Method for preparing cells for expressing chimeric antigen receptor from cryopreserved cells

Abstract

The invention provides a method for preparing cells expressing a chimeric antigen receptor from cryopreserved cells. The method comprises the following steps: (1) resuscitating the cryopreserved cells; (2) activating the cells by contacting the cells with an activating agent; (3) contacting the cell with a CAR-encoding nucleic acid molecule, the CAR-encoding nucleic acid molecule being on a non-viral vector, to introduce the nucleic acid molecule into the cell; and (4) harvesting cells. According to the invention, the high-quality CAR-T cell is successfully prepared from the cryopreserved PBMC by using an electroporation process, and compared with the CAR-T prepared from the fresh PBMC, the CAR-T cell has the same performance, which proves that the immune cell therapy can be realized through the cryopreservation of the PBMC.

Description

Technical Field

[0001] The present invention relates to the technical field of immunocyte therapy, and more particularly to a method for preparing cells expressing chimeric antigen receptors from cryopreserved cells. Background Art

[0002] Chimeric antigen receptor (CAR) is a genetically recombinant receptor. Generally, a chimeric antigen receptor includes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, and can be transduced into different immune cells, such as T lymphocytes and NK cells. Multiple CAR-T cell products have been launched for the treatment of hematological tumors.

[0003] Generally speaking, CAR-T cells are derived from T cells isolated from a patient's peripheral blood mononuclear cells and are prepared through steps such as sorting, activation, and introduction of the CAR gene. The efficacy of CAR-T therapy depends to a large extent on the quality of T cells. Therefore, the quality of a patient's immune cells is closely related to the efficacy of treatment. Many studies have shown that the composition of a patient's immune cells is significantly different from that of healthy people. Healthy people may have a larger proportion of CD4+ T cells than patients, and have greater expansion ability and more types of TCRs during CAR-T cell culture. And TCR has an important function in immune responses. T cells isolated from healthy individuals are less differentiated and better maintain the stem cell-like state of cells during culture. In order to well preserve the characteristics of T cells and retain the therapeutic effect of CAR-T, extracting PBMCs at the healthy stage and cryopreserving them for future use would be an effective method.

[0004] It has been reported that cryopreserved PBMCs are used to prepare CAR-T cells through a lentiviral process. However, due to high costs, complexity, and major safety issues, the production of CAR-T products still faces challenges. Non-viral transduction is inexpensive and easy to operate, and its efficiency is equivalent to or better than that of viral transduction, so it has received increasing attention. However, there is currently no report on successfully preparing CAR-T cells from cryopreserved PBMCs through a non-viral method. Summary of the Invention

[0005] The object of the present invention is to provide a method for preparing cells expressing chimeric antigen receptors from cryopreserved cells through non-viral delivery technology.

[0006] The present invention provides a method for preparing cells expressing chimeric antigen receptors from cryopreserved cells, the method comprising: (1) thawing the cryopreserved cells; (2) contacting the cells with an activator for activation; (3) contacting the cells with a nucleic acid molecule encoding CAR, the nucleic acid molecule encoding CAR being on a non-viral vector to introduce the nucleic acid molecule into the cells; (4) harvesting the cells.

[0007] In some embodiments, the cryopreserved cells are cryopreserved PBMCs or T cells (such as CD3+, CD4+ and / or CD8+ T cells).

[0008] In some embodiments, the cells expressing a chimeric antigen receptor are CAR-T cells.

[0009] In some embodiments, the cryopreserved cells are resuscitated using a serum-free medium, which may contain serum or serum substitutes, growth factors, etc., such as containing 2% FBS, IL-7, IL-15, etc. The cryopreserved cells can also be resuscitated using a resuscitation solution without medium, such as a PBS solution containing 0.5% HSA and 2 mM EDTA.

[0010] In some embodiments, the cryopreserved cells are resuscitated using a serum-free medium, which may contain one or more of an apoptosis protein inhibitor and an insulin-transferrin-selenium additive.

[0011] In some embodiments, the action pathways of the apoptosis protein inhibitor include one or more of p-JAK2, p-STAT3, caspase-3, Bax, and ROCK.

[0012] In some embodiments, the apoptosis protein inhibitor is selected from one or more of kaempferol-3-O-rutinoside, Ac-DEVD-CHO, and RevitaCell.

[0013] In some embodiments, the cells in step (1) are PBMCs, and step (1) further includes a step of sorting the resuscitated cells by antibody sorting or flow cytometry sorting, such as sorting using CD4 / CD8 magnetic beads. In some embodiments, the sorted cells are CD3+ T cells.

[0014] In some embodiments, the activator includes one or more selected from the following: CD3 antibody, CD28 antibody, 4-1BB antibody, 4-1BBL antigen.

[0015] In some embodiments, the activator is selected from the group consisting of: CD3 antibody, CD3 antibody and CD28 antibody, CD3 antibody and 4-1BB antibody, and CD3 antibody and 4-1BBL antigen.

[0016] In some embodiments, the activator is a CD3 antibody and a CD28 antibody fixed on magnetic beads; preferably, the activator is Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads and / or CTSDynabeads CD3 / 28.

[0017] In some embodiments, the final concentration of the activator is 1-20 μg / mL.

[0018] In some embodiments, the concentration ratio of the activator to the immune cells is 1-20 μg / mL: 2.45-2.8×10 8 immune cells, preferably 5-10 μg / mL: 2.5-2.6×10 8 immune cells.

[0019] In some embodiments, the CAR comprises an optional signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.

[0020] In some embodiments, the antigen-binding domain targets one or more of the following antigens: CD19, CD20, CD22, BCMA, mesothelin (MSLN), EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, folate receptor α, ErbB (such as ERBB2), Her2 / neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, FAP, podoplanin, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4.

[0021] In some embodiments, the nucleic acid molecule encoding the CAR is DNA and the non-viral vector is a plasmid vector.

[0022] In some embodiments, the nucleic acid molecule encoding the CAR is RNA, such as mRNA or saRNA, and the non-viral vector is an LNP, LPX, VLP, inorganic nanoparticle, or exosome.

[0023] In some embodiments, the non-viral vector is a plasmid vector containing a transposon, the transposon contains a nucleic acid molecule encoding the CAR, and the cell in step (3) is further contacted with a transposase or a nucleic acid molecule encoding the transposase.

[0024] The transposon and the transposase belong to the same transposon system, and the transposon system is selected from: Tol1 transposon system, Tol2 transposon system, Frog Prince transposon system, Minos transposon system, Hsmar1 transposon system, Helraiser transposon system, ZB transposon system, BZ transposon system, Intruder transposon system, SPINON transposon system, TcBuster transposon system, Passer transposon system, JL transposon system, Yabusame-1 transposon system, Uribo2 transposon system, PiggyBac (PB) transposon system, SleepingBeauty (SB) transposon system, and various variants or derivatives of the above transposon systems.

[0025] In some embodiments, the transposon system is the PB transposon system, the BZ transposon system, or the JL transposon system.

[0026] In some embodiments, the nucleic acid molecule encoding the transposase is DNA or RNA.

[0027] In some embodiments, the cell is contacted with a transposase or a nucleic acid molecule encoding the transposase, and cell transduction is carried out by electroporation.

[0028] In some embodiments, the introduction is carried out by electroporation.

[0029] In some embodiments, step (3) includes: contacting the cell with a DNA vector containing the JL transposon and mRNA encoding the JL transposase, and the JL transposon includes a CAR gene expression cassette and terminal inverted repeats located on both sides of the CAR gene expression cassette.

[0030] In some embodiments, the amino acid sequence of the JL transposase is as shown in SEQ ID NO:5.

[0031] In some embodiments, the terminal inverted repeats are as shown in SEQ ID NO:6 (3' ITR) and SEQ ID NO:7 (5' ITR).

[0032] In some embodiments, the DNA vector is an antibiotic-free microplasmid vector, and the antibiotic-free microplasmid vector contains a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein contains the following sequences: (1) the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having one or more mutations of E24D, I36V, and V43I compared with SEQ ID NO: 14; or (2) the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, and N54K compared with SEQ ID NO: 17; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0033] In a preferred embodiment, the amino acid sequence of the antitoxin protein is any one of SEQ ID NOs: 14-20.

