central memory cells supports the effectiveness of multivalent expression of anti-CD3 single-chain antibodies in enhancing T cell function.

[0134] The activation effects of these three activators on resting T cells were detected using a cell proliferation tracking fluorescent probe CFSE. The experimental process and results are attached. Figure 7 .

[0135] By the attached Figure 7 The data show that the controls were Dynabeads and K562-aCD3-aCD28 cell lines. The CFSE fluorescent probe method was used to effectively detect the activation effects of the three activators on resting T cells. The multivalent anti-CD3 single-chain antibody activator showed the highest T cell proliferation ratio (45.3%), indicating that it has a strong activation ability.

[0136] These experimental results showed that the ability of K562-aCD3(H)-aCD28 cell line to activate T cells was better than that of Dynabeads and K562-aCD3-aCD28 cell line, verifying that multivalent expression of anti-CD3 single-chain antibody can enhance T cell activation and proliferation.

[0137] Example 6

[0138] Detect the effect of multivalent expression of anti-CD3 single-chain antibody on the function of CAR-T cells.

[0139] Using Dynabeads and K562-aCD3-aCD28 cell lines as controls, the ability of K562-aCD3(H)-aCD28 cell lines to activate peripheral blood T cells and prepare anti-CD19 CAR-T cells was tested. The CAR expression level, proliferation, and differentiation phenotype of CAR-T cells were detected and recorded by fluorescently labeled antibodies and flow cytometry. The results are shown in the attached Figure 8 .

[0140] CAR expression level is an important indicator to measure the success of CAR-T cell preparation. High CAR expression usually means that more T cells have been successfully transduced and expressed CAR molecules, thus having stronger targeted killing ability. The proliferation ability of CAR-T cells is the key to their continued function in vivo. Good proliferation ability means that CAR-T cells can rapidly proliferate in vivo and reach sufficient numbers to eliminate tumor cells; the differentiation phenotype of CAR-T cells is closely related to their function. T cells at different differentiation stages have different effector functions and persistence. Figure 8 The data shows that Figure 8 In the figure, A is the CAR positive rate, B is the clustering phenotype of CAR-T cells, and the controls are Dynabeads and K562-aCD3-aCD28 cell lines. CAR-T cells activated by K562-aCD3(H)-aCD28 cell line showed higher CAR expression, indicating that multivalent expression of anti-CD3 single-chain antibodies enhanced the transduction efficiency or stability of CAR; CAR-T cells activated by K562-aCD3(H)-aCD28 cell line proliferated faster or had a higher proliferation ratio, indicating that multivalent expression of anti-CD3 single-chain antibodies promoted the proliferation of T cells; CAR-T cells activated by K562-aCD3(H)-aCD28 cell line contained a higher proportion of memory T cells or had a more ideal differentiation phenotype distribution, indicating that multivalent expression of anti-CD3 single-chain antibodies was beneficial to the long-term survival and functional maintenance of CAR-T cells.

[0141] After the anti-CD19 CAR-T cells entered the resting state, the anti-CD19 CAR-T cells generated by three different activators were subjected to an in vitro short-term killing test. The target cells expressing CD19 and the target cells expressing Meso were mixed in equal proportions and co-cultured with the anti-CD19 CAR-T cells at different effector-target ratios. After 48 hours, the residual target cells were detected by flow cytometry and the killing effect was statistically analyzed. The results are attached. Fig. 9 .

[0142] By the attached Fig. 9 The data shows that Fig. 9 In the figure, A is the in vitro short-term killing rate, B is the specific killing rate, and the controls are Dynabeads and K562-aCD3-aCD28 cell lines. The in vitro short-term killing rate of anti-CD19CAR-T cells generated by K562-aCD3(H)-aCD28 cell line activation was the highest when the effector-target ratio was 2:1.

[0143] These experimental results show that the K562-aCD3(H)-aCD28 cell line has a better ability to improve the function of CAR-T cells when preparing CAR-T cells than Dynabeads and K562-aCD3-aCD28 cell lines, proving that the activator of multivalent expression of anti-CD3 single-chain antibody can not only activate and proliferate T cells, but also improve the function and quality of CAR-T cells.

[0144] Example 7

[0145] Based on the multivalent expression of anti-CD3 single-chain antibodies, it was verified that multi-signal integration can further enhance T cell activation and the quality of CAR-T cells.

[0146] The K562-aCD3(H)-aCD28 cell line obtained in the construction process of the present invention was used as a control to detect the ability of the K562-aCD3(H)-aCD28-mbIL21 cell line to activate peripheral blood T cells and generate anti-DR5 CAR-T cells. The activation level of T cells and the CAR expression level, proliferation, and differentiation phenotype of anti-DR5 CAR-T cells were detected and recorded by fluorescently labeled antibodies and flow cytometry. The results are shown in the attached Fig.10 .

[0147] By the attached Fig.10 The data shows that Fig.10 In the figure, A is the expression of activation markers on T cells, B is the CAR positivity rate, C is the differentiation phenotype after activation, D is the expansion fold, and the control is the K562-aCD3(H)-aCD28 cell line. The K562-aCD3(H)-aCD28-mbIL21 cell line can induce higher expression of activation markers, indicating that the addition of IL-21 enhances T cell activation; CAR-T cells stimulated by the K562-aCD3(H)-aCD28-mbIL21 cell line show higher CAR expression, indicating that the integration of IL-21 promotes the expression and stability of the CAR gene; CAR-T cells stimulated by the K562-aCD3(H)-aCD28-mbIL21 cell line will show a faster proliferation rate and a higher proliferation multiple; K562-aCD3(H)-aCD28-mbIL21 cell line can induce a higher proportion of memory T cells, indicating that the addition of IL-21 is beneficial to the long-term survival and functional maintenance of CAR-T cells; indicating that on the basis of multivalent expression of anti-CD3 single-chain antibody to enhance antigen recognition signals, the integration of IL-21-mediated cytokine signals not only further enhances T cell activation, but also significantly improves the proliferation and function of CAR-T cells.

[0148] Using Dynabeads and K562-aCD3(H)-aCD28-mbIL21 cell lines as controls, it was verified that the K3H28-21H cell line of the present invention has a stronger ability to activate peripheral blood T cells and prepare higher quality anti-CD19 CAR-T cells. The results are shown in the attached Fig.11 .

[0149] By the attached Fig.11 The data shows that Fig.11 In the figure, A is the expression of activation markers on T cells, B is the CAR positive rate, and the controls are Dynabeads and K562-aCD3(H)-aCD28-mbIL21 cell lines. After 20 hours of activation, the CD25-CD69+ activity of T cells activated by K3H28-21H reached 50%, indicating that K3H28-21H can effectively activate T cells in a short time; compared with the control activators Dynabeads and K562-aCD3(H) / aCD28 / mbIL-21, K3H28-21H showed a stronger activation effect; K3H28-21H performed well in activating T cells, and the integration of multiple signals can further enhance the activation of T cells and the function of CAR-T cells, which will be conducive to the preparation of higher quality CAR-T cells. High-quality CAR-T cells usually have higher CAR expression levels, stronger proliferation ability and better killing effect. The above experiments show that the artificial antigen-presenting cells constructed by the present invention can produce higher-quality CAR-T cells when preparing anti-CD19 CAR-T cells and anti-DR5 CAR-T cells, verifying that the K3H28-21H cell line has a wide range of applicability and has the potential to prepare a variety of high-quality CAR-T cells.

[0150] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. An artificial antigen presenting cell, characterized in that The artificial antigen presenting cells can activate T cells in PBMC in vitro; the artificial antigen presenting cells use the HOTAgs tool to introduce multivalency and stably express multivalent membrane-bound activating antibodies, membrane-bound activating antibodies, natural co-stimulatory ligands or molecules, and membrane-bound cytokines; the membrane-bound activating antibodies and membrane-bound cytokines are stably expressed on the T cell membrane using transmembrane domains; The membrane-bound activating antibody is selected from any one of an anti-CD3 single-chain antibody and an anti-CD28 single-chain antibody; The HOTags tool is selected from HOTag3 or HOTag6, and is used to introduce multivalency to increase the local concentration of membrane-bound activating antibodies.

2. The artificial antigen presenting cell according to claim 1, characterized in that The artificial antigen presenting cells are K562 cells that use HOTAg3 hexamer and CD8 transmembrane domain to express multivalent membrane-bound anti-CD3 single-chain antibody, membrane-bound anti-CD28 single-chain antibody, natural co-stimulatory ligand HVEML and membrane-bound cytokine IL-21; The amino acid sequence of the anti-CD3 single-chain antibody is shown in SEQ ID NO.1, containing a signal peptide located at positions 1 to 19 of its amino acid sequence, and the signal peptide sequence is shown in SEQ ID NO.2; the amino acid sequence of the anti-CD28 single-chain antibody is shown in SEQ ID NO.3, containing a signal peptide located at positions 1 to 21 of its amino acid sequence, and the signal peptide sequence is shown in SEQ ID NO.4; the amino acid sequence of the cytokine IL-21 is shown in SEQ ID NO.5; the amino acid sequence of the co-stimulatory ligand HVEML is shown in SEQ ID NO.6; the amino acid sequence of the HOTAg3 hexamer is shown in SEQ ID NO.7; and the amino acid sequence of the CD8 transmembrane domain is shown in SEQ ID NO.

8.

3. An expression vector, characterized in that: The expression vector is used to prepare the artificial antigen-presenting cell according to any one of claims 1 to 2; The expression vector includes a gene encoding a multivalent membrane-bound activating antibody, a gene encoding a membrane-bound activating antibody, a gene encoding a membrane-bound cytokine, a gene encoding a transmembrane region, or a gene encoding a natural co-stimulatory ligand or molecule.