[0034] In some embodiments, the replicon is R6K.

[0035] In some embodiments, the electroporation conditions are to transfer the mixture containing nucleic acid, cells, and electroporation solution into an electroporation cuvette, place it in a Lonza Nucleofactor 4D or Maxcyte electroporator, and select the program numbered FI-115 or Resting T / Expand T4 for electroporation.

[0036] In some embodiments, the method for preparing cells expressing a chimeric antigen receptor from cryopreserved cells according to the present invention adopts the preparation process of rapid CAR-T.

[0037] The method further satisfies at least any one of the following conditions (a)-(c):

[0038] (a) Step (3) is carried out together with step (2), or is carried out no later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2, or 1 hour after the start of step (1);

[0039] (b) Step (4) is carried out no later than 48, 36, 30, 24, 18, 12, 6, 3, 2, or 1 hour after the start of step (3)

[0040] (c) Step (4) is carried out no later than 72, 60, 48, 36, 30, 24, 20, 18, or 12 hours after the start of step (2).

[0041] In some embodiments, the method for preparing cells expressing chimeric antigen receptors from cryopreserved cells according to the present invention adopts the preparation process of ordinary CAR-T. The activation time is 12 - 84 hours, preferably 24 - 72 hours, more preferably 24, 48 or 72 hours.

[0042] In some embodiments, evaluated by the number of live cells, compared with the cells at the start of step (2), the cells from step (4) do not expand or expand by no more than 5%, 10%, 20%, 30%, 40%, 50% or 100%.

[0043] In some embodiments, the method for preparing cells expressing chimeric antigen receptors from cryopreserved cells according to the present invention adopts the preparation process of ordinary CAR-T. The activation time is 12 - 84 hours, preferably 24 - 72 hours, more preferably 24, 48 or 72 hours.

[0044] In some embodiments, the culture time is at least 3 days, preferably at least 5 days, more preferably 3 - 13 days.

[0045] In some embodiments, the culture medium is AIM-V medium with or without 5% serum or its substitute. Preferably, the medium further contains cytokines, such as IL-7 and / or IL-15. Preferably, the final concentration of IL-7 is 1 - 25 ng / mL, and the final concentration of IL-15 is 1 - 25 ng / mL. Preferably, the cytokines are added to the medium 0 - 6 hours after the start of the culture.

[0046] The present invention also provides cells expressing chimeric antigen receptors prepared by the preparation method of any of the embodiments.

[0047] The present invention also provides the use of the cells expressing CAR in the preparation of drugs for treating and / or preventing malignant tumors.

[0048] In some embodiments, the tumor is a solid cancer, such as selected from: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain cancer, thymoma, sarcoma, malignant epithelial tumor (carcinoma), uterine cancer, renal cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer, or one or more of their metastatic cancers. In some embodiments, the cancer is a liquid cancer, such as selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorder, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasmacytic myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or lymphoma of undetermined classification. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is the plasmid map of P19V21 plasmid;

[0050] Figure 2 It is the plasmid map of MSLN CAR too plasmid;

[0051] Figure 3 It is the plasmid map of pCpGfree MCS-0637 empty microplasmid, including the nucleotide sequence of antitoxin 0637 and R6K replicon;

[0052] Figure 4 This is the pCpGfree MCS-43009 empty miniplasmid map, including the nucleotide sequence of antitoxin 43009 and the R6K replicon.

[0053] Figures 5 - 7 The CAR-T method was prepared using the PB transposon system. Compared with fresh PBMC (0M), the frozen-thawed PBMCs were compared in terms of cell survival rate, cell phenotype, and T cell differentiation status. The data are expressed as mean values, and each point represents the data of an individual. The Friedman test was used for the inter-group significance test. P value ≤ 0.05 was significant, and the P value was marked at the top of the graph *P ≤ 0.05.

[0054] Figure 8 The process of preparing CAR-T using the PB transposon system.

[0055] Figures 9 - 13 Comparison of cell viability, cell expansion, CD3+ cell percentage, CD4+T to CD8+T cell ratio, and CAR+ cell percentage of fresh and frozen PBMCs in the process of preparing CAR-T cell manufacturing using the PB transposon system. Data are expressed as mean values, and each point represents the data of an individual. The Friedman test was used for the significance test between groups. P value ≤ 0.05 was significant, and the P value was marked at the top of the graph *P ≤ 0.05.

[0056] Figures 14 - 15 Comparison of T cell differentiation status and exhaustion characteristics of CAR-T products prepared by PB transposon system and from frozen and fresh PBMC. Data are expressed as mean values, each point represents the data of an individual, and the significance test between groups was performed by Friedman test, with P value ≤ 0.05 as significant, and P value marked at the top of the graph *P ≤ 0.05.

[0057] Figure 16 Comparison of cytokine secretion (IL-13, IL-10, IL-6, IL-5, IL-4, IL-12, IFN-γ and TNF-α) of CAR-T cells prepared by fresh and frozen PBMCs stimulated with SKOV3 cells for the preparation of CAR-T using the PB transposon system. Data are expressed as mean values, each point represents the data of an individual, and the significance test between groups was performed using the Friedman test. P value ≤ 0.05 was considered significant, and the P value was marked at the top of the graph *P ≤ 0.05.

[0058] Figure 17For the preparation of CAR-T using the PB transposon system, the killing ability curves of CAR-T cells prepared from fresh and frozen PBMCs with different effector-to-target ratios of 4:1 (left) or 2:1 (right) against SKOV3 cells at 24 hours. Data are presented as mean values, each point represents the data of an individual, and the significance test between groups was performed using the Friedman test. A P value ≤ 0.05 was considered significant, and the P values are marked at the top of the graph *P ≤ 0.05. Detailed implementation manners

[0059] Definitions

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0061] The term "chimeric antigen receptor" (CAR) is an artificially engineered receptor that can anchor a specific molecule (such as an antibody) that recognizes an antigen on the surface of a tumor cell to an immune cell (such as a T cell), enabling the immune cell to recognize a tumor antigen or a viral antigen and kill the tumor cell or the virus-infected cell. A CAR generally sequentially comprises an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, a hinge region, a transmembrane region, and an intracellular signaling region. Generally, the polypeptide that binds to a tumor cell membrane antigen can bind to a membrane antigen widely expressed by tumor cells with moderate affinity. The polypeptide that binds to a tumor cell membrane antigen can be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single-chain antibody, a single-domain antibody, a Fab fragment, an F(ab')2 fragment, and an Fv fragment.

[0062] The term "single-chain antibody" (scFv) refers to an antibody fragment formed by linking the amino acid sequences of the variable region of the antibody light chain (VL region) and the variable region of the antibody heavy chain (VH region) via a hinge, and having the ability to bind an antigen. In certain embodiments, the single-chain antibody (scFv) of interest is derived from an antibody of interest. The antibody of interest can be a human antibody, including human-mouse chimeric antibodies and humanized antibodies. The antibody can be secreted or membrane-anchored; preferably, it is membrane-anchored.

[0063] The terms "single-domain antibody", "variable domain of the heavy chain of a heavy-chain antibody", "VHH", "nanobody", "single variable domain" are used interchangeably and all refer to a single-domain polypeptide or protein that specifically recognizes and binds to an antigen. A single-domain antibody is the variable region of a heavy-chain antibody. Generally, a single-domain antibody contains three CDRs and four FRs. A single-domain antibody is the smallest functional antigen-binding fragment. Generally, after obtaining an antibody that is naturally lacking the light chain and the first constant region of the heavy chain (CH1), the variable region of the antibody heavy chain is then cloned to construct a single-domain antibody consisting of only one variable region of the heavy chain.

[0064] The term "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell.

[0065] The term "vector" refers to any element that can transfer and / or transport a nucleic acid composition to a host cell, enter the host cell and / or reach a specific location and / or compartment within the host cell, such as plasmids, phages, transposons, cosmids, chromosomes, artificial chromosomes (YAC or BAC), viruses, virus capsids, virions, etc.

[0066] The term "viral vector" refers to a vector that utilizes the molecular mechanism of a virus to deliver its genome into other cells for infection, and mediates gene transfer. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0067] The term "non-viral vector" refers to a vector that uses non-viral means to mediate gene transfer, including plasmid vectors, non-viral materials (such as LNP, LPX, VLP, inorganic nanoparticles, exosomes, etc.).

[0068] The following provides an exemplary description of the methods herein.

[0069] A method for preparing cells expressing a chimeric antigen receptor from cryopreserved cells, the method comprising: (1) thawing the cryopreserved cells, with or without sorting the thawed cells; (2) contacting the cells with an activator for activation; (3) contacting the cells with a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule encoding a CAR is on a non-viral vector to introduce the nucleic acid molecule into the cells; (4) harvesting the cells.

[0070] Cell cryopreservation

[0071] Fresh leukapheresis products are obtained from a subject, which can be a healthy individual or a cancer patient. After obtaining the cells, a cryopreservation solution is added, and cell cryopreservation is performed by direct cooling or programmed cooling.

[0072] The cryopreservation solution can be a combination of cryopreservation solutions well-known in the art. For example, the cryopreservation solution includes a diluent and a cryoprotectant. The diluent is selected from one or more of compound electrolyte injection solution, 0.9% NaCl injection solution, and sodium lactate Ringer's injection solution. The cryoprotectant is selected from one or more of dextran glucose injection solution, human albumin solution, dimethyl sulfoxide, ethylene glycol, trehalose, sucrose, and glucose. In a specific embodiment, the cryopreservation solution includes: 40 - 80% v / v compound electrolyte injection solution, 5 - 20% v / v dextran 40 glucose injection solution, 10 - 30% v / v human albumin solution, and 5 - 10% v / v dimethyl sulfoxide. The cryopreservation solution can include other components, such as vitamins, serum, etc. In one embodiment, the concentration of the vitamin is 1 - 20 mg / ml, preferably 1 - 15 mg / ml, more preferably 5 - 10 mg / ml.

[0073] Cell cryopreservation can directly cool down to -80°C and then be stored in a liquid nitrogen tank; or cool down successively at 2-8°C, -20°C, and -80°C, and finally be stored in a liquid nitrogen tank.

[0074] The cryopreservation time can be more than 3 months, such as 3 months, 6 months, 9 months, 12 months, 2 years, 3 years, 5 years, or more than 10 years.

[0075] Cell recovery

[0076] The general steps of cell recovery are to take out the cryopreserved cells, preheat and thaw them, and then add a cell recovery solution to the cryopreserved cells. The cryopreserved cells can be recovered using a serum-free medium, and the serum-free medium can contain serum or serum substitutes, growth factors, etc., such as containing 2% FBS, IL-7, IL-15, etc. The cryopreserved cells can also be recovered using a recovery solution without a medium, such as a PBS solution containing 0.5% HSA and 2 mM EDTA.

[0077] In addition, in order to promote cell recovery, improve cell viability and activity, other components can also be added to the medium. For example, the recovery solution in any embodiment of CN201810129440.5 or the culture in any embodiment of CN202311000790.9. In some embodiments, the cryopreserved cells are recovered using a serum-free medium, and the serum-free medium can contain one or more of an apoptosis protein inhibitor and an insulin-transferrin-selenium additive. The action pathways of the apoptosis protein inhibitor include one or more of p-JAK2, p-STAT3, caspase-3, Bax, and ROCK. The apoptosis protein inhibitor is selected from one or more of kaempferol-3-O-rutinoside, Ac-DEVD-CHO, and RevitaCell. The concentration of kaempferol-3-O-rutinoside is 10 μg / mL - 100 μg / mL, preferably 50 μg / mL - 100 μg / mL. The concentration of Ac-DEVD-CHO is 10 - 100 μM, preferably 40 - 60 μM. The dilution factor of RevitaCell is 50 - 250 times, preferably 100 - 200 times.

[0078] Cell sorting

[0079] The cryopreserved cells can be PBMC, T cells, or sorted T cells. T cells can be CD3+, CD4+, and / or CD8+ T cells. Depending on the type of cells, sorting or non-sorting steps can be included. Sorting is performed by antibody sorting or flow cytometry sorting, such as sorting using CD4 / CD8 magnetic beads. In some embodiments, the sorted cells are CD3+ T cells.

[0080] Cell activation

[0081] In this article, an "activator" is used to activate immune cells, which helps to alleviate cell damage during cell preparation and improve cell viability and survival rate. In theory, any reagent known in the art that can be used for immune cell activation can be used. In an exemplary embodiment where the immune cell is a T cell (especially a CD3+ T cell), the activator includes one or more selected from the following: CD3 antibody, CD28 antibody, 4-1BB antibody, 4-1BBL antigen.

[0082] The activation process includes incubating immune cells under conditions where the activator contacts the cells. In the incubation mixture, the final concentration of the activator, the concentration of the immune cells, and their ratio are not limited. For example, the activator can be coated on a solid-phase carrier, and the final concentration of the coating solution can be 1-20 μg / ml, such as 1, 5, 10, 15, or 20 μg / ml. The concentration ratio of the activator to the immune cells can be 1-20 μg / ml: 2.45-2.8×10 8 immune cells. The activation temperature is any temperature suitable for the growth of immune cells, preferably 27-45 °C, more preferably 37 °C. Usually, the activation is carried out in an environment containing CO2, such as 5% CO2.

[0083] The medium required for activation incubation can be any commercial or self-made medium suitable for the growth of immune cells (such as T cells). In one or more embodiments, activation incubation is carried out using AIM-V medium with or without 5% serum or its substitute. Preferably, the medium also contains cytokines, such as IL-7 and / or IL-15; preferably, the final concentration of IL-7 is 1-50 ng / mL, and the final concentration of IL-15 is 1-50 ng / mL.

[0084] The activator can exist in the incubation mixture in the form of a solute, or it can be immobilized on a solid-phase carrier. Solid-phase carriers that can be used to immobilize activators (such as antibodies) are well known in the art, such as magnetic beads or the container wall. In some embodiments, the activator is a CD3 antibody and a CD28 antibody immobilized on magnetic beads; preferably, the activator is Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads and / or CTS Dynabeads CD3 / 28. In some embodiments, the activator is a CD3 antibody immobilized on the container wall, a CD3 antibody and a CD28 antibody, a CD3 antibody and a 4-1BB antibody, or a CD3 antibody and a 4-1BBL antigen; preferably, the container is a T75 flask.

[0085] In the rapid CAR-T preparation process, the activation in step (2) can be carried out simultaneously with step (3), or the activation time of step (2) is less than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour.

[0086] In the ordinary CAR-T preparation process, in a specific embodiment, the sorted T cells are electroporated 24 - 72 hours after activation with a CD3 antibody-coated container, preferably activated for 48 hours; the sorted T cells are electroporated 24 - 72 hours after activation with a CD3 antibody-coated container and a CD28 antibody, preferably activated for 48 hours; the sorted T cells are electroporated 24 - 72 hours after activation with a CD3 antibody-coated container and a 4-1BBL antigen, preferably activated for 48 hours; the sorted T cells are electroporated 24 - 72 hours after activation with a CD3 antibody-coated container and a 4-1BB antibody; the sorted T cells are electroporated 24 - 72 hours after activation with Miltenyi MACS GMPTransAct CD3 / 28 magnetic beads coated with a CD3 antibody and a CD28 antibody, preferably activated for 48 - 72 hours; the sorted T cells are electroporated 24 - 72 hours after activation with Dynabeads CD3 / 28 magnetic beads coated with a CD3 antibody and a CD28 antibody, preferably activated for 48 - 72 hours. In one or more embodiments, the final concentration of Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads is 1 mL / 1×10 8 T cells, 4 mL / 1×10 8 T cells, or 8 mL / 1×10 8 T cells. In one or more embodiments, the ratio of CTSDynabeads CD3 / 28 magnetic beads to T cells is 1:1.

[0087] Chimeric antigen receptor

[0088] In some embodiments, the CAR comprises an optional signal peptide, an antigen-binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory signal domain, and an intracellular signal domain.

[0089] In some embodiments, the signal peptide is selected from a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, and a light chain signal peptide.

[0090] In some embodiments, the antigen-binding domain targets any one or more of the following antigens: CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, folate receptor alpha, ErbB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, podoplanin, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globoH, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4.

[0091] In some embodiments, the hinge region is selected from the extracellular hinge region of CD8, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4.