4. The expression vector according to claim 3, characterized in that The expression vector comprises (1) A gene expression vector containing a coding sequence of a promoter SFFV for controlling the expression of an anti-CD3 single-chain antibody, a coding sequence of an activating antibody anti-CD3 single-chain antibody, a coding sequence of a CD8 transmembrane domain, a coding sequence of a HOTAg3 hexamer for controlling the multivalent expression of an anti-CD3 single-chain antibody, a coding sequence of BFP, and a post-transcriptional regulatory element WPRE for enhancing gene expression efficiency, which are sequentially connected; (2) a gene expression vector containing the coding sequence of the promoter SFFV for controlling the expression of the anti-CD28 single-chain antibody, the coding sequence of the activating antibody anti-CD28 single-chain antibody, the coding sequence of the CD8 transmembrane domain, the coding sequence of GFP, and the post-transcriptional regulatory element WPRE for enhancing gene expression efficiency, which are sequentially connected; (3) a gene expression vector containing the coding sequence of the promoter SFFV for controlling the expression of the cytokine IL-21, the coding sequence of the cytokine IL-21, the coding sequence of the CD8 transmembrane domain, the coding sequence of mCherry, and the post-transcriptional regulatory element WPRE for enhancing the gene expression efficiency, which are sequentially connected; (4) a gene expression vector containing the coding sequence of the promoter SFFV for controlling the expression of the co-stimulatory ligand HVEML, the coding sequence of the co-stimulatory ligand HVEML, the coding sequence of the IRES connected to the puromycin resistance gene, the coding sequence of the puromycin resistance gene and the post-transcriptional regulatory element WPRE for enhancing the gene expression efficiency, which are sequentially connected; The amino acid sequence of the BFP is shown in SEQ ID NO.9; the sequence of the GFP is shown in SEQ ID NO.10; the sequence of the mCherry is shown in SEQ ID NO.11; the amino acid sequence of the puromycin resistance gene is shown in SEQ ID NO.12; and the nucleotide sequence of the IRES is shown in SEQ ID NO.

13.

5. A recombinant virus, characterized in that: The recombinant lentivirus is prepared by co-transfecting HEK 293T cells with the expression vector according to any one of claims 3 to 4 and a packaging helper plasmid.

6. A method for constructing an artificial antigen presenting cell according to any one of claims 1 to 2, characterized in that: The method comprises introducing a recombinant virus as claimed in claim 5 into a host cell, and obtaining the positive clone by screening with antibiotics or fluorescent markers; the host cell is selected from any one of K562 cells, HEK293T cells, Jurkat cells, CHO cells, Raji cells or other cell lines that are easy to be genetically modified.

7. A culture medium, characterized in that The culture medium comprises the artificial antigen presenting cells according to any one of claims 1 to 2; the culture medium also comprises exogenous cytokine IL-2 with a final concentration of 200 IU / mL and fetal bovine serum with a volume percentage of 10%, and the culture medium is RPMI-1640 culture medium.

8. Use of the artificial antigen presenting cell according to any one of claims 1 to 2, the expression vector according to any one of claims 3 to 4, the recombinant virus according to claim 5, and the culture medium according to claim 7 in the preparation of a T cell-based tumor immunotherapy product.

9. A method for preparing a T cell tumor immunity product, characterized in that: Specifically, it includes activating peripheral T lymphocytes and culturing CAR-T cells with a high positive rate and maintaining a low differentiation state. The conditions for activating peripheral T lymphocytes are: peripheral blood T lymphocytes and lethally irradiated artificial antigen presenting cells according to any one of claims 1 to 2 are co-cultured in a ratio of 2:

1.

10. A method for preparing a T cell tumor immunity product according to claim 9, characterized in that: The tumor cells are hematological malignant tumor cells or solid tumor cells, wherein the hematological malignant tumor cells are selected from any one of human chronic myeloid leukemia cell line K562 cells, human B cell lymphoma cell line Raji cells, human Burkitt lymphoma cell line Daudi cells, and precursor B cell acute lymphoblastic leukemia cell line NALM-6 cells; the solid tumor cells are selected from any one of human epidermal cancer cell line A431 cells, human ovarian cancer cell line SKOV3 cells, human colorectal cancer cell line LoVo cells, non-small cell lung cancer cell line A549 cells, glioblastoma cell line U87 cells, triple-negative breast cancer cell line MDA-MB-231 cells, hepatocellular carcinoma cell line HepG2 cells, colorectal cancer cell line HT-29 cells, and prostate cancer cell line PC-3 cells; The CAR-T cells are selected according to the targeted antigen: any one of anti-CD19 CAR-T cells, anti-CD20 CAR-T cells, anti-BCMACAR-T cells, anti-HER2 CAR-T cells, anti-EGFR CAR-T cells, anti-Mesothelin CAR-T cells, anti-CD22 CAR-T cells, anti-CEA CAR-T cells, and anti-PSMA CAR-T cells; the CAR-T cells are selected according to the CAR structure: any one of the first-generation CAR containing an antigen domain and an activation signal domain, the second-generation CAR containing an antigen domain and a co-stimulatory domain, the third-generation CAR containing two co-stimulatory domains, the fourth-generation CAR containing an inducibly expressed cytokine gene, and the fifth-generation CAR fused with multiple signal transduction domains.

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