[0092] In some embodiments, the transmembrane domain comprises the transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0093] In some embodiments, the intracellular co-stimulatory signaling domain comprises the intracellular domain derived from CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulator, and DNAX-activating protein 10.

[0094] In some embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain.

[0095] In some embodiments, the immune cell is a mesothelin-targeting CAR-T cell. The structure of the CAR is as follows: from the N-terminus to the C-terminus, it sequentially contains a CD8α signal peptide, mesothelin VHH No. 1444, a CD8α hinge region, a CD28 transmembrane region and an intracellular co-stimulatory signaling region, and a CD3ζ intracellular signaling domain; the amino acid sequence of mesothelin VHH No. 1444 is as shown in SEQ ID NO: 36, and the amino acid sequence of the CAR is as shown in SEQ ID NO: 37.

[0096] Plasmid vector

[0097] In some embodiments, the nucleic acid molecule encoding the CAR is DNA, and the non-viral vector is a plasmid vector.

[0098] Vectors generally contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. Such sequences (collectively referred to as "flanking sequences" in certain embodiments) generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting nucleic acids encoding the antibody to be expressed, and optional marker elements. See, for example, WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193.

[0099] When the nucleic acid molecule encoding the CAR is DNA, the nucleic acid molecule is usually integrated into the cell genome by gene editing techniques to stably express the CAR gene. Gene editing techniques include, but are not limited to, homologous recombination; gene editing techniques based on zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR, such as those using Cas9 or cpf1), meganucleases, integrases, recombinases, and transposases.

[0100] Transposons and transposases

[0101] In some embodiments, the non-viral vector is a plasmid vector containing a transposon, and the transposon contains a nucleic acid molecule encoding the CAR.

[0102] DNA transposons can transpose through a non-replicative "cut-and-paste" mechanism. This requires the transposase to recognize two terminal inverted repeats (ITRs), which can cut its target, thereby releasing the DNA transposon from its donor template. After being excised, the DNA transposon can then integrate into the recipient DNA that has been cut by the same transposase.

[0103] The transposon and the corresponding transposase form a transposon system. According to the type of the transposon system, the transposase and the transposon containing the corresponding ITR sequence are selected. The nucleic acid molecule encoding the CAR is located between the ITR sequences in the transposon. In some embodiments, outside the ITR sequences at both ends of the transposon DNA sequence, there is a cleavage site sequence for the transposase, and the cleavage site sequence is TA (nucleotide sequence).

[0104] In some embodiments, the cells in step (2) are also contacted with a transposase or a nucleic acid molecule encoding a transposase. In some embodiments, the nucleic acid molecule encoding the transposase is DNA or RNA. In some embodiments, step (2) causes the cells to contact a plasmid vector containing a nucleic acid molecule encoding a transposase and a transposon. In some embodiments, step (2) causes the cells to contact a plasmid vector containing a nucleic acid molecule encoding a transposase and a plasmid vector containing a transposon.

[0105] The transposon system is selected from: Tol1 transposon system, Tol2 transposon system, Frog Prince transposon system, Minos transposon system, Hsmar1 transposon system, Helraiser transposon system, ZB transposon system, BZ transposon system, Intruder transposon system, SPINON transposon system, TcBuster transposon system, Passer transposon system, JL transposon system, Yabusame-1 transposon system, Uribo2 transposon system, PiggyBac (PB) transposon system, SleepingBeauty (SB) transposon system, and various variants or derivatives of the above transposon systems.

[0106] The ZB transposon system is the ZB transposon system described in any embodiment of patent CN201510429987.3, and the entire content of this application is incorporated herein by reference. A specific embodiment of the ZB transposon system variant is the BZ transposon system, which is the BZ transposon system described in any embodiment of patent CN202211150935.9, and the entire content of this application is incorporated herein by reference. The BZ transposon system includes a BZ transposase and a BZ transposon containing an ITR sequence recognizable by the BZ transposase.

[0107] The BZ transposase is a transposase having any one or more sets of the following mutations compared with SEQ ID NO: 1:

[0108] Q71R\H110R,

[0109] Q71R\Q79R\H110R,

[0110] G216A\Q71R\Q79R\H110R,

[0111] H208V\Q71R\Q79R\H110R,

[0112] H208V\G216A\Q71R\Q79R\H110R, F21K\D22A\Q71R\H110R,

[0113] N005S\F21K / D22A\Q71R\Q79R\H110R, K120S\N125L\Q71R\Q79R\H110R, G216A\H208V\G189A\Q71R\Q79R\H110R, G216A\H208V\K251T\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138K\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138R\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\K134A\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\Q79R\H110R, G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\Q79R\H110R, G216A\Q71R\H110R,

[0114] H208V\Q71R\H110R,

[0115] H208V\G216A\Q71R\H110R,

[0116] G216A\H208V\G189A\Q71R\H110R, G216A\H208V\K251T\Q71R\H110R, G216A\H208V\K251T\G189A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\Q71R\H110R, G216A\H208V\K251T\G189A\Q138R\Q71R\H110R, G216A\H208V\K251T\G189A\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R, G216A\H208V\K251T\G189A\Q138K\Q71R\H110R,

[0117] G216A\H208V\K251T\G189A\Q138R\Q71R\H110R,

[0118] G216A\H208V\K251T\G189A\K134A\Q71R\H110R,

[0119] G216A\H208V\K251T\G189A\Q138K\K134A\Q71R\H110R,

[0120] G216A\H208V\K251T\G189A\Q138R\K134A\Q71R\H110R,

[0121] G216A\H208V\K251T\G189A\Q138K\V144E\Q71R\H110R,

[0122] G216A\H208V\K251T\G189A\Q138K\K137T\Q71R\H110R, or

[0123] N005S\F21K / D22A\Q71R\H110R。

[0124] G216A\H208V,

[0125] G216A\H208V\G189A,

[0126] G216A\H208V\K251T,

[0127] G216A\H208V\K251T\G189A,

[0128] G216A\H208V\K251T\G189A\Q138K,

[0129] G216A\H208V\K251T\G189A\Q138R,

[0130] G216A\H208V\K251T\G189A\K134A,

[0131] G216A\H208V\K251T\G189A\Q138K\K134A,

[0132] G216A\H208V\K251T\G189A\Q138R\K134A,

[0133] G216A\H208V\K251T\G189A\Q138K\V144E, or

[0134] G216A\H208V\K251T\G189A\Q138K\K137T.

[0135] Among them, the first group of mutations Q71R\H110R means that the BZ transposase contains the mutation sites Q71R and H110R compared with SEQ ID NO: 1, and the other groups of mutations are similar.

[0136] The BZ transposon contains a nucleic acid molecule encoding a CAR and ITR sequences recognizable by the BZ transposase at both ends of the nucleic acid molecule encoding the CAR. The ITR sequences are as shown in SEQ ID NO: 2 or 3, or compared with SEQ ID NO: 2 or 3, the CpG motif therein is mutated to TpG or CpA.

[0137] The Passer (PS) transposon system is the PS transposon system described in any embodiment of Patent CN201910366530.0, and the entire content of this application is incorporated herein by reference. A specific embodiment of the PS transposon system variant is the JL transposon system, which is the JL transposon system described in any embodiment of CN202310081106.8, and the entire content of this application is incorporated herein by reference.

[0138] In some embodiments, the JL transposon system includes a JL transposase and a JL transposon containing ITR sequences recognizable by the JL transposase.

[0139] In some embodiments, the JL transposase is a mutant transposase of the PS transposase as shown in SEQ ID NO: 4, having one or more of the following mutations compared to the PS transposase as shown in SEQ ID NO: 4: TQS57-59KKA, T129R, T129K, I98K, TQ57-58RK, TQ57-58RK\T129K, TQ57-58RK\T129R, E32K, E32K\T129K, E32K\T129R, TQ57-58RK\I98K, TQ57-58RK\I98K\T129K, TQS57-59KKA\I98K, TQS57-59KKA\I98K\T129K, R123H, Q136K, K16R, E47K, TQ57-58RR, E32K\T57R\Q58R, T57R, T57K, Q58K, Q58R, S59A, M95L, Y46Q, A8S, T187K, I35V, N199H, N193S, T350S, Q22K, T368E, N213D, H24R, T150A, H165D, K55R, K73R, L228M, E335S, K159H, V359L, T129Q, H215K, R51K, A84L, Q69E, I284L, K45R, H215E, H215Q, I237V.

[0140] In some embodiments, the JL transposase is a transposase fused with a functional polypeptide on a wild-type PS transposase or a mutant transposase containing the above mutations, and the functional polypeptide is a DNA sequence-specific or non-specific binding domain and / or a nuclear localization signal domain. The DNA sequence-specific or non-specific binding domain includes a leucine zipper domain, a CRISPR / Cas domain, a TALE domain, a zinc finger domain, an AAV Rep DNA binding domain, or any combination thereof. The nuclear localization signal domain includes SV40 NLS, C-myc NLS, TAF1 NLS, TP53 NLS, STAT3 NLS, or any combination thereof.

[0141] The JL transposon contains a nucleic acid molecule encoding a CAR, and ITR sequences recognizable by the JL transposase at both ends of the nucleic acid molecule encoding the CAR. The ITR sequences are as shown in any one of SEQ ID NO: 6-13.

[0142] In some embodiments, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

[0143] In some embodiments, the plasmid vectors of the transposons include, but are not limited to, conventional circular DNA plasmids, linear DNA plasmids, minicircle plasmids, nanoplasmids, Doggybone, and other DNA forms that do not contain antibiotic or / and replicon DNA sequences. In some embodiments, the DNA vector is a DNA microvector, and the DNA backbone sequence of the microvector does not contain an antibiotic expression cassette and is preferably limited to a length within 600 bp, and / or does not contain CpG DNA motifs. In some embodiments, the DNA vector is an antibiotic-free minicircle plasmid, that is, a minicircle plasmid without an antibiotic resistance gene (a minicircle plasmid without an antibiotic expression cassette), also known as a tiny or tiniplasmid. The antibiotic-free minicircle plasmids applicable to the present invention can refer to patent application 202310072956., the entire content of which is incorporated herein by reference.

[0144] In some embodiments, the antibiotic-free minicircle plasmid contains a nucleotide sequence encoding an antitoxin protein and a replicon; the amino acid sequence of the antitoxin protein contains the following sequences: (1) the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having one or more mutations of E24D, I35V, V43I compared with SEQ ID NO: 14; or (2) the amino acid sequence shown in SEQ ID NO: 17, or an amino acid sequence having one or more mutations of T6I, T43A, K47E, A50S, E51D, G52A, N54K compared with SEQ ID NO: 17; the length of the replicon is ≤800 bp, preferably ≤600 bp or ≤300 bp.

[0145] In some embodiments, the amino acid sequence of the antitoxin protein is any one of SEQ ID NOs: 14-20.

[0146] In some embodiments, the replicon is selected from ColE1, ColE2, pMB1, pSC101, RSF, R6K, pUC57, RK2, and p15A; preferably R6K or pUC57.

[0147] In some embodiments, the length of the plasmid backbone of the antibiotic-free minicircle plasmid is ≤1000 bp, preferably ≤900 bp, ≤800 bp or ≤600 bp.

[0148] In some embodiments, the nucleotide sequence encoding the antitoxin protein does not contain CpG motifs. Preferably, the nucleotide sequence encoding the antitoxin protein is as shown in SEQ ID NO: 21 or 22.

[0149] In some embodiments, the nucleotide sequence of the replicon does not contain CpG motifs.

[0150] In a preferred embodiment, the length of the backbone sequence of the antibiotic-free microplasmid is ≤600 bp, and the replicon is an R6K replicon without CpG motifs. The nucleotide sequence of the R6K replicon without CpG motifs is shown in SEQ ID NO: 23.

[0151] In some embodiments, the nucleotide sequence of the antibiotic-free microplasmid (empty vector) is shown in SEQ ID NO: 24 or 25; the map structure is as Figure 3 or Figure 4.

[0152] Cell transduction

[0153] Step (3) Contact the cells with a nucleic acid molecule encoding a CAR to introduce the nucleic acid molecule into the cells.

[0154] In some embodiments, the nucleic acid molecule encoding a CAR is RNA, such as mRNA, saRNA, and the non-viral vector is LNP, LPX, VLP, inorganic nanoparticles or exosomes. The RNA molecule encoding a CAR is transfected into cells through a non-viral vector and can be used for transient expression of CAR. In some embodiments, the RNA molecule encoding a CAR can also be introduced into cells directly by electroporation without a vector.

[0155] In some embodiments, in step (3), the cells are contacted with a plasmid vector of a transposon and a transposase or mRNA encoding a transposase. The plasmid vector of the transposon contains a CAR gene expression cassette and ITR sequences recognizable by the transposase located at both ends of the CAR gene expression cassette. The CAR gene expression cassette may contain gene functional elements such as a promoter, a nucleic acid molecule encoding a CAR, and a polyA signal sequence.

[0156] In some embodiments, in step (3), the cells are contacted with a plasmid vector of a transposon. The plasmid vector of the transposon contains a CAR gene expression cassette, ITR sequences recognizable by the transposase located at both ends of the CAR gene expression cassette, and a nucleic acid molecule encoding a transposase. At this time, the transposon and the nucleic acid molecule encoding a transposase are located on the same plasmid vector.

[0157] In some embodiments, in step (3), the cells are contacted with a plasmid vector of a transposon and a plasmid vector of a transposase. The plasmid vector of the transposon contains a CAR gene expression cassette and ITR sequences recognizable by the transposase located at both ends of the CAR gene expression cassette. The plasmid vector of the transposase contains a transposase gene expression cassette. At this time, the transposon and the nucleic acid molecule encoding a transposase are located on different plasmid vectors.

[0158] In some embodiments, step (3) includes: contacting the cells with a DNA vector containing a JL transposon and an mRNA encoding a JL transposase, wherein the JL transposon includes a CAR gene expression cassette and terminal inverted repeats located on both sides of the CAR gene expression cassette.

[0159] In some embodiments, the amino acid sequence of the JL transposase is as shown in SEQ ID NO:5.

[0160] In some embodiments, the terminal inverted repeats are as shown in SEQ ID NO:6 (3' ITR) and SEQ ID NO:7 (5' ITR).

[0161] In some embodiments, the plasmid map of the DNA vector containing the JL transposon is as Figure 2 shown.

[0162] In some embodiments, in step (3), when contacting the cells with a nucleic acid molecule encoding a CAR, the nucleic acid molecule encoding a CAR is introduced into the cells, and the introduction includes transfecting the cells by means of electroporation, microinjection, calcium phosphate precipitation, cationic polymers, dendrimers, liposomes, lipid nanoparticles (LNP), particle bombardment, fugene, direct acoustic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, nucleofection or any combination thereof.

[0163] In some embodiments, the introduction includes contacting the cells with an mRNA encoding a transposase and a plasmid containing a transposon. Preferably, the dosage of the mRNA is 1 - 30 μg per 1×10 7 cells, and the dosage of the plasmid is 0.1 - 5 μg per 1×10 7 cells. The most preferred dosage of the mRNA is 15 μg per 1×10 7 cells, and the dosage of the plasmid is 4 - 5 μg per 1×10 7 cells.

[0164] In some embodiments, the cells are contacted with a nucleic acid molecule encoding a CAR no later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour after the cells start to be contacted with an activator.

[0165] In some embodiments, the contact is adding a transposon plasmid containing a nucleic acid molecule encoding a CAR and an mRNA encoding a transposase to the culture medium of the cells and the activator after the activation in step (2) is completed, and then introducing the transposon plasmid containing a nucleic acid molecule encoding a CAR and the mRNA encoding a transposase into the cells by electroporation.

[0166] In some embodiments, after the activation in step (2) is completed, the activator is removed from the culture medium, and then a transposon plasmid containing a nucleic acid molecule encoding a CAR and an mRNA encoding a transposase are added to the culture medium containing the activated cells.

[0167] In some embodiments, the cell can express a therapeutic agent and / or contain a coding sequence of a therapeutic agent, and in step (3), the cell is further contacted with a nucleic acid molecule of the therapeutic agent to introduce the nucleic acid molecule of the therapeutic agent into the cell.

[0168] In some embodiments, the nucleic acid molecule of the therapeutic agent is also located on the plasmid vector of the transposon.

[0169] In some embodiments, the nucleic acid molecule of the therapeutic agent and the nucleic acid molecule encoding a CAR are located in the same transposon plasmid vector. The gene expression cassette of the therapeutic agent and the gene expression cassette of the CAR can be linked by a cleavable linker (such as a 2A linker) and located between the ITRs at both ends; or, the gene expression cassette of the therapeutic agent and the gene expression cassette of the CAR are respectively located between two sets of ITRs.

[0170] In some embodiments, the nucleic acid molecule of the therapeutic agent and the nucleic acid molecule encoding a CAR are located in different transposon plasmid vectors. The transposon plasmid vector containing the nucleic acid molecule of the therapeutic agent is similar in structure to the above-mentioned transposon plasmid vector containing the nucleic acid molecule encoding a CAR, and the only difference is that the gene expression cassette of the CAR is replaced with the gene expression cassette of the therapeutic agent.

[0171] In some embodiments, the nucleic acid molecule of the therapeutic agent and the nucleic acid molecule encoding a CAR are located in different transposon plasmid vectors.

[0172] In some embodiments, in step (2), the cell is contacted with a plasmid vector containing a nucleic acid molecule of a therapeutic agent, a plasmid vector containing a nucleic acid molecule encoding a CAR, and a transposase or an mRNA encoding a transposase to introduce the nucleic acid molecule encoding a CAR and the nucleic acid molecule of the therapeutic agent into the cell simultaneously.

[0173] In some embodiments, the therapeutic agent is an antibody (such as a single-chain antibody, a single-domain antibody, a bispecific antibody) or a cytokine.

[0174] In some embodiments, the therapeutic agent is an immune checkpoint inhibitor.

[0175] In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that targets any one or more of PD-1, LAG-3, TIM3, B7-H1, CD160, P1H, 2B4, CEACAM (such as CEACAM-1, CEACAM-3, and / or CEACAM-5), TIGIT, CTLA-4, BTLA, and LAIR1.

[0176] In some embodiments, the therapeutic agent is an antibody that targets PD-1, preferably a single-domain antibody that targets PD-1. The sequence of the single-domain antibody that targets PD-1 is the single-domain antibody that targets PD-1 described in any embodiment of Patent CN202011582908.X, the entire content of which is incorporated herein by reference.

[0177] In some embodiments, the sequence of the single-domain antibody that targets PD-1 is as shown in any of SEQ ID NO:26-29.

[0178] In some embodiments, the therapeutic agent is an antibody that targets CTLA-4, preferably a single-domain antibody that targets CTLA-4. The sequence of the single-domain antibody that targets CTLA-4 is the single-domain antibody that targets CTLA-4 described in any embodiment of Patent CN202111152925.4, the entire content of which is incorporated herein by reference.

[0179] In some embodiments, the sequence of the single-domain antibody that targets CTLA-4 is as shown in SEQ ID NO:30.

[0180] In some embodiments, the therapeutic agent is a bispecific antibody that contains a first functional region that targets PD-1 and a second functional region that targets CTLA4. In some embodiments, the bispecific antibody is the bispecific antibody described in any embodiment of Patent CNCN202310338674.1, the entire content of which is incorporated herein by reference.

[0181] In some embodiments, the first functional region and the second functional region in the bispecific antibody are fused through a linker, and the linker is (GGSGG)p or (G4S)mGn, where m, n, and p are each independently positive integers from 1 to 10.

[0182] In some embodiments, the bispecific antibody further contains an Fc region and / or a cmyc-his tag; for example, the Fc region is the Fc region of IgG1, IgG2, IgG3, or IgG4.

[0183] In some embodiments, the sequence of the bispecific antibody is as shown in any of SEQ ID NO:31-34.

[0184] Cytokine

[0185] In some embodiments, step (2) and / or (3) is carried out in a cell culture medium (such as a serum-free medium) containing IL-2, IL-15, IL-6, an LSD1 inhibitor, or a MALT1 inhibitor. In some embodiments, steps (2) and (3) are carried out in a cell culture medium (such as a serum-free medium) containing IL-7, IL-21, or a combination thereof. In some embodiments, step (2) and / or (3) is carried out in a cell culture medium (such as a serum-free medium) containing IL-2, IL-15, IL-21, IL-7, IL-6, an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof. In some embodiments, the cell culture medium is a serum-free medium containing a serum replacement (SR).

[0186] Harvest cells

[0187] After introducing the nucleic acid molecule encoding the CAR into the cells, a cell culture step may also be included before harvesting. The culture can be carried out using any conditions and media suitable for the growth of immune cells known in the art. Exemplarily, the electroporated T cells of the present invention are cultured at 37 °C and 5% CO2 in AIM-V medium with or without 5% serum or its replacement. The medium may also contain cytokines, such as IL-7 and / or IL-15; preferably, the final concentration of IL-7 is 1-25 ng / mL, and the final concentration of IL-15 is 1-25 ng / mL; preferably, the cytokines are added to the medium 0-6 hours after the start of the culture.

[0188] In the rapid CAR-T process, the cells are cultured with substantially no expansion before harvesting, and the culture time is less than 48, 36, 30, 24, 18, 12, 6, 3, 2, or 1 hour.

[0189] Step (4) is no later than 48, 36, 30, 24, 18, 12, 6, 3, 2, or 1 hour after the start of step (3); or step (4) is no later than 72, 60, 48, 36, 30, 24, 20, 18, 12 hours after the start of step (2).

[0190] In the conventional CAR-T process, the electroporated immune cells can be expanded in culture. The culture time is at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, preferably 3-13 days.

[0191] In some embodiments, the method is carried out in a closed system. In some embodiments, the entire processes of sorting, activation, transduction, culture and harvesting are carried out in a closed system.

[0192] Drug composition

[0193] The present invention also provides cells expressing a chimeric antigen receptor prepared by the preparation method of any one of the embodiments.

[0194] The present invention also provides the use of the cells expressing a CAR in the preparation of a medicament for treating or preventing a malignant tumor.

[0195] In some embodiments, the tumor is a solid cancer, such as selected from: mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, kidney cancer, liver cancer, brain cancer, thymoma, sarcoma, malignant epithelial tumor (carcinoma), uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer, or one or more of their metastatic cancers. In some embodiments, the cancer is a liquid cancer, such as selected from: chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B cell acute lymphoblastic leukemia (BALL), T cell acute lymphoblastic leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorder, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, heavy chain disease, plasmacytic myeloma, solitary plasmacytoma of bone, extramedullary plasmacytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, ALK+ large B cell lymphoma, large B cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B cell lymphoma, acute myeloid leukemia (AML), or lymphoma of undetermined classification.

[0196] The CAR-expressing cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as relevant cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention can comprise CAR-expressing cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable excipients (such as carriers, diluents or excipients). Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0197] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The quantity and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease.

[0198] When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual variations in the condition of the patient (subject). The cells can be administered by infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient's signs of disease and thus adjusting the treatment.

[0199] Administration of the subject compositions can be effected in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally to a patient. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the CAR-expressing cell compositions of the present invention are preferably administered by intravenous injection. The compositions of CAR-expressing cells can be directly injected into tumors, lymph nodes or sites of infection.

[0200] The present invention will be illustrated by way of specific examples hereinafter. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are conventional materials and methods in the art unless otherwise specified.

[0201] Experimental methods

[0202] The cell phenotypes of PBMC and CAR-T were determined using a multi-color flow cytometer. Anti-human CD45 antibody (Biolegend), anti-human CD16 antibody (Thermo), anti-human CD3 antibody (Biolegend), and anti-human CD56 antibody (Biolegend) were used to detect the phenotypes of T cells, NK cells, and B cells. Anti-human CD3 antibody (BD Biosciences), anti-human CD4 antibody (Biolegend), and anti-human CD8 antibody (Thermo) were used to evaluate the purity of the enriched T cells. Biotin-binding protein and streptavidin PE (BD Biosciences) antibodies from MSLN were used to evaluate the CAR transduction efficiency. To study the differentiation of CAR-T cells during culture, anti-human CD45RO antibody (Biolegend) and anti-human CCR7 antibody (Biolegend) were used. Anti-human PD-1 antibody (BD Biosciences), anti-human LAG-3 antibody (Biolegend), and anti-human TIM-3 antibody (BD Biosciences) were used to evaluate CAR-T cell exhaustion. The cells to be tested were washed in phosphate-buffered saline (PBS) and incubated with the respective antibodies at 4°C for 15 minutes. Subsequently, the cells were washed with 4 mL of PBS and resuspended in 300 μl of PBS. Flow cytometry analysis was performed using a CytoFLEX S instrument from Beckman. The collected data were analyzed using Kaluza analysis software.

[0203] Real-time cell analysis was used to detect the cytotoxicity of CAR-T. First, 1×10 4 target cells were seeded per well in an E-96-cell plate (Agilent). Subsequently, the plate was placed in an xCElligence RTCA SP instrument (Agilent) to monitor the growth of tumor cells. Once the cell index exceeded 1, the following steps were performed. Within 16 - 24 hours after CAR-T cell resuscitation, two different effector-to-target ratios (E:T ratios: 4:1, 2:1) and CAR positivity rates were considered. Then the corresponding number of positive cells was seeded into the E-96-cell plate previously seeded with tumor cells. Subsequently, the plate was placed in an xCElligence RTCA SP instrument (Agilent) to monitor the cytotoxic effect. After 24 hours, the cytotoxicity data were analyzed.

[0204] For the cytokine release assay, 1×10 4 target cells were co-cultured with CAR-T cells at an E:T ratio of 4:1 for 24 hours. The detection of cytokines IL-5, IL-13, IL-2, IL-6, IL-10, IFN-γ, TNF-α, and IL-4 was performed using the LEGENDplexTM multi-analyte flow assay kit manual.

[0205] Example

[0206] Example 1, Cryopreservation, Sorting and Recovery of T Cells

[0207] 1. Collect 50 - 60 mL of leukapheresis samples from 4 healthy donors using Spectra Optia, and isolate peripheral blood mononuclear cells (PBMCs) by Ficoll density gradient centrifugation. The PBMCs are divided into 4 parts. One part is immediately made into CAR - T cells (0M), and the other part is cryopreserved in liquid nitrogen for 3 months (3M), 6 months (6M), and 12 months (12M) using self - made cryopreservation medium before making CAR - T cells.

[0208] The cryopreserved cells are resuscitated using EDTA / PBS (containing 5‰ HSA). T cell sorting is performed using Miltenyi CD4 / CD8 magnetic beads. 1×10 9 WBC is added to 200 μL of CD4 magnetic beads and 200 μL of CD8 magnetic beads. After incubation for 30 min, CD4+T and CD8+T cells are screened through an LS sorting column.

[0209] 2. After sorting, the cells are seeded into culture flasks containing medium (AIM - V + 5% SR) at a cell density of 1E6 / mL, and IL - 7 & IL - 15 (final concentrations of 25 ng / mL & 25 ng / mL) and Miltenyi MACS GMP TransAct CD3 / 28 magnetic beads are added. The cells are placed at 37℃ and 5% CO2 for 2 days.

[0210] Example 2, Preparation of CAR - T Using PB Transposon System

[0211] Take the activated T cells from Example 1, 1*10 7 cells / group, add piggybac enzyme mRNA at 320 μg / mL and plasmid P19V21 expressing the MSLN CAR sequence at 90 μg / mL. Transfer the mixture to an electroporation cuvette, place it in a Lonza Nucleofactor 4D or Maxcyte electroporator, and select the program numbered FI - 115 or Resting T / Expand T4 for electroporation; transfer the electroporated cell suspension to a T75 culture flask (the culture medium is AIM - V medium containing 5% SR, 25 ng / mL IL7, and 25 ng / mL il15), mix well, and place it at 37℃. The culture medium is supplemented every 2 - 3 days to ensure the optimal growth conditions of the cells. On the 11th day, after washing the cells with physiological saline, add cryopreservation solution and store in liquid nitrogen.

[0212] Among them, the amino acid sequence of the piggybac enzyme is shown in SEQ ID NO: 39. The plasmid map of plasmid P19V21 is as Figure 1 shown, and the sequence is shown in SEQ ID NO: 35. The structure of MSLN CAR is as follows: from the N-terminus to the C-terminus, it sequentially contains the CD8α signal peptide, mesothelin VHH No. 1444, CD8α hinge region, CD28 transmembrane region and intracellular co-stimulatory signal region, and CD3ζ intracellular signal domain; the amino acid sequence of mesothelin VHH No. 1444 is shown in SEQ ID NO: 36, and the amino acid sequence of MSLN CAR is shown in SEQ ID NO: 37.

[0213] Example 3. Preparation of CAR-T using the JL transposon system

[0214] Take the T cells activated in Example 1, 1×10 7 cells / group, and add the plasmid dosage of the plasmid expressing MSLN CAR at 5 μg / mL and the mRNA dosage of JL enzyme at 2.5 μg / mL. Transfer the mixture to an electroporation cuvette, place it in a Lonza Nucleofactor 4D or Maxcyte electroporator, and select the program numbered FI-115 or Resting T / Expand T4 for electroporation; transfer the electroporated cell suspension to a T75 culture flask (the culture medium is AIM-V medium containing 5% SR), mix well, place it at 37°C, and perform medium exchange every 2-3 days to ensure the optimal growth conditions of the cells. On the 11th day, wash the cells with saline, add cryopreservation solution and store them in liquid nitrogen.

[0215] Among them, the plasmid map of the plasmid expressing MSLN CAR is as Figure 2 shown, and the sequence is as SEQ ID NO: 38; except for the transposon, the plasmid backbone includes the nucleotide sequences of R6K replicon and antitoxin protein 0637. The nucleotide sequence of R6K replicon is shown in SEQ ID NO: 23, and the nucleotide sequence of antitoxin protein 0637 is shown in SEQ ID NO: 21. The structure of MSLN CAR is the same as in step 3, and the amino acid sequence of JL enzyme is shown in SEQ ID NO: 5.

[0216] Example 4. Preparation of rapid CAR-T using the JL transposon system

[0217] Take the T cells activated for 5 h, 1×10 7Cells / group, add 5 μg / mL of the plasmid expressing MSLN CAR and 2.5 μg / mL of JL enzyme mRNA. Transfer the mixture to an electroporation cuvette, place it in a Lonza Nucleofactor 4D or Maxcyte electroporator, and select the program numbered FI-115 or Resting T / Expand T4 for electroporation; transfer the electroporated cell suspension to a T75 culture flask (the culture medium is AIM-V medium containing 5% SR), mix well, place it at 37°C and 5% CO2 for no more than 24 h, and harvest CAR-T cells. From cell sorting to harvesting CAR-T cells, the total duration of the product preparation cycle is 24 h respectively, including 3 h for sorting, 5 h for activation, 1 h for electroporation, 13 h for culture, and 2 h for formulation.

[0218] Example 5, PBMC cell viability and phenotype

[0219] The cell viability of PBMC is a key factor directly affecting the success rate of CAR-T cell manufacturing. The present invention compared the effects of different freezing times on the cell viability of PBMC. After fresh group (0M), freezing for 3 months (3M), freezing for 6 months (6M), and freezing for 12 months (12M), the survival rates of PBMC were 96.59%, 91.45%, 91.43%, and 89.53% ( Figure 5 ). The survival rate decreased slightly after freezing, but there was no significant difference in cell viability between frozen-thawed PBMC and fresh PBMC. In addition, the cell phenotypes of PBMC after different freezing preservation times were also compared. Multicolor flow cytometry analysis was performed on PBMC samples to determine the proportions of T cells, B cells, NK cells, and the CD4 / CD8 ratio. The results showed that freezing preservation had no significant effect on the proportions of T cells, NK cells, B cells, and the CD4 / CD8 ratio ( Figure 6 ). Interestingly, the proportions of NK cells and B cells decreased slightly after freezing. This may be related to the cell state during flow cytometry analysis, but it does not affect the preparation of CAR-T cells obtained by modifying CD3+ T cells. Finally, the differentiation status of CD3+ T cells in fresh and frozen-thawed PBMC was detected by CD45RO and CCR7 staining. The results showed that compared with fresh PBMCs, the proportions of Tn (CD45RO-CCR7+) and Tem (CD45RO+CCR7-) in T cells increased significantly after freezing preservation for 12M ( Figure 7 ). Some studies have shown that Tn and Tem cells can enhance the activation ability, persistence, and effector function of CAR-T cells, and improve the efficacy of CAR-T cell therapy. In summary, the cryopreservation process itself does not have a negative impact on the cell viability and subsets of PBMC.

[0220] Example 6, Viability, Expansion, and Phenotype of CAR-T Cells Prepared with the PB Transposon System

[0221] CAR-T cells were generated from fresh or cryopreserved PBMCs through the CD4 / CD8 magnetic bead separation process, 48-hour TransAct TM activation, followed by electroporation with the CAR vector, and culturing the cells for 9 days ( Figure 8 ). First, the present invention tested the effect of cryopreservation on cell viability. Compared to CAR-T cell cultures initiated with fresh PBMCs, cryopreserved-thawed PBMC-initiated CAR-T cells did not show significant differences in cell viability ( Figure 9 ). Previous studies have shown the effect of cryopreservation on cell proliferation. Therefore, we evaluated the proliferation levels of cryopreserved and fresh PBMCs on days -2, 0, 3, 7, and 11 of production. Our results showed that cryopreservation decreased the proliferation of non-virally produced CAR-T cells, but the differences were not significant ( Figure 10 ). Evaluation of the final product phenotype showed no significant differences in mean CD3 purity ( Figure 11 ), mean CD4:CD8 cell ratio ( Figure 12 ), and mean CAR-T transduction efficiency ( Figure 13 ). Therefore, the study demonstrated that the non-viral transposon electroporation process was successful from cryopreserved PBMCs used to manufacture CAR-T cells and showed no significant differences in cell viability, expansion, and cell phenotype when juxtaposed with fresh PBMCs.

[0222] Example 7, Differentiation and Exhaustion Characteristics of CAR-T Cells Prepared with the PB Transposon System

[0223] The proportion of naive T and memory T in CAR-T cell products is a key indicator for evaluating their function. The T cell differentiation status was investigated by staining cells with fluorescent dye-labeled CD45RO and CCR7-specific antibodies. This analysis showed that the components of TEM (CD45RO+CCR7-) and TCM (CD45RO+CCR7+) changed less, while the components of Tn (CD45RO-CCR7+) and Teff (CD45RO-CCR7-) increased significantly ( Figure 14 ). Further analysis was performed to determine whether cryopreservation affected the persistence of CAR-T cell function. Exhaustion signals of CAR-T cells: PD1+, TIM3+, and LAG3+ were detected. The results showed that the proportions of PD1+ and TIM3+ cells were basically not affected by cryopreservation. An increase in LAG3+ levels was observed in CAR-T cells prepared from cryopreserved PBMCs, but the difference was not significant compared to the fresh group ( Figure 15) These findings suggest that cryopreservation does not accelerate the exhaustion of CAR-T cells. Therefore, all results may indicate that the functions of cryopreserved and fresh PBMC-based CAR-T preparations are similar.

[0224] Example 8, Efficacy and Safety of CAR-T Prepared by PB Transposon System

[0225] The efficacy and safety of CAR-T cell products are intrinsically linked to the secretion of cytokines. Excessive secretion of cytokines after tumor cell stimulation may trigger cytokine storms. Therefore, this invention compared the levels of certain cytokines in CAR-T cell products, including IL-13, IL-10, IL-6, IL-5, IL-4, IL-12, IFN-γ, and TNF-α. The research results showed that in the cryopreserved PBMC group, the levels of IL-13, IL-10, IL-4, IFN-γ, and TNF-α were significantly reduced, while the levels of IL-6, IL-5, and IL-12 remained basically unchanged ( Figure 16 ). Next, the killing ability of CAR-T cells against tumor cells was carried out. CAR-T cells were incubated with SKOV3 tumor cells, and the subsequent growth of tumor cells was monitored by real-time cell analysis. The research results showed that there was no significant difference in the cytotoxicity of CAR-T cells from fresh or cryopreserved PBMC at effector-to-target ratios of 4:1 and 2:1 ( Figure 17 ). Therefore, all results indicate that CAR-T cells manufactured with cryopreserved PBMC have no side effects on CAR-T cytotoxicity.

Claims

1. A method for preparing cells expressing chimeric antigen receptors from cryopreserved cells, characterized in that, The method includes: (1) resuscitating cryopreserved cells; (2) contacting the cells with an activator for activation; (3) contacting the cells with a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule encoding a CAR is on a non-viral vector to introduce the nucleic acid molecule into the cells; (4) harvesting the cells.

2. The method according to claim 1, characterized in that The cryopreserved cells are cryopreserved PBMCs or T cells, and the cells expressing a chimeric antigen receptor are CAR-T cells.

3. The method according to claim 1 or 2, characterized in that, The cryopreserved cells are resuscitated using a serum-free medium, and the serum-free medium may contain serum or a serum substitute; Preferably, the serum-free medium further contains one or more of an apoptosis protein inhibitor and an insulin-transferrin-selenium additive, and the action pathways of the apoptosis protein inhibitor include one or more of p-JAK2, p-STAT3, caspase-3, Bax, and ROCK.

4. The method according to any one of claims 1 to 3, characterized in that, The cells in step (1) are PBMCs, and in step (1), sorting of the resuscitated cells is further included, and the sorting is performed using CD4 / CD8 magnetic beads to sort out CD3+ T cells.

5. The method according to any one of claims 1-4, characterized in that, The activator is selected from: a CD3 antibody, a CD28 antibody, a 4-1BB antibody, and a 4-1BBL antigen; Preferably, the activator is selected from the following group: a CD3 antibody, a CD3 antibody and a CD28 antibody, a CD3 antibody and a 4-1BB antibody, and a CD3 antibody and a 4-1BBL antigen.

6. The method according to any one of claims 1-5, characterized in that, The nucleic acid molecule encoding a CAR is DNA, and the non-viral vector is a plasmid vector; Or the nucleic acid molecule encoding a CAR is RNA, such as mRNA or saRNA, and the non-viral vector is an LNP, an LPX, a VLP, an inorganic nanoparticle, or an exosome.

7. The method according to any one of claims 1 to 6, characterized in that, The non-viral vector is a plasmid vector containing a transposon, the transposon contains a nucleic acid molecule encoding a CAR, and the cells in step (3) are further contacted with a transposase or a nucleic acid molecule encoding a transposase; The transposon and the transposase belong to the same transposon system. Preferably, the transposon system is selected from: Tol1 transposon system, Tol2 transposon system, Frog Prince transposon system, Minos transposon system, Hsmar1 transposon system, Helraiser transposon system, ZB transposon system, BZ transposon system, Intruder transposon system, SPINON transposon system, TcBuster transposon system, Passer transposon system, JL transposon system, Yabusame-1 transposon system, Uribo2 transposon system, PiggyBac (PB) transposon system, SleepingBeauty (SB) transposon system, and various variants or derivatives of the above transposon systems; More preferably, the transposon system is a PB transposon system, a BZ transposon system, or a JL transposon system.

8. The method according to any one of claims 1 to 7, characterized in that, The introduction is performed by electroporation.

9. The method according to any one of claims 1-8, characterized in that, Wherein, The method further satisfies at least any one or more of the following conditions (a)-(c): (a) Step (3) is carried out together with step (2) or carried out no later than 48, 36, 24, 20, 16, 12, 8, 5, 4, 3, 2 or 1 hour after the start of step (2), (b) Step (4) is carried out no later than 48, 36, 30, 24, 18, 12, 6, 3, 2 or 1 hour after the start of step (3); (c) Step (4) is carried out no later than 72, 60, 48, 36, 30, 24, 20, 18, 12 hours after the start of step (2).

10. The method according to any one of claims 1-8, characterized in that, In step (2), the activation time is 12 - 84 hours, preferably 24 - 72 hours, more preferably 24, 48 or 72 hours; And / or, in step (4), a cell culture step is further included before harvesting, and the culture time is 3 - 10 days.

11. Cells expressing a chimeric antigen receptor prepared by the preparation method according to any one of claims 1 - 10.

12. Use of the cells expressing a chimeric antigen receptor according to claim 11 in the preparation of a medicament for treating and / or preventing malignant tumors.

